John P Konhilas

Interests

No activities entered.

Courses

Scholarly Contributions

Chapters

• Lopez-Pier, M., Lipovka, Y., & Konhilas, J. P. (2016). Inherited Cardiomyopathies. In Cardiomyopathies. Intech Publishing.

Journals/Publications

• Hoyer-Kimura, C., Konhilas, J., Pires, P., Banek, C., Dennis, M., Salcedo, V., & Hay, M. (2023).

  A novel therapeutic Ang(1-7) analogue improves neurovascular function in mice with vascular contributions to cognitive impairment and dementia

  . Physiology, 38(S1). doi:10.1152/physiol.2023.38.s1.5735136
• Hoyer-Kimura, C., Langlas, P., Lopez-Schultz, M., Salcedo, V., Fricks, J., & Konhilas, J. (2023).

  Sex, age and menopause differentially impact gut barrier function and proteomic profile

  . Physiology, 38(S1). doi:10.1152/physiol.2023.38.s1.5735182
• Skaria, R. S., Lopez‐Pier, M. A., Kathuria, B. S., Leber, C. J., Langlais, P. R., Aras, S. G., Khalpey, Z. I., Hitscherich, P. G., Chnari, E., Long, M., Churko, J. M., Runyan, R. B., & Konhilas, J. P. (2023).

  Epicardial placement of human placental membrane protects from heart injury in a swine model of myocardial infarction

  . Physiological Reports, 11(20). doi:10.14814/phy2.15838
• Hay, M., Sweitzer, N., Konhilas, J., Hoyer‐Kimura, C., & Ryan, L. (2022). Circulating neurodegeneration biomarkers neurofilament light protein and p‐tau181 are increased in individuals with heart failure at risk for vascular cognitive impairment. Alzheimer's & Dementia, 18(S5). doi:10.1002/alz.059920
• Keresztes, A., Olson, K., Nguyen, P., Lopez-Pier, M. A., Hecksel, R., Barker, N. K., Liu, Z., Hruby, V., Konhilas, J., Langlais, P. R., & Streicher, J. M. (2022). Antagonism of the mu-delta opioid receptor heterodimer enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein kinase II signaling. Pain, 163(1), 146-158.
  More info

  The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.
• Streicher, J. M., Langlais, P. R., Konhilas, J. P., Hruby, V., Liu, Z., Barker, N. K., Hecksel, R., Lopez-Pier, M. A., Nguyen, P., Olson, K., & Keresztes, A. (2022). Antagonism of the Mu-Delta Opioid Receptor Heterodimer Enhances Opioid Anti-Nociception by Activating Src and CaMKII Signaling. Pain.
• Celli, B., McCormack, M., Rahman, I., Schnellmann, R. G., Langlais, P. R., Konhilas, J. P., Zmijewski, J., Casanova, C., Ledford, J., Tesfaigzi, Y., Sauler, M., Doubleday, K., Hansel, N. N., Hersh, C. P., Kinney, G. L., Fawzy, A., Owen, C. A., Cordoba-Lanus, E., Cleveland, K., , Zhang, D., et al. (2020). Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment. American Journal of Respiratory and Critical Care Medicine.
• Hay, M., Ryan, L., Huentelman, M., Konhilas, J., Hoyer-Kimura, C., Beach, T. G., Serrano, G. E., Reiman, E. M., Blennow, K., Zetterberg, H., & Parthasarathy, S. (2021). Serum Neurofilament Light is elevated in COVID-19 Positive Adults in the ICU and is associated with Co-Morbid Cardiovascular Disease, Neurological Complications, and Acuity of Illness. Cardiology and cardiovascular medicine, 5(5), 551-565.
  More info

  In critically ill COVID-19 patients, the risk of long-term neurological consequences is just beginning to be appreciated. While recent studies have identified that there is an increase in structural injury to the nervous system in critically ill COVID-19 patients, there is little known about the relationship of COVID-19 neurological damage to the systemic inflammatory diseases also observed in COVID-19 patients. The purpose of this pilot observational study was to examine the relationships between serum neurofilament light protein (NfL, a measure of neuronal injury) and co-morbid cardiovascular disease (CVD) and neurological complications in COVID-19 positive patients admitted to the intensive care unit (ICU). In this observational study of one-hundred patients who were admitted to the ICU in Tucson, Arizona between April and August 2020, 89 were positive for COVID-19 (COVID-pos) and 11 was COVID-negative (COVID-neg). A healthy control group (n=8) was examined for comparison. The primary outcomes and measures were subject demographics, serum NfL, presence and extent of CVD, diabetes, sequential organ failure assessment score (SOFA), presence of neurological complications, and blood chemistry panel data. COVID-pos patients in the ICU had significantly higher mean levels of Nfl (229.6 ± 163 pg/ml) compared to COVID-neg ICU patients (19.3 ± 5.6 pg/ml), Welch's t-test, p =.01 and healthy controls (12.3 ± 3.1 pg/ml), Welch's t-test p =.005. Levels of Nfl in COVID-pos ICU patients were significantly higher in patients with concomitant CVD and diabetes (n=35, log Nfl 1.6±.09), and correlated with higher SOFA scores (r=.5, p =.001). These findings suggest that in severe COVID-19 disease, the central neuronal and axonal damage in these patients may be driven, in part, by the level of systemic cardiovascular disease and peripheral inflammation. Understanding the contributions of systemic inflammatory disease to central neurological degeneration in these COVID-19 survivors will be important to the design of interventional therapies to prevent long-term neurological and cognitive dysfunction.
• Hoyer-Kimura, C., Konhilas, J. P., Mansour, H. M., Polt, R., Doyle, K. P., Billheimer, D., & Hay, M. (2021). Neurofilament light: a possible prognostic biomarker for treatment of vascular contributions to cognitive impairment and dementia. Journal of neuroinflammation, 18(1), 236.
  More info

  Decreased cerebral blood flow and systemic inflammation during heart failure (HF) increase the risk for vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer disease-related dementias (ADRD). We previously demonstrated that PNA5, a novel glycosylated angiotensin 1-7 (Ang-(1-7)) Mas receptor (MasR) agonist peptide, is an effective therapy to rescue cognitive impairment in our preclinical model of VCID. Neurofilament light (NfL) protein concentration is correlated with cognitive impairment and elevated in neurodegenerative diseases, hypoxic brain injury, and cardiac disease. The goal of the present study was to determine (1) if treatment with Ang-(1-7)/MasR agonists can rescue cognitive impairment and decrease VCID-induced increases in NfL levels as compared to HF-saline treated mice and, (2) if NfL levels correlate with measures of cognitive function and brain cytokines in our VCID model.
• Iwanski, J., Kazmouz, S. G., Li, S., Stansfield, B., Salem, T. T., Perez-Miller, S., Kazui, T., Jena, L., Uhrlaub, J. L., Lick, S., Nikolich-Žugich, J., Konhilas, J. P., Gregorio, C. C., Khanna, M., Campos, S. K., & Churko, J. M. (2021). Antihypertensive drug treatment and susceptibility to SARS-CoV-2 infection in human PSC-derived cardiomyocytes and primary endothelial cells. Stem cell reports, 16(10), 2459-2472.
  More info

  The pathogenicity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been attributed to its ability to enter through the membrane-bound angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, it has been heavily speculated that angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) therapy may modulate SARS-CoV-2 infection. In this study, exposure of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and human endothelial cells (hECs) to SARS-CoV-2 identified significant differences in protein coding genes involved in immunity, viral response, and cardiomyocyte/endothelial structure. Specifically, transcriptome changes were identified in the tumor necrosis factor (TNF), interferon α/β, and mitogen-activated protein kinase (MAPK) (hPSC-CMs) as well as nuclear factor kappa-B (NF-κB) (hECs) signaling pathways. However, pre-treatment of hPSC-CMs or hECs with two widely prescribed antihypertensive medications, losartan and lisinopril, did not affect the susceptibility of either cell type to SARS-CoV-2 infection. These findings demonstrate the toxic effects of SARS-CoV-2 in hPSC-CMs/hECs and, taken together with newly emerging multicenter trials, suggest that antihypertensive drug treatment alone does not alter SARS-CoV-2 infection.
• Konhilas, J. P., Hoyer-Kimura, C. H., Behm, C., Fricks, J. P., & Duca, F. (2021). Abstract P481: Short-term Synbiotic, B420 And Oligofructose, Treatment Reverse High-fat-diet Related Pathologies In Ischemic Reperfusion Mouse Models. Circulation Research, 129(Suppl_1). doi:10.1161/res.129.suppl_1.p481
• Konhilas, J. P., Solomon, A. E., Hoyer-Kimura, C., Kangath, A., & Duca, F. (2021). Abstract P366: The Impact Of Estrogen Signaling On Gut Epithelial Cells. Circulation Research, 129(Suppl_1). doi:10.1161/res.129.suppl_1.p366
• Lopez-Pier, M. A., Koppinger, M. P., Harris, P. R., Cannon, D. K., Skaria, R. S., Hurwitz, B. L., Watts, G., Aras, S., Slepian, M. J., & Konhilas, J. P. (2021). An adaptable and non-invasive method for tracking Bifidobacterium animalis subspecies lactis 420 in the mouse gut. Journal of microbiological methods, 189, 106302.
  More info

  Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota, intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome. The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract.
• McCormack, M., Schnellmann, R., Konhilas, J., Ledford, J. G., Hansel, N. N., Fawzy, A., Polverino, F., Wu, T. D., Rojas-Quintero, J., Wang, X., Mayo, J., Tomchaney, M., Tram, J., Packard, S., Zhang, D., Cleveland, K. H., Cordoba-Lanus, E., Owen, C. A., Kinney, G. L., , Hersh, C. P., et al. (2021). Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment. American Journal of Respiratory and Critical Care Medicine, 204(6), 651-666. doi:10.1164/rccm.202012-4510oc
• Polverino, F., Wu, T. D., Rojas-Quintero, J., Wang, X., Mayo, J., Tomchaney, M., Tram, J., Packard, S., Zhang, D., Cleveland, K. H., Cordoba-Lanus, E., Owen, C. A., Fawzy, A., Kinney, G. L., Hersh, C. P., Hansel, N. N., Doubleday, K., Sauler, M., Tesfaigzi, Y., , Ledford, J. G., et al. (2021). Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment. American journal of respiratory and critical care medicine, 204(6), 651-666.
  More info

  Cigarette smoke (CS) inhalation triggers oxidative stress and inflammation, leading to accelerated lung aging, apoptosis, and emphysema, as well as systemic pathologies. Metformin is beneficial for protecting against aging-related diseases. We sought to investigate whether metformin may ameliorate CS-induced pathologies of emphysematous chronic obstructive pulmonary disease (COPD). Mice were exposed chronically to CS and fed metformin-enriched chow for the second half of exposure. Lung, kidney, and muscle pathologies, lung proteostasis, endoplasmic reticulum (ER) stress, mitochondrial function, and mediators of metformin effects and/or were studied. We evaluated the association of metformin use with indices of emphysema progression over 5 years of follow-up among the COPDGene (Genetic Epidemiology of COPD) study participants. The association of metformin use with the percentage of emphysema and adjusted lung density was estimated by using a linear mixed model. Metformin protected against CS-induced pulmonary inflammation and airspace enlargement; small airway remodeling, glomerular shrinkage, oxidative stress, apoptosis, telomere damage, aging, dysmetabolism and ; and ER stress. The AMPK (AMP-activated protein kinase) pathway was central to metformin's protective action. Within COPDGene, participants receiving metformin compared with those not receiving it had a slower progression of emphysema (-0.92%; 95% confidence interval [CI], -1.7% to -0.14%;  = 0.02) and a slower adjusted lung density decrease (2.2 g/L; 95% CI, 0.43 to 4.0 g/L;  = 0.01). Metformin protected against CS-induced lung, renal, and muscle injury; mitochondrial dysfunction; and unfolded protein responses and ER stress in mice. In humans, metformin use was associated with lesser emphysema progression over time. Our results provide a rationale for clinical trials testing the efficacy of metformin in limiting emphysema progression and its systemic consequences.
• Watts, G., Slepian, M. J., Skaria, R. S., Lopez-pier, M. A., Koppinger, M. P., Konhilas, J. P., Hurwitz, B. L., Harris, P. R., Cannon, D. K., & Aras, S. (2021). An adaptable and non-invasive method for tracking Bifidobacterium animalis subspecies lactis 420 in the mouse gut.. Journal of microbiological methods, 106302. doi:10.1016/j.mimet.2021.106302
  More info

  Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota, intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome. The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract.
• Konhilas, J. P., Sanchez, J. N., Regan, J. A., Constantopoulos, E., Lopez-Pier, M., Cannon, D. K., Skaria, R., McKee, L. A., Chen, H., Lipovka, Y., Pollow, D., & Brooks, H. L. (2020). Using 4-vinylcyclohexene diepoxide as a model of menopause for cardiovascular disease. American journal of physiology. Heart and circulatory physiology, 318(6), H1461-H1473.
  More info

  There is a sharp rise in cardiovascular disease (CVD) risk and progression with the onset of menopause. The 4-vinylcyclohexene diepoxide (VCD) model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. We hypothesized that menopausal female mice were more susceptible to CVD than pre- or perimenopausal females. Female mice were treated with VCD or vehicle for 20 consecutive days. Premenopausal, perimenopausal, and menopausal mice were administered angiotensin II (ANG II) or subjected to ischemia-reperfusion (I/R). Menopausal females were more susceptible to pathological ANG II-induced cardiac remodeling and cardiac injury from a myocardial infarction (MI), while perimenopausal, like premenopausal, females remained protected. Specifically, ANG II significantly elevated diastolic (130.9 ± 6.0 vs. 114.7 ± 6.2 mmHg) and systolic (156.9 ± 4.8 vs. 141.7 ± 5.0 mmHg) blood pressure and normalized cardiac mass (15.9 ± 1.0 vs. 7.7 ± 1.5%) to a greater extent in menopausal females compared with controls, whereas perimenopausal females demonstrated a similar elevation of diastolic (93.7 ± 2.9 vs. 100.5 ± 4.1 mmHg) and systolic (155.9 ± 7.3 vs. 152.3 ± 6.5 mmHg) blood pressure and normalized cardiac mass (8.3 ± 2.1 vs. 7.5 ± 1.4%) compared with controls. Similarly, menopausal females demonstrated a threefold increase in fibrosis measured by Picrosirus red staining. Finally, hearts of menopausal females (41 ± 5%) showed larger infarct sizes following I/R injury than perimenopausal (18.0 ± 5.6%) and premenopausal (16.2 ± 3.3, 20.1 ± 4.8%) groups. Using the VCD model of menopause, we provide evidence that menopausal females were more susceptible to pathological cardiac remodeling. We suggest that the VCD model of menopause may be critical to better elucidate cellular and molecular mechanisms underlying the transition to CVD susceptibility in menopausal women. Before menopause, women are protected against cardiovascular disease (CVD) compared with age-matched men; this protection is gradually lost after menopause. We present the first evidence that demonstrates menopausal females are more susceptible to pathological cardiac remodeling while perimenopausal and cycling females are not. The VCD model permits appropriate examination of how increased susceptibility to the pathological process of cardiac remodeling accelerates from pre- to perimenopause to menopause.
• Koppinger, M. P., Lopez-Pier, M. A., Skaria, R., Harris, P. R., & Konhilas, J. P. (2020). attenuates cardiac injury without lowering cholesterol in low-density lipoprotein receptor-deficient mice fed standard chow. American journal of physiology. Heart and circulatory physiology, 319(1), H32-H41.
  More info

  Disruption of the normal gut microbiome (dysbiosis) is implicated in the progression and severity of myriad disorders, including hypercholesterolemia and cardiovascular disease. Probiotics attenuate and reverse gut dysbiosis to improve cardiovascular risk factors like hypertension and hypercholesterolemia. is a well-studied lactic acid-producing probiotic with known cholesterol-lowering properties and anti-inflammatory effects. In the present study, we hypothesized that delivered to hypercholesterolemic low-density lipoprotein receptor knockout (LDLr KO) mice will reduce cholesterol levels and minimize cardiac injury from an ischemic insult. [1 × 10 or 50 × 10 colony-forming units (CFU)/day] was administered by oral gavage to wild-type mice and LDLr KO for up to 6 wk followed by an ischemia-reperfusion (I/R) protocol. After 4 wk of gavage, total serum cholesterol in wild-type mice receiving saline was 113.5 ± 5.6 mg/dL compared with 113.3 ± 6.8 and 101.9 ± 7.5 mg/dL in mice receiving 1 × 10 or 50 × 10 CFU/day, respectively. Over the same time frame, administration of at 1 × 10 or 50 × 10 CFU/day did not lower total serum cholesterol (283.0 ± 11.1, 263.3 ± 5.0, and 253.1 ± 7.0 mg/dL; saline, 1 × 10 or 50 × 10 CFU/day, respectively) in LDLr KO mice. Despite no impact on total serum cholesterol, administration significantly attenuated cardiac injury following I/R, as evidenced by smaller infarct sizes compared with controls in both wild-type and LDLr KO groups. In conclusion, daily significantly protected against cardiac injury without lowering cholesterol levels, suggesting anti-inflammatory properties of uncoupled from improvements in serum cholesterol. We demonstrated that daily delivery of to wild-type and hypercholesterolemic lipoprotein receptor knockout mice attenuated cardiac injury following ischemia-reperfusion without lowering total serum cholesterol in the short term. In addition, we validated protection against cardiac injury using histology and immunohistochemistry techniques. offers promise as a probiotic to mitigate ischemic cardiac injury.
• Lipovka, Y., Konhilas, J. P., Skaria, R., Sanchez, J. N., Regan, J. A., Pollow, D. P., Mckee, L. A., Lopez-pier, M. A., Lipovka, Y., Konhilas, J. P., Constantopoulos, E., Chen, H., Cannon, D. K., & Brooks, H. L. (2020). Using 4-vinylcyclohexene diepoxide as a model of menopause for cardiovascular disease.. American journal of physiology. Heart and circulatory physiology, 318(6), H1461-H1473. doi:10.1152/ajpheart.00555.2019
  More info

  There is a sharp rise in cardiovascular disease (CVD) risk and progression with the onset of menopause. The 4-vinylcyclohexene diepoxide (VCD) model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. We hypothesized that menopausal female mice were more susceptible to CVD than pre- or perimenopausal females. Female mice were treated with VCD or vehicle for 20 consecutive days. Premenopausal, perimenopausal, and menopausal mice were administered angiotensin II (ANG II) or subjected to ischemia-reperfusion (I/R). Menopausal females were more susceptible to pathological ANG II-induced cardiac remodeling and cardiac injury from a myocardial infarction (MI), while perimenopausal, like premenopausal, females remained protected. Specifically, ANG II significantly elevated diastolic (130.9 ± 6.0 vs. 114.7 ± 6.2 mmHg) and systolic (156.9 ± 4.8 vs. 141.7 ± 5.0 mmHg) blood pressure and normalized cardiac mass (15.9 ± 1.0 vs. 7.7 ± 1.5%) to a greater extent in menopausal females compared with controls, whereas perimenopausal females demonstrated a similar elevation of diastolic (93.7 ± 2.9 vs. 100.5 ± 4.1 mmHg) and systolic (155.9 ± 7.3 vs. 152.3 ± 6.5 mmHg) blood pressure and normalized cardiac mass (8.3 ± 2.1 vs. 7.5 ± 1.4%) compared with controls. Similarly, menopausal females demonstrated a threefold increase in fibrosis measured by Picrosirus red staining. Finally, hearts of menopausal females (41 ± 5%) showed larger infarct sizes following I/R injury than perimenopausal (18.0 ± 5.6%) and premenopausal (16.2 ± 3.3, 20.1 ± 4.8%) groups. Using the VCD model of menopause, we provide evidence that menopausal females were more susceptible to pathological cardiac remodeling. We suggest that the VCD model of menopause may be critical to better elucidate cellular and molecular mechanisms underlying the transition to CVD susceptibility in menopausal women.NEW & NOTEWORTHY Before menopause, women are protected against cardiovascular disease (CVD) compared with age-matched men; this protection is gradually lost after menopause. We present the first evidence that demonstrates menopausal females are more susceptible to pathological cardiac remodeling while perimenopausal and cycling females are not. The VCD model permits appropriate examination of how increased susceptibility to the pathological process of cardiac remodeling accelerates from pre- to perimenopause to menopause.
• Skaria, R., Parvaneh, S., Zhou, S., Kim, J., Wanjiru, S., Devers, G., Konhilas, J., & Khalpey, Z. (2020). Path to precision: prevention of post-operative atrial fibrillation. Journal of thoracic disease, 12(5), 2735-2746.
  More info

  Development of post-operative atrial fibrillation (POAF) following open-heart surgery is a significant clinical and economic burden. Despite advancements in medical therapies, the incidence of POAF remains elevated at 25-40%. Early work focused on detecting arrhythmias from electrocardiograms as well as identifying pre-operative risk factors from medical records. However, further progress has been stagnant, and a deeper understanding of pathogenesis and significant influences is warranted. With the advent of more complex machine learning (ML) algorithms and high-throughput sequencing, we have an unprecedented ability to capture and predict POAF in real-time. Integration of multimodal heterogeneous data and application of ML can generate a paradigm shift for diagnosis and treatment. This will require a concerted effort to consolidate and streamline real-time data. Herein, we will review the current literature and emerging opportunities aimed at predictive targets and new insights into the mechanisms underlying long-term sequelae of POAF.
• Harris, P. R., Keen, D. A., Constantopoulos, E., Weninger, S. N., Hines, E., Koppinger, M. P., Khalpey, Z. I., & Konhilas, J. P. (2019). Fluid type influences acute hydration and muscle performance recovery in human subjects. Journal of the International Society of Sports Nutrition, 16(1), 15.
  More info

  Exercise and heat trigger dehydration and an increase in extracellular fluid osmolality, leading to deficits in exercise performance and thermoregulation. Evidence from previous studies supports the potential for deep-ocean mineral water to improve recovery of exercise performance post-exercise. We therefore wished to determine whether acute rehydration and muscle strength recovery was enhanced by deep-ocean mineral water following a dehydrating exercise, compared to a sports drink or mountain spring water. We hypothesized that muscle strength would decrease as a result of dehydrating exercise, and that recovery of muscle strength and hydration would depend on the type of rehydrating fluid.
• Hay, M., Polt, R., Heien, M. L., Vanderah, T. W., Largent-Milnes, T. M., Rodgers, K., Falk, T., Bartlett, M. J., Doyle, K. P., & Konhilas, J. P. (2019). A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction. The Journal of pharmacology and experimental therapeutics, 369(1), 9-25.
  More info

  Increasing evidence indicates that decreased brain blood flow, increased reactive oxygen species (ROS) production, and proinflammatory mechanisms accelerate neurodegenerative disease progression such as that seen in vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias. There is a critical clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension, and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated angiotensin-(1-7) peptide Ang-1-6-O-Ser-Glc-NH2 (PNA5) as a therapy to treat VCID and to investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration compared with the native angiotensin-(1-7) peptide. Moreover, after treatment with 1.0/mg/kg, s.c., for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of the Mas receptor results in a dose-dependent inhibition of ROS in human endothelial cells. Last, PNA5 treatment decreased VCID/HF-induced activation of brain microglia/macrophages and inhibited circulating tumor necrosis factor , interleukin (IL)-7, and granulocyte cell-stimulating factor serum levels while increasing that of the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammation-related brain diseases.
• Avery, R. J., Runyan, R. B., Konhilas, J. P., Yu, S. K., Runyan, R. B., Konhilas, J. P., Khalpey, Z. I., Cherukuri, G., & Avery, R. J. (2018). Remodeling Failing Human Myocardium With Hybrid Cell/Matrix and Transmyocardial Revascularization.. ASAIO journal (American Society for Artificial Internal Organs : 1992), 64(5), e130-e133. doi:10.1097/mat.0000000000000727
  More info

  Given the limited treatment options for advanced heart failure, the intrinsic regenerative properties of stem cells have been evaluated for myocardial remodeling. Previous stem cells techniques for myocardiocyte remodeling have been limited by the low cellular retention. Presented is a hybrid approach for remodeling infarcted myocardium through implantation of allogeneic human amniotic fluid-derived mesenchymal stem cells within micronized human allograft-derived liquid matrix during the performance of transmyocardial revascularization (TMR). Given the induced increase in vascular density from TMR, we hypothesize that it may serve as a therapeutic delivery system for stem cell placement into damaged myocardium. We present a patient with ischemic cardiomyopathy and refractory angina, who clinically improved after this hybrid therapy of intraoperative TMR and placement of amniotic fluid-derived mesenchymal stem cells and liquid matrix within the TMR channels. Noninvasive testing of myocardial viability biomarkers utilizing both cardiac magnetic resonance imaging and thallium imaging supported the clinical improvement in cardiac symptom may be related to ventricular remodeling in a region of infarct with subsequent functional improvement.
• Chung, A. G., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M., Figueroa, A. G., Becktel, D. A., Antony Day, W., Konhilas, J. P., McKay, B. S., Nguyen, T. V., & Doyle, K. P. (2018). Liquefaction of the Brain following Stroke Shares a Similar Molecular and Morphological Profile with Atherosclerosis and Mediates Secondary Neurodegeneration in an Osteopontin-Dependent Mechanism. eNeuro, 5(5).
  More info

  Here we used mouse models of heart and brain ischemia to compare the inflammatory response to ischemia in the heart, a protein rich organ, to the inflammatory response to ischemia in the brain, a lipid rich organ. We report that ischemia-induced inflammation resolves between one and four weeks in the heart compared to between eight and 24 weeks in the brain. Importantly, we discovered that a second burst of inflammation occurs in the brain between four and eight weeks following ischemia, which coincided with the appearance of cholesterol crystals within the infarct. This second wave shares a similar cellular and molecular profile with atherosclerosis and is characterized by high levels of osteopontin (OPN) and matrix metalloproteinases (MMPs). In order to test the role of OPN in areas of liquefactive necrosis, OPN mice were subjected to brain ischemia. We found that at seven weeks following stroke, the expression of pro-inflammatory proteins and MMPs was profoundly reduced in the infarct of the OPN mice, although the number of cholesterol crystals was increased. OPN mice exhibited faster recovery of motor function and a higher number of neuronal nuclei (NeuN) positive cells in the peri-infarct area at seven weeks following stroke. Based on these findings we propose that the brain liquefies after stroke because phagocytic cells in the infarct are unable to efficiently clear cholesterol rich myelin debris, and that this leads to the perpetuation of an OPN-dependent inflammatory response characterized by high levels of degradative enzymes.
• Doyle, K. P., Nguyen, T. V., Mckay, B. S., Konhilas, J. P., Day, A. W., Figueroa, A. G., Hayes, M., Constantopoulos, E., Zbesko, J. C., Frye, J. B., Chung, A. G., Chung, A. G., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M., Figueroa, A. G., Day, A. W., Konhilas, J. P., , Mckay, B. S., et al. (2018). Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis and mediates secondary neurodegeneration in an osteopontin-dependent mechanism. eNeuro, 5(5), ENEURO.0076-18.2018.
• Lopez-Pier, M. A., Lipovka, Y., Koppinger, M. P., Harris, P. R., & Konhilas, J. P. (2018). The clinical impact of estrogen loss on cardiovascular disease in menopausal females. Medical research archives, 6(2).
  More info

  According to the CDC (2017), more women than men have died from heart disease over the last 20-25 years. On the contrary, premenopausal women are protected against heart and cardiovascular disease (CVD) compared to men. Following menopause, there is sharp rise in CVD mortality and morbidity in women compared to men indicating that women lose protection against CVD during menopause. This loss of CVD protection in women drives the CDC statistics. Life expectance of women has now reached 82 (almost 35 years longer than at the turn of the 20 century). Yet, women typically undergo menopause at 50-60 years of age, which means that women spend over 40% of their life in menopause. Therefore, menopausal women, and associated CVD risk, must be considered as distinct from an aging or senescent woman. Despite longstanding knowledge that premenopausal women are protected from CVD, our fundamental understanding regarding the shift in CVD risk with menopause remains inadequate and impedes our ability to develop sex-specific therapeutic strategies to combat menopausal susceptibility to CVD. This review provides a critical overview of clinical trials attempting to address CVD susceptibility postmenopausal using hormone replacement therapy. Next, we outline key deficiencies in pre-clinical menopause models and introduce an alternative to overcome these deficiencies. Finally, we discuss a novel connection between AMPK and estrogen-dependent pathways that may serve as a potential solution to increased CVD susceptibility in menopausal women.
• Pappas, C. T., Farman, G. P., Mayfield, R. M., Konhilas, J. P., & Gregorio, C. C. (2018). Cardiac-specific knockout of Lmod2 results in a severe reduction in myofilament force production and rapid cardiac failure. Journal of molecular and cellular cardiology, 122, 88-97.
  More info

  Leiomodin-2 (Lmod2) is a striated muscle-specific actin binding protein that is implicated in assembly of thin filaments. The necessity of Lmod2 in the adult mouse and role it plays in the mechanics of contraction are unknown. To answer these questions, we generated cardiac-specific conditional Lmod2 knockout mice (cKO). These mice die within a week of induction of the knockout with severe left ventricular systolic dysfunction and little change in cardiac morphology. Cardiac trabeculae isolated from cKO mice have a significant decrease in maximum force production and a blunting of myofilament length-dependent activation. Thin filaments are non-uniform and substantially reduced in length in cKO hearts, affecting the functional overlap of the thick and thin filaments. Remarkably, we also found that Lmod2 levels are directly linked to thin filament length and cardiac function in vivo, with a low amount (
• Pier, M. A., & Konhilas, J. P. (2018). Abstract 559: AMP-Activated Protein Kinase And Estrogen-Dependent Mechanisms Underlying Increased Susceptibility To Cardiovascular Disease During Menopause. Circulation Research, 123(Suppl_1). doi:10.1161/res.123.suppl_1.559
• Schipper, D. A., Louis, A. V., Dicken, D. S., Johnson, K., Smolenski, R. T., Black, S. M., Runyan, R., Konhilas, J., Garcia, J. G., & Khalpey, Z. (2018). Improved metabolism and redox state with a novel preservation solution: implications for donor lungs after cardiac death (DCD). Pulmonary circulation, 7(2), 494-504.
  More info

  Lungs donated after cardiac death (DCD) are an underutilized resource for a dwindling donor lung transplant pool. Our study investigates the potential of a novel preservation solution, Somah, to better preserve statically stored DCD lungs, for an extended time period, when compared to low-potassium dextran solution (LPD). We hypothesize that Somah is a metabolically superior organ preservation solution for hypothermic statically stored porcine DCD lungs, possibly improving lung transplant outcomes. Porcine DCD lungs (n = 3 per group) were flushed with and submerged in cold preservation solution. The lungs were stored up to 12 h, and samples were taken from lung tissue and the preservation medium throughout. Metabolomic and redox potential were analyzed using high performance liquid chromatography, mass spectrometry, and RedoxSYS®, comparing substrate and pathway utilization in both preservation solutions. Glutathione reduction was seen in Somah but not in LPD during preservation. Carnitine, carnosine, and n-acetylcarnosine levels were elevated in the Somah medium compared with LPD throughout. Biopsies of Somah exposed lungs demonstrated similar trends after 2 h, up to 12 h. Adenosine gradually decreased in Somah medium over 12 h, but not in LPD. An inversely proportional increase in inosine was found in Somah. Higher oxidative stress levels were measured in LPD. Our study suggests suboptimal metabolic preservation in lungs stored in LPD. LPD had poor antioxidant potential, cytoprotection, and an insufficient redox potential. These findings may have immediate clinical implications for human organs; however, further investigation is needed to evaluate DCD lung preservation in Somah as a viable option for transplant.
• Avery, R. J., Yu, S. K., Cherukuri, G., Runyan, R. B., Konhilas, J., & Khalpey, Z. I. (2017). Remodeling Failing Human Myocardium With Hybrid Cell/Matrix and Transmyocardial Revascularization. ASAIO journal (American Society for Artificial Internal Organs : 1992).
  More info

  Given the limited treatment options for advanced heart failure, the intrinsic regenerative properties of stem cells have been evaluated for myocardial remodeling. Previous stem cells techniques for myocardiocyte remodeling have been limited by the low cellular retention. Presented is a hybrid approach for remodeling infarcted myocardium through implantation of allogeneic human amniotic fluid-derived mesenchymal stem cells within micronized human allograft-derived liquid matrix during the performance of transmyocardial revascularization (TMR). Given the induced increase in vascular density from TMR, we hypothesize that it may serve as a therapeutic delivery system for stem cell placement into damaged myocardium. We present a patient with ischemic cardiomyopathy and refractory angina, who clinically improved after this hybrid therapy of intraoperative TMR and placement of amniotic fluid-derived mesenchymal stem cells and liquid matrix within the TMR channels. Noninvasive testing of myocardial viability biomarkers utilizing both cardiac magnetic resonance imaging and thallium imaging supported the clinical improvement in cardiac symptom may be related to ventricular remodeling in a region of infarct with subsequent functional improvement.
• Avery, R., Billheimer, D., Runyan, R. B., Konhilas, J. P., Schipper, D., Runyan, R. B., Rao, P., Pilikian, T., Marsh, K. M., Konhilas, J. P., Khalpey, Z., Johnson, K., Hallmark, B., Fisher, J. M., Duncker, D. J., Billheimer, D., & Avery, R. (2017). Abstract 14670: Human Amniotic Patch Placement During Cardiac Surgery Reduces Post-Operative Atrial Fibrillation. Circulation.
  More info

  Introduction: Atrial fibrillation after cardiac surgery occurs in approximately one third of patients and is associated with increased morbidity and mortality. Inflammation and oxidative stress may...
• Danilo, C. A., Constantopoulos, E., McKee, L. A., Chen, H., Regan, J. A., Lipovka, Y., Lahtinen, S., Stenman, L. K., Nguyen, T. V., Doyle, K. P., Slepian, M. J., Khalpey, Z. I., & Konhilas, J. P. (2017). Bifidobacterium animalis subsp. lactis 420 mitigates the pathological impact of myocardial infarction in the mouse. Beneficial microbes, 8(2), 257-269.
  More info

  There is a growing appreciation that our microbial environment in the gut plays a critical role in the maintenance of health and the pathogenesis of disease. Probiotic, beneficial gut microbes, administration can directly attenuate cardiac injury and post-myocardial infarction (MI) remodelling, yet the mechanisms of cardioprotection are unknown. We hypothesised that administration of Bifidobacterium animalis subsp. lactis 420 (B420), a probiotic with known anti-inflammatory properties, to mice will mitigate the pathological impact of MI, and that anti-inflammatory T regulatory (Treg) immune cells are necessary to impart protection against MI as a result of B420 administration. Wild-type male mice were administered B420, saline or Lactobacillus salivarius 33 (Ls-33) by gavage daily for 14 or 35 days, and underwent ischemia/reperfusion (I/R). Pretreatment with B420 for 10 or 28 days attenuated cardiac injury from I/R and reduced levels of inflammatory markers. Depletion of Treg cells by administration of anti-CD25 monoclonal antibodies eliminated B420-mediated cardio-protection. Further cytokine analysis revealed a shift from a pro-inflammatory to an anti-inflammatory environment in the probiotic treated post-MI hearts compared to controls. To summarise, B420 administration mitigates the pathological impact of MI. Next, we show that Treg immune cells are necessary to mediate B420-mediated protection against MI. Finally, we identify putative cellular, epigenetic and/or post-translational mechanisms of B420-mediated protection against MI.
• Danilo, C. A., Constantopoulos, E., McKee, L. A., Chen, H., Regan, J. A., Lipovka, Y., Lahtinen, S., Stenman, L. K., Nguyen, T., Doyle, K., Slepian, M. J., Khalpey, Z. I., & Konhilas, J. P. (2017). Bifidobacterium animalis subsp. lactis 420 mitigates the pathological impact of myocardial infarction in the mouse.. Benef Microbes, 8(2), 257-269. doi:10.3920/BM2016.0119
• Hay, M., Vanderah, T. W., Samareh-Jahani, F., Constantopoulos, E., Uprety, A. R., Barnes, C. A., & Konhilas, J. (2017). Cognitive impairment in heart failure: A protective role for angiotensin-(1-7). Behavioral neuroscience, 131(1), 99-114.
  More info

  Patients with congestive heart failure (CHF) have increased hospital readmission rates and mortality if they are concomitantly diagnosed with cognitive decline and memory loss. Accordingly, we developed a preclinical model of CHF-induced cognitive impairment with the goal of developing novel protective therapies against CHF related cognitive decline. CHF was induced by ligation of the left coronary artery to instigate a myocardial infarction (MI). By 4- and 8-weeks post-MI, CHF mice had approximately a 50% and 70% decline in ejection fraction as measured by echocardiography. At both 4- and 8-weeks post-MI, spatial memory performance in CHF mice as tested using the Morris water task was significantly impaired as compared with sham. In addition, CHF mice had significantly worse performance on object recognition when compared with shams as measured by discrimination ratios during the novel object recognition NOR task. At 8-weeks post-MI, a subgroup of CHF mice were given Angiotensin (Ang)-(1-7) (50mcg/kg/hr) subcutaneously for 4 weeks. Following 3 weeks treatment with systemic Ang-(1-7), the CHF mice NOR discrimination ratios were similar to shams and significantly better than the performance of CHF mice treated with saline. Ang-(1-7) also improved spatial memory in CHF mice as compared with shams. Ang-(1-7) had no effect on cardiac function. Inflammatory biomarker studies from plasma revealed a pattern of neuroprotection that may underlie the observed improvements in cognition. These results demonstrate a preclinical mouse model of CHF that exhibits both spatial memory and object recognition dysfunction. Furthermore, this CHF-induced cognitive impairment is attenuated by treatment with systemic Ang-(1-7). (PsycINFO Database Record
• Kazui, T., Tran, P. L., Pilikian, T. R., Marsh, K. M., Runyan, R., Konhilas, J., Smith, R., & Khalpey, Z. I. (2017). A dual therapy of off-pump temporary left ventricular extracorporeal device and amniotic stem cell for cardiogenic shock. Journal of cardiothoracic surgery, 12(1), 80.
  More info

  Temporary mechanical circulatory support device without sternotomy has been highly advocated for severe cardiogenic shock patient but little is known when coupled with amniotic stem cell therapy.
• Khalpey, Z. I., Kazui, T., Runyan, R. B., Konhilas, J. P., Konhilas, J. P., Runyan, R. B., Kazui, T., & Khalpey, Z. I. (2017). A dual therapy of off-pump temporary left ventricular extracorporeal device and amniotic stem cell for cardiogenic shock. J Cardiothorac Surg, 7(12), 80.
• Konhilas, J. P., Gregorio, C. C., Mayfield, R. M., Strom, J., Royal Harris, P., Koppinger, M., & Lopez-Pier, M. A. (2017). Abstract 267: Activation of Non-canonical Estrogen-dependent Pathways to Mitigate Pathological Cardiac Remodeling. Circulation Research, 121(suppl_1). doi:10.1161/res.121.suppl_1.267
• Luckey, S. W., Haines, C. D., Konhilas, J. P., Luczak, E. D., Messmer-Kratzsch, A., & Leinwand, L. A. (2017). Cyclin D2 is a critical mediator of exercise-induced cardiac hypertrophy. Experimental biology and medicine (Maywood, N.J.), 242(18), 1820-1830.
  More info

  A number of signaling pathways underlying pathological cardiac hypertrophy have been identified. However, few studies have probed the functional significance of these signaling pathways in the context of exercise or physiological pathways. Exercise studies were performed on females from six different genetic mouse models that have been shown to exhibit alterations in pathological cardiac adaptation and hypertrophy. These include mice expressing constitutively active glycogen synthase kinase-3β (GSK-3βS9A), an inhibitor of CaMK II (AC3-I), both GSK-3βS9A and AC3-I (GSK-3βS9A/AC3-I), constitutively active Akt (myrAkt), mice deficient in MAPK/ERK kinase kinase-1 (MEKK1-/-), and mice deficient in cyclin D2 (cyclin D2-/-). Voluntary wheel running performance was similar to NTG littermates for five of the mouse lines. Exercise induced significant cardiac growth in all mouse models except the cyclin D2-/- mice. Cardiac function was not impacted in the cyclin D2-/- mice and studies using a phospho-antibody array identified six proteins with increased phosphorylation (greater than 150%) and nine proteins with decreased phosphorylation (greater than 33% decrease) in the hearts of exercised cyclin D2-/- mice compared to exercised NTG littermate controls. Our results demonstrate that unlike the other hypertrophic signaling molecules tested here, cyclin D2 is an important regulator of both pathologic and physiological hypertrophy. Impact statement This research is relevant as the hypertrophic signaling pathways tested here have only been characterized for their role in pathological hypertrophy, and not in the context of exercise or physiological hypertrophy. By using the same transgenic mouse lines utilized in previous studies, our findings provide a novel and important understanding for the role of these signaling pathways in physiological hypertrophy. We found that alterations in the signaling pathways tested here had no impact on exercise performance. Exercise induced cardiac growth in all of the transgenic mice except for the mice deficient in cyclin D2. In the cyclin D2 null mice, cardiac function was not impacted even though the hypertrophic response was blunted and a number of signaling pathways are differentially regulated by exercise. These data provide the field with an understanding that cyclin D2 is a key mediator of physiological hypertrophy.
• Vega, R. B., Konhilas, J. P., Kelly, D. P., & Leinwand, L. A. (2017). Molecular Mechanisms Underlying Cardiac Adaptation to Exercise. Cell metabolism, 25(5), 1012-1026.
  More info

  Exercise elicits coordinated multi-organ responses including skeletal muscle, vasculature, heart, and lung. In the short term, the output of the heart increases to meet the demand of strenuous exercise. Long-term exercise instigates remodeling of the heart including growth and adaptive molecular and cellular re-programming. Signaling pathways such as the insulin-like growth factor 1/PI3K/Akt pathway mediate many of these responses. Exercise-induced, or physiologic, cardiac growth contrasts with growth elicited by pathological stimuli such as hypertension. Comparing the molecular and cellular underpinnings of physiologic and pathologic cardiac growth has unveiled phenotype-specific signaling pathways and transcriptional regulatory programs. Studies suggest that exercise pathways likely antagonize pathological pathways, and exercise training is often recommended for patients with chronic stable heart failure or following myocardial infarction. Herein, we summarize the current understanding of the structural and functional cardiac responses to exercise as well as signaling pathways and downstream effector molecules responsible for these adaptations.
• Behunin, S. M., Lopez-Pier, M. A., Mayfield, R. M., Danilo, C. A., Lipovka, Y., Birch, C., Lehman, S., Tardiff, J. C., Gregorio, C. C., & Konhilas, J. P. (2016). Liver Kinase B1 complex acts as a novel modifier of myofilament function and localizes to the Z-disk in cardiac myocytes. Archives of biochemistry and biophysics.
  More info

  Contractile perturbations downstream of Ca(2+) binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy "sensing" is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2-6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular "energy" sensing in the heart.
• Birch, C. L., Behunin, S. M., Lopez-Pier, M. A., Danilo, C., Lipovka, Y., Saripalli, C., Granzier, H., & Konhilas, J. P. (2016). Sex dimorphisms of crossbridge cycling kinetics in transgenic hypertrophic cardiomyopathy mice. American journal of physiology. Heart and circulatory physiology, 311(1), H125-36.
  More info

  Familial hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere and may lead to hypertrophic, dilated, restrictive, and/or arrhythmogenic cardiomyopathy, congestive heart failure, or sudden cardiac death. We hypothesized that hearts from transgenic HCM mice harboring a mutant myosin heavy chain increase the energetic cost of contraction in a sex-specific manner. To do this, we assessed Ca(2+) sensitivity of tension and crossbridge kinetics in demembranated cardiac trabeculas from male and female wild-type (WT) and HCM hearts at an early time point (2 mo of age). We found a significant effect of sex on Ca(2+) sensitivity such that male, but not female, HCM mice displayed a decrease in Ca(2+) sensitivity compared with WT counterparts. The HCM transgene and sex significantly impacted the rate of force redevelopment by a rapid release-restretch protocol and tension cost by the ATPase-tension relationship. In each of these measures, HCM male trabeculas displayed a gain-of-function when compared with WT counterparts. In addition, cardiac remodeling measured by echocardiography, histology, morphometry, and posttranslational modifications demonstrated sex- and HCM-specific effects. In conclusion, female and male HCM mice display sex dimorphic crossbridge kinetics accompanied by sex- and HCM-dependent cardiac remodeling at the morphometric, histological, and cellular level.
• Keen, D. A., Constantopoulos, E., & Konhilas, J. P. (2016). The impact of post-exercise hydration with deep-ocean mineral water on rehydration and exercise performance. Journal of the International Society of Sports Nutrition, 13, 17.
  More info

  Dehydration caused by prolonged exercise impairs thermoregulation, endurance and exercise performance. Evidence from animal and human studies validates the potential of desalinated deep-ocean mineral water to positively impact physiological and pathophysiological conditions. Here, we hypothesize that deep-ocean mineral water drawn from a depth of 915 m off the Kona, HI coast enhances recovery of hydration and exercise performance following a dehydrating exercise protocol compared to mountain spring water and a carbohydrate-based sports drink.
• Konhilas, J. P. (2016). Abstract 444: Probiotic Administration Mitigates the Detrimental Effects of Myocardial Infarction in Mice. Circulation Research, 119.
  More info

  Advances in sequencing and bioinformatics technologies have allowed unprecedented characterization of the gut microbiome. As a result, there is a growing appreciation that our microbial environment plays a critical role in the maintenance of health and the pathogenesis of disease. Accordingly, recent evidence suggests a role for gut microbiota in modulating cardiovascular disease and cardiac injury. We hypothesized that administration of the probiotic, Bifidobacterium animalis subsp. lactis 420 (B420), to mice will mitigate the pathological impact of ischemic heart disease (IHD), and that anti-inflammatory T regulatory (T reg ) immune cells are necessary to impart protection against IHD as a result of B420 administration. Pretreatment with B420 for 14 or 35 days attenuated cardiac injury from ischemia/reperfusion or permanent coronary ligation. Infarcted hearts from B420 treated animals displayed a significant reduction in pro-inflammatory markers and an increase in anti-inflammatory regulatory T cells (T reg ). We further show that T reg immune cells are necessary players to communicate this protection by B420 administration. This protection is due, at least in part, to an increase in anti-inflammatory M2 type macrophages in B420 treated animals. These results suggest that administration of the probiotic B420 protects against cardiac injury and that regulatory T cells mediate this effect. Modulation of the inflammatory response by administration of a specific strain of probiotic may offer a rational, safe, and cost effective way to prevent inflammatory damage after cardiac injury.
• Konhilas, J. P., Brooks, H. L., Sanchez, J. N., Konhilas, J. P., Pollow, D. P., Romero-aleshire, M. J., Romero-Aleshire, M. J., Pollow, D. P., Sanchez, J. N., Konhilas, J. P., Constantopoulos, E., Constantopoulos, E., & Brooks, H. L. (2016). Abstract P601: Menopausal Female Mice are Hypersensitive to Pathological Cardiac Remodeling. Hypertension, 68.
  More info

  Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. Loss of estrogen is detrimental; yet, estrogen replacement as cardioprotective remains controversial. Gonadectomized rodents demonstrate benefit of estrogen replacement for CVD. Human studies that show benefit of hormone replacement therapy are challenged by studies that do not show benefit. A novel model of menopause utilizing 4-vinylcyclohexene diepoxide (VCD) induces gradual ovarian failure, preserving the “perimenopause” transitional period and androgen secreting capacity of residual ovarian tissue. We hypothesize that menopausal females are hypersensitive to CVD-induced pathological cardiac remodeling. To address this hypothesis, we instigated menopause in 2 month-old females by daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days); control mice received sesame oil as vehicle. Vaginal cytology was used to monitor estrous cycles and determine when cycling ceased. Mice were considered menopausal after 15 consecutive days in persistent diestrus, non-cycling. Female mice from cycling, perimenopausal and menopausal groups received the hypertensive agent angiotensin II (Ang II, 800 ng/kg/min via alzet s.q. mini-pump, 14 days). Menopause did not impact systolic blood pressure (SBP) measured via tail cuff (control: 115.1±6.0 mmHg, n=7; menopause: 115.0±5.2 mmHg, n=10). Ang II infusion induced a significant exacerbation of hypertension (SBP) in menopausal females (156.9±4.8 mmHg; n=10) compared to Ang II controls (114.7±5.0 mmHg; n=8). This hypersensitivity to Ang II-induced hypertension was attenuated by estrogen supplementation started during perimenopause (0.1 mg, 60 day release). In addition, menopausal females demonstrated worsened pathological cardiac remodeling measured by functional (echocardiography), cellular (myocardial fibrosis) and molecular (fetal gene program) assessments. Using a novel model of menopause (VCD) combined with infusion of the hypertensive agent Ang II, we demonstrated that menopausal mice are more susceptible, or hypersensitive to pathological cardiac remodeling compared to cycling and perimenopausal mice.
• Lipovka, Y., & Konhilas, J. P. (2016). The complex nature of oestrogen signalling in breast cancer: enemy or ally?. Bioscience reports, 36(3).
  More info

  The pleiotropic nature of oestradiol, the main oestrogen found in women, has been well described in the literature. Oestradiol is positioned to play a unique role since it can respond to environmental, genetic and non-genetic cues to affect genetic expression and cellular signalling. In breast cancer, oestradiol signalling has a dual effect, promoting or inhibiting cancer growth. The potential impact of oestradiol on tumorigenesis depends on the molecular and cellular characteristics of the breast cancer cell. In this review, we provide a broad survey discussing the cellular and molecular consequences of oestrogen signalling in breast cancer. First, we review the structure of the classical oestrogen receptors and resultant transcriptional (genomic) and non-transcriptional (non-genomic) signalling. We then discuss the nature of oestradiol signalling in breast cancer including the specific receptors that initiate these signalling cascades as well as potential outcomes, such as cancer growth, proliferation and angiogenesis. Finally, we examine cellular and molecular mechanisms underlying the dimorphic effect of oestrogen signalling in breast cancer.
• Paidy, S. R., Runyan, R. B., Konhilas, J. P., Runyan, R. B., Pilikian, T. R., Paidy, S. R., Marsh, K. M., Konhilas, J. P., Khalpey, Z., Hemphill, C., Ferng, A. S., & Connell, A. M. (2016). Human Amniotic Membrane Promotes Antimicrobial Microenvironment in a Device-Related Infection. Journal of Biomedical Science and Engineering, 09(2), 122-126. doi:10.4236/jbise.2016.92008
  More info

  Hospital inquired post-operative infections are a leading cause of morbidity and mortality in patients receiving left ventricular assist devices. We present the case of a patient who acquired Candida albicans and Staphylococci infections following left ventricular assist device implantation. Use of standard antibiotics caused the infection to worsen, thus an amniotic membrane patch was used as a last-resort treatment. Within 6 weeks of the amniotic membrane patch treatment, the infection resolved, the wound appeared exceptionally clean upon inspection, and granulation tissue was present. After the infection resolved, a successful latissimusdorsi tissue flap procedure was completed and the patient made a full recovery.
• Schreiber, C. A., Konhilas, J. P., Ikeda, Y., Holditch, S. J., Burnett, J. C., & Bongers, C. A. (2016). 172. B-Type Natriuretic Peptide Gene Therapy as a Novel Early Treatment for Familial Hypertrophic Cardiomyopathy. Molecular Therapy, 24, S67. doi:10.1016/s1525-0016(16)32981-1
  More info

  Hypertrophic cardiomyopathy (HCM) is the most common familial cardiomyopathy, with the prevalence between 0.2 to 0.5%. HCM presents as an increase in left ventricular mass with thickening of the interventricular septum (IVS), myocellular disarray, interstitial fibrosis, heart rhythm abnormalities, and sudden death, necessitating therapeutic intervention. HCM is commonly caused by mutations in genes MYH7, MYBPC3, TNNT2, and TNNI3.The cardiac hormone, B-type natriuretic peptide (BNP) is a guanylyl cyclase A (GC-A) agonist. Physiological effects of BNP include natriuresis, vasodilation and blood pressure-lowering action. Additionally, BNP shows direct cardioprotective properties including anti-hypertrophic and anti-fibrotic effects, independently of its anti-hypertensive effects. We have recently found that genetic BNP ablation results in progressive cardiac hypertrophy with upregulation in HCM-associated genes in rats. In the current study, we tested the hypothesis that sustained BNP treatment with novel gene delivery of BNP may preserve cardiac function and structure in HCM in a mouse model of HCM.We used transgenic HCM mice expressing a mutated α-myosin heavy chain. Adeno-associated virus serotype 9 (AAV9) vectors were utilized to exploit their natural myocardial tropism. Three groups of mice were treated via tail-vein injections at four weeks of age (week one of study); Group 1, No HCM control (C57bl/6, no transgenic α-myosin heavy chain, n=6); Group 2, Untreated HCM mice with transgenic α-myosin heavy chain (PBS treated, n=10); Group 3, AAV9-BNP vector-treated HCM mice (n=6). To observe therapeutic benefit we assayed non-invasive blood pressure, myocardial architecture and remodeling (echocardiography), exercise capacity (treadmills), and any cardiorenal interplay through renal parameters. At 6, 9, 12, 15 and 25 weeks after AAV vector administration, left ventricular mass by echo was significantly higher in the untreated HCM group than the AAV-BNP-treated HCM and wildtype C57bl/6 mice. Significant increases in IVS and posterior wall thickness were also observed in control HCM group. At 11, 19, and 25 weeks after AAV vector administration, exercise capacity, monitored by treadmill tolerance, was significantly lower in the control HCM group than the AAV-BNP-treated HCM mice. No significant changes were seen in urine outputs. HCMAAV9-BNP treated mice showed a trend towards a lower blood pressure than both C57bl/6 and HCM mice.Our data demonstrate that AAV9-mediated BNP over-expression blocks progressive left ventricular enlargement and improves exercise capacity in a mouse model of HCM. The present study supports the BNP/GC-A/cGMP axis as a novel therapeutic target for the treatment of HCM.
• Behunin, S. M., Lopez-Pier, M. A., Birch, C. L., McKee, L. A., Danilo, C., Khalpey, Z., & Konhilas, J. P. (2015). LKB1/Mo25/STRAD uniquely impacts sarcomeric contractile function and posttranslational modification. Biophysical journal, 108(6), 1484-94.
  More info

  The myocardium undergoes extensive metabolic and energetic remodeling during the progression of cardiac disease. Central to remodeling are changes in the adenine nucleotide pool. Fluctuations in these pools can activate AMP-activated protein kinase (AMPK), the central regulator of cellular energetics. Binding of AMP to AMPK not only allosterically activates AMPK but also promotes phosphorylation of AMPK by an upstream kinase complex, LKB1/Mo25/STRAD (liver kinase B 1, mouse protein 25, STE-related adaptor protein). AMPK phosphorylation by the LKB1 complex results in a substantial increase in AMPK activity. Molecular targeting by the LKB1 complex depends on subcellular localization and transcriptional expression. Yet, little is known about the ability of the LKB1 complex to modulate targeting of AMPK after activation. Accordingly, we hypothesized that differing stoichiometric ratios of LKB1 activator complex to AMPK would uniquely impact myofilament function. Demembranated rat cardiac trabeculae were incubated with varying ratios of the LKB1 complex to AMPK or the LKB1 complex alone. After incubation, we measured the Ca(2+) sensitivity of tension, rate constant for tension redevelopment, maximum tension generation, length-dependent activation, cooperativity, and sarcomeric protein phosphorylation status. We found that the Ca(2+) sensitivity of tension and cross-bridge dynamics were dependent on the LKB1 complex/AMPK ratio. We also found that the LKB1 complex desensitizes and suppresses myofilament function independently of AMPK. A phospho-proteomic analysis of myofilament proteins revealed site-specific changes in cardiac Troponin I (cTnI) phosphorylation, as well as a unique distribution of cTnI phosphospecies that were dependent on the LKB1 complex/ AMPK ratio. Fibers treated with the LKB1 complex alone did not alter cTnI phosphorylation or phosphospecies distribution. However, LKB1 complex treatment independent of AMPK increased phosphorylation of myosin-binding protein C. Therefore, we conclude that the LKB1/AMPK signaling axis is able to alter muscle function through multiple mechanisms.
• Khalpey, Z., Qu, N., Hemphill, C., Louis, A. V., Ferng, A. S., Son, T. G., Stavoe, K., Penick, K., Tran, P. L., Konhilas, J., Lagrand, D. S., & Garcia, J. G. (2015). Rapid porcine lung decellularization using a novel organ regenerative control acquisition bioreactor. ASAIO journal (American Society for Artificial Internal Organs : 1992), 61(1), 71-7.
  More info

  To regenerate discarded lungs that would not normally be used for transplant, ex vivo reseeding after decellularization may produce organs suitable for clinical transplantation and therefore close the donor gap. Organ regenerative control acquisition (Harvard Biosciences, Holliston, MA), a novel bioreactor system that simulates physiological conditions, was used to evaluate a method of rapid decellularization. Although most current decellularization methods are 24-72 hours, we hypothesized that perfusing porcine lungs with detergents at higher pressures for less time would yield comparable bioscaffolds suitable for future experimentation. Methods involved perfusion of 1% Triton X-100 (Triton) and 0.1% sodium dodecyl sulfate at varied physiological flow rates. Architecture of native and decellularized lungs was analyzed with hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Dry gas and liquid ventilation techniques were introduced. Our 7 hour decellularization procedure removes nuclear material while maintaining architecture. Bioscaffolds have the microarchitecture for reseeding of stem cells. Hematoxylin and eosin staining suggested removal of nuclear material, whereas SEM and TEM imaging demonstrated total removal of cells with structural architecture preserved. This process can lead to clinical implementation, thereby increasing the availability of human lungs for transplantation.
• Konhilas, J. P., Chen, H., Luczak, E., McKee, L. A., Regan, J., Watson, P. A., Stauffer, B. L., Khalpey, Z. I., Mckinsey, T. A., Horn, T., LaFleur, B., & Leinwand, L. A. (2015). Diet and sex modify exercise and cardiac adaptation in the mouse. American journal of physiology. Heart and circulatory physiology, 308(2), H135-45.
  More info

  The heart adapts to exercise stimuli in a sex-dimorphic manner when mice are fed the traditional soy-based chow. Females undergo more voluntary exercise (4 wk) than males and exhibit more cardiac hypertrophy per kilometer run (18, 32). We have found that diet plays a critical role in cage wheel exercise and cardiac adaptation to the exercise stimulus in this sex dimorphism. Specifically, feeding male mice a casein-based, soy-free diet increases daily running distance over soy-fed counterparts to equal that of females. Moreover, casein-fed males have a greater capacity to increase their cardiac mass in response to exercise compared with soy-fed males. To further explore the biochemical mechanisms for these differences, we performed a candidate-based RT-PCR screen on genes previously implicated in diet- or exercise-based cardiac hypertrophy. Of the genes screened, many exhibit significant exercise, diet, or sex effects but only transforming growth factor-β1 shows a significant three-way interaction with no genes showing a two-way interaction. Finally, we show that the expression and activity of adenosine monophosphate-activated kinase-α2 and acetyl-CoA carboxylase is dependent on exercise, diet, and sex.
• Konhilas, J. P., Nikolich-zugich, J., Uhrlaub, J. L., Romero-aleshire, M. J., Pollow, D. P., Nikolich-zugich, J., Konhilas, J. P., & Brooks, H. L. (2015). Abstract P618: Foxp3+ Regulatory T cell Depletion Eliminates Ang II-Induced Hypertension Resistance in Female Mice. Hypertension, 66.
  More info

  Compared to males, premenopausal females are resistant to the development of Ang II hypertension. In males, Ang II induces hypertension, in part, through mechanisms requiring T effector lymphocytes. Recently, our lab has demonstrated that females can prevent the T lymphocyte-dependent increase in blood pressure (SBP and MAP) and expression of pro-inflammatory cytokines in the kidney in response to Ang II infusion. Because Foxp3 + T regulatory cells suppress the pro-inflammatory and hypertensive actions of T effector cells, we sought to determine whether Foxp3 + T regulatory cells contribute to this resistance in females. Premenopausal (8 week old) 129SVE female mice were infused with Ang II (800ng/kg/min, 14d) and received 4 doses of the anti-CD25 antibody PC-61 to transiently deplete Foxp3 + T regulatory cells (every 84 hours beginning 12 hours prior to Ang II infusion, 250μg/dose, i.p., vehicle control). Blood pressure was measured before and after Ang II infusion via non-invasive tail cuff. Ang II induced a significant increase in systolic blood pressure in Foxp3 + -depleted mice, while resistance was retained in vehicle-treated mice (Con Δ5 ± 5mmHg, Ang II Δ10 ± 7mmHg, PC-61 Δ28 ± 9 * mmHg, * p + splenic T cells compared to control (Con 1.7x10 6 cells, Ang II 2.3x10 6 cells, PC-61 8.3x10 5* cells, * P + and CD4 + T cell counts. The number of Foxp3 + T cells residing in the kidney was also significantly reduced by PC-61 (Con 1,152 ± 368 cells, Ang II 686 ± 389 cells, PC-61 210 ± 35 * cells, * P + -depleted mice (MCP-1- Con 1.0 ± 0.1, Ang II 1.6 ± 0.4, PC-61 1.8 ± 0.2 * ; ENaC-α- Con 1.0 ± 0.1, Ang II 1.6 ± 0.2, PC-61 2.1 ± 0.1 * , * P + T regulatory cells play a significant role in mediating the resistance to Ang II hypertension in premenopausal female mice, and may influence renal inflammation and sodium retention during chronic Ang II infusion.
• Li, F., Buck, D., De Winter, J., Kolb, J., Meng, H., Birch, C., Slater, R., Escobar, Y. N., Smith, J. E., Yang, L., Konhilas, J., Lawlor, M. W., Ottenheijm, C., & Granzier, H. L. (2015). Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy. Human molecular genetics, 24(18), 5219-33.
  More info

  Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (
• Lipovka, Y., & Konhilas, J. P. (2015). AMP-Activated Protein Kinase Signalling in Cancer and Cardiac Hypertrophy. Cardiovascular pharmacology: open access, 4(3).
  More info

  The AMP-protein kinase (AMPK) pathway is very versatile as it regulates cellular energetic homeostasis in many different tissue types. An appreciation for the importance of AMPK signalling and regulation in cardiovascular and tumor biology is increasing. Recently, a link has been established between anti-cancer therapy and susceptibility to cardiac disease. It has been shown that some anti-cancer drugs lead to an increased risk of cardiac disease, underlined by de-regulation of AMPK signalling. This review explores the AMPK signalling axis in both cardiac and tumor metabolism. We then examine off-target AMPK inhibition by cancer drugs and how this may translate into increased risk of cardiovascular disease. Finally, we discuss the implication of deregulated AMPK signalling during different stages of cardiac hypertrophy. Better understanding of the molecular pathways behind pathological processes will lead to the development of more effective therapeutics for cancer and cardiovascular diseases.
• Lipovka, Y., Chen, H., Vagner, J., Price, T. J., Tsao, T., & Konhilas, J. P. (2015). Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase. Bioscience reports, 35(5).
  More info

  Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα.
• Pollow, D. P., Romero-Aleshire, M. J., Sanchez, J. N., Konhilas, J. P., & Brooks, H. L. (2015). ANG II-induced hypertension in the VCD mouse model of menopause is prevented by estrogen replacement during perimenopause. American journal of physiology. Regulatory, integrative and comparative physiology, 309(12), R1546-52.
  More info

  Premenopausal females are resistant to the development of hypertension, and this protection is lost after the onset of menopause, resulting in a sharp increase in disease onset and severity. However, it is unknown how a fluctuating ovarian hormone environment during the transition from perimenopause to menopause impacts the onset of hypertension, and whether interventions during perimenopause prevent disease onset after menopause. A gradual transition to menopause was induced by repeated daily injections of 4-vinylcyclohexene diepoxide (VCD). ANG II (800 ng·kg(-1)·min(-1)) was infused into perimenopausal and menopausal female mice for 14 days. A separate cohort of mice received 17β-estradiol replacement during perimenopause. ANG II infusion produced significantly higher mean arterial pressure (MAP) in menopausal vs. cycling females, and 17β-estradiol replacement prevented this increase. In contrast, MAP was not significantly different when ANG II was infused into perimenopausal and cycling females, suggesting that female resistance to ANG II-induced hypertension is intact during perimenopause. ANG II infusion caused a significant glomerular hypertrophy, and hypertrophy was not impacted by hormonal status. Expression levels of aquaporin-2 (AQP2), a collecting duct protein, have been suggested to reflect blood pressure. AQP2 protein expression was significantly downregulated in the renal cortex of the ANG II-infused menopause group, where blood pressure was increased. AQP2 expression levels were restored to control levels with 17β-estradiol replacement. This study indicates that the changing hormonal environment in the VCD model of menopause impacts the severity of ANG II-induced hypertension. These data highlight the utility of the ovary-intact VCD model of menopause as a clinically relevant model to investigate the physiological mechanisms of hypertension that occur in women during the transition into menopause.
• Chen, H., Perez, J. N., Constantopoulos, E., McKee, L., Regan, J., Hoyer, P. B., Brooks, H. L., & Konhilas, J. (2014). A method to study the impact of chemically-induced ovarian failure on exercise capacity and cardiac adaptation in mice. Journal of visualized experiments : JoVE.
  More info

  The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.
• Khalpey, Z., Janardhanan, R., Konhilas, J., & Hemphill, C. (2014). First in man: adipose-derived stromal vascular fraction cells may promote restorative cardiac function. The American journal of medicine, 127(5), e11-2.
• Konhilas, J. P., & Behunin, S. M. (2014). The Interaction of AMP-Activated Protein Kinase and its Upstream Activator, Lkb1/Mo25/Strad, Modifies Contractile Function in Rat Cardiac Trabeculae. Biophysical Journal, 106(2), 767a-768a. doi:10.1016/j.bpj.2013.11.4218
  More info

  AMP-activated protein kinase (AMPK) is an energetic regulator that is activated by alterations in cellular AMP. I, as well as others, have shown that AMPK modifies both metabolic and contractile function of the heart. However, to become fully activated, it is necessary for AMPK to be phosphorylated by the upstream AMPK kinase complex, LKB1/MO25/STRAD. Yet, the relationship between this upstream kinase complex, AMPK activation, and myofilament contractile function is not known. We hypothesize that a high LKB1/MO25/STRAD to AMPK ratio will have a different impact on myofilament function than a low LKB1/MO25/STRAD to AMPK ratio. Accordingly, demembranated rat cardiac trabeculae were pre-incubated with varying ratios of LKB1/MO25/STRAD to AMPK and contractile function was measured. Surprisingly, at sarcomere length of 2.2 μm, cardiac fibers pre-incubated with the LKB1/MO25/STRAD complex alone are desensitized to Ca2+ (EC50 4.16 ±0.12 μM [n=6] vs 2.56±0.11 μM [n=13] p
• Konhilas, J. P., & Birch, C. (2014). The R403Q Mutation Alters Isometric and Energetic Properties in 2 Month Mice. Biophysical Journal, 106(2), 561a. doi:10.1016/j.bpj.2013.11.3118
  More info

  Introduction: Familial hypertrophic cardiomyopathy (FHC) is a primary disease of the sarcomere. The R403Q mutation resides at the actin-interaction site on myosin and leads to progressive hypertrophic cardiomyopathy that can result in sudden cardiac death or congestive heart failure. Previous studies examining the functional impact of the R403Q mutation give inconsistent results such as either reduced or enhanced actin-activated ATPase. We hypothesized that the R403Q mutation intrinsically increases the energetic cost of contraction.Methods: To do this, we assessed Ca2+-sensitivity of tension and cross-bridge kinetics in de-membranated cardiac trabeculae from wild-type (WT) and R403Q hearts at 2 months of age. Assuming a two-state model of cross-bridge binding and un-binding, we measured cross-bridge kinetics using 2 methods: (1) the rate of ATP hydrolysis (off-rate) by NADH oxidation that is enzymatically and stoichiometrically coupled to ATP consumption, and (2) the rate of force redevelopment (ktr; on-rate plus off-rate) by a rapid release-restretch protocol.Results: Male R403Q mice display a decrease in Ca2+ sensitivity (2.461μM ± 0.072) compared to WT males and females (2.189 μM ± 0.059 and 2.166 μM ±0.044, respectively) at SL 2.0μm. R403Q males display increased tension cost at SL 2.0μm when compared to WT counterparts (9.56 ± 0.68 and 6.82 ±1.07 pmol s-1 mm-1 mN-1, respectively). At maximal activation, the rate of force redevelopment (ktr) is not significantly different between R403Q and WT males (40.26±6.86 and 30.26±3.95 s-1).Conclusions: Although maximal ktr is not significantly different between WT and R403Q males, an increase in ATP hydrolysis (off rate) suggests an alteration in cross-bridge kinetics. Importantly, future studies will explore the relationship between submaximal activation and ktr. Along with no overt pathology at 2 months, the R403Q mutation alters the energetic cost of contraction and isometric properties.
• Konhilas, J. P., & Birch, C. (2014). The R403Q mutation alters isometric and energetic properties in 2 month mice (1081.1). The FASEB Journal, 28.
  More info

  Familial hypertrophic cardiomyopathy is a primary disease of the sarcomere. The R403Q mutation resides at the actin-interaction site on myosin and leads to progressive hypertrophic cardiomyopathy that can lead to sudden cardiac death or congestive heart failure. We hypothesized that the R403Q mutation intrinsically increases the energetic cost of contraction. To do this, we assessed Ca2+-sensitivity of tension and cross-bridge kinetics in de-membranated cardiac trabeculae from wild-type (WT) and R403Q hearts at 2 months of age. We measured cross-bridge kinetics using 2 methods: (1) the rate of ATP hydrolysis (off-rate) by NADH oxidation, and (2) the rate of force redevelopment (ktr) by a rapid release-restretch protocol. Male R403Q mice display a decrease in Ca2+ sensitivity compared to WT males and females at SL 2.0μm. R403Q males display increased tension cost at SL 2.0μm when compared to WT counterparts. At maximal activation, the rate of force redevelopment is not significantly different between R403Q...
• Konhilas, J. P., Pollow, D. P., Perez, J. N., Konhilas, J. P., Constantopoulos, E., & Brooks, H. L. (2014). Menopause Impairs Cardiovascular Resilience and Blood Pressure Regulation: 1296 Board #36 May 29, 8. Medicine and Science in Sports and Exercise, 46, 334. doi:10.1249/01.mss.0000494185.77614.53
• Lipovka, Y., & Konhilas, J. P. (2014). Estradiol Activates AMPK through Interaction with Estrogen Receptor Beta. Biophysical Journal, 106(2), 595a. doi:10.1016/j.bpj.2013.11.3295
  More info

  In industrialized countries, the prevalence of congestive heart failure (CHF) is increasing steadily and has become one of the leading causes of hospitalization. In addition, the risk of cardiovascular disease increases in post-menopausal women. Yet, the association between estrogen and the risk of CHF has not been adequately studied.Recently, AMP-kinase (AMPK) has emerged as prominent player in the development of cardiac hypertrophy and heart failure. Our on-going studies indicate that AMPK activation is deregulated during menopause potentially through downregulation of total AMPK protein. The aim of this study is to determine the mechanistic link between Estradiol and AMPK activation.We show that Estradiol (E2) upregulates AMPK activity in several cell lines including mouse c2c12 myotubes and human ductal breast epithelial tumor cells (T47D). In addition, E2 treatment of neonatal rat cardiomyocytes (NRCM) blocks the cellular hypertrophy induced by phenylephrine (PE) treatment, to the same extent as the AMPK activator AICAR. These data suggest that Estradiol attenuates the PE-induced increase in NRCM size, at least partially, through AMPK pathway activation. Co-immunoprecipitation experiments were done to explore if there is a physical interaction between estrogen receptors and AMPK. Our co-immunoprecipitation data in NRCM and female mice hearts suggests that Estrogen Receptor Beta (ERβ) associates with AMPK and MO25, a component of the upstream AMPK activation complex. We conclude that the well-established E2 attenuation of NRCM hypertrophy may be partially mediated through activation of AMPK. Moreover, the mechanism of E2-dependent AMPK is through direct interaction of ERβ with components of the AMPK signaling axis including AMPK and MO25. Additional studies are needed to determine site-specificity of this interaction and if this interaction underlies the mechanism of Estradiol-induced AMPK activation. In addition, future studies will further explore the role of ERβ in myocyte hypertrophy.
• Lipovka, Y., & Konhilas, J. P. (2014). Estradiol activates AMPK through interaction with extrogen receptor beta (15.4). The FASEB Journal, 28.
  More info

  The susceptibility and progression of cardiovascular disease (CVD) increases in post-menopausal women. AMP-kinase (AMPK) has emerged as prominent player as a cellular energetic regulator that is kn...
• Lopez-pier, M. A., Konhilas, J. P., & Behunin, S. M. (2014). LKB1 and MO25 Demonstrate Significant Interaction with Myofilament Proteins. Biophysical Journal, 106(2), 768a. doi:10.1016/j.bpj.2013.11.4219
  More info

  AMP-activated protein kinase (AMPK) is an energetic regulator in the heart that is activated by alterations in energetic status. AMPK has been shown to alter both metabolic status as well as contractile function in the heart. However to become fully activated, the upstream kinase complex LKB1/MO25/STRAD must phosphorylate AMPK. Recent evidence from our lab clearly illustrates that pre-incubation of demembranated cardiac trabeculae with the LKB1/MO25/STRAD complex (alone) can alter Ca2+-sensitivity of isometric tension development. The mechanism underlying the ability of the LKB1/MO25/STRAD complex to alter myofilament contractility is currently unknown. Accordingly, we hypothesize that the LKB1/MO25/STRAD complex alters myofilament contractility through a direct interaction with myofilament proteins. To test this hypothesis, cardiac trabeculae or cut papillary tissue was harvested from 2 month old male Sprague-Dawley rats following a Krebs-Henselet perfusion. Cardiac trabeculae or papillary strips were demembranated overnight and washed. Myofibrils were isolated and solubilized. Using western blot analysis we show that MO25 remained associated with myofibrillar proteins. Next, we incubated demembranated muscle strips with the LKB1/STRAD/M025 complex (0.02 U/μL) for thirty minutes at 30 C. Following incubation, myofibrils were isolated and solubilized. By western blot analysis, we show a significant increase in the amount of Mo25 as well as LKB1 associated with myofibrillar proteins over un-incubated controls (p=0.0354 and p=
• Lopez-pier, M. A., Konhilas, J. P., & Behunin, S. M. (2014). The energy regulating upstream kinase complex LKB1/MO25/STRAD is a potential novel regulator of thin filament function (1081.3). The FASEB Journal, 28.
  More info

  AMP-activated protein kinase (AMPK) is a cellular energetic regulator that is known to modify both metabolic and contractile function in the heart. Phosphorylation by an upstream kinase complex (LKB1/MO25/STRAD; LMS) is required for AMPK activation. Yet, the relationship between the LMS, AMPK activation, and myofilament function is unknown. Accordingly, we hypothesized that the upstream kinase complex can regulate myofilament function independently or in combination with AMPK. To do this, demembranated (skinned) rat cardiac trabeculae were incubated with either the LMS or with varying ratios of the LMS with AMPK and myofilament function was measured. Skinned rat trabeculae treated with the LMS alone were desensitized to Ca2+ and had lowered maximum tension. Adding increasing amounts of AMPK in combination with LMS reversed this effect increasing Ca2+-sensitivity of tension and maximum tension. We furthered hypothesized that the mechanism underlying this relationship is mediated through direct interaction ...
• Konhilas, J. P., & Behunin, S. M. (2013). Abstract 227: Phosphorylation Patterning Determined by AMP-Activated Kinase, the LKB1/MO25/STRAD Complex, and Protein Phosphatase 1 Alters Contractile Function in Cardiac Rat Trabeculae. Circulation Research, 113.
  More info

  Post-translational modifications (PTM) of myofilament proteins alter contractile function of the heart in healthy as well as diseased myocardium and the patterning of PTMs can influence cardiac disease progression. PTM of the thin filament regulatory protein cardiac troponin I (cTnI) is known to modify contractile properties, including steady-state Ca2+ sensitivity of force and crossbridge cycling rates. Accordingly, the purpose of this study was to determine the effect of cTnI PTM patterning on myofilament function. Therefore, I hypothesize that the impact of cTnI PTM on contractile function will depend on the relative phosphorylation levels. To do this, demembranated rat cardiac trabeculae from 2 month-old male Sprague-Dawley rats were treated with AMP activated kinase (AMPK) (0.005 U/ µL), Protein Phosphatase 1 (PP1) (1U/µL), and the upstream AMPK kinase, the LKB1/MO25/STRAD complex (0.2 mU/µL). Fibers that were incubated with activated AMPK displayed an increase in Ca2+ sensitivity compared to untreated control fibers (EC50 1.41±0.08 μM [n=2] vs. 2.52±0.43 μM [n=9] p
• Konhilas, J. P., & Birch, C. (2013). Abstract 345: R403Q Mutation Increases the Rate of Force Redevelopment in 2 Month Mice. Circulation Research, 113.
  More info

  Familial hypertrophic cardiomyopathy is a primary disease of the sarcomere. The R403Q mutation resides at the actin-interaction site on myosin and leads to progressive hypertrophic cardiomyopathy which progresses towards heart failure. Along with deteriorating cardiac function, these hearts experience an overall change in metabolic landscape, suggesting altered energetic function in hearts that express the R403Q mutation. We tested the hypothesis that the R403Q mutation intrinsically increases the energetic cost of contraction. To do this, we determined myofilament function in demembranated cardiac trabeculae from male wild-type (WT) and R403Q mice at 2 months of age, prior to overt signs of cardiac pathology. Firstly, steady-state Ca2+ sensitivity of force generation was not significantly different between male R403Q (n=4) and WT counterparts (n=2) consistent with previous findings. Secondly, the rate of force redevelopment (ktr) in skinned cardiac tissue was measured following unloaded isotonic shortening and a rapid re-stretch to 15% of the original muscle length at a sarcomere length of 2.0μm. R403Q mice display an increased rate of force redevelopment (49.89 s-1 ± 8.13, n = 4) compared to WT counterparts (24.52 ± 4.29, n = 6) at maximal activation indicating an increase in the apparent rates of crossbridges entering and leaving force-generating states (p < 0.05). In conclusion, the R403Q mutation does not impact steady-state Ca2+ sensitivity of force but increases total crossbridge cycling rate suggesting a higher energy cost of force generation. Future studies are aimed at determining the energetic cost of contraction in R403Q hearts and how this increased energetic cost leads to hypertrophic cardiomyopathy.
• Konhilas, J. P., Pollow, D. P., Perez, J., Konhilas, J. P., Constantopoulos, E., Brooks, H. L., & Booth, A. (2013). Postmenopausal response to angiotensin II-induced hypertension is blunted during perimenopause: a study in the accelerated ovarian failure (AOF) model of menopause. The FASEB Journal, 27.
• Konhilas, J., Chen, H., Hwang, H., McKee, L. A., Perez, J. N., Regan, J. A., Constantopoulos, E., Lafleur, B., & Konhilas, J. -. (2013). Temporal and morphological impact of pressure overload in transgenic FHC mice. Frontiers in physiology, 4.
  More info

  Although familial hypertrophic cardiomyopathy (FHC) is characterized as cardiac disease in the absence of overt stressors, disease penetrance, and pathological progression largely depend on modifying factors. Accordingly, pressure overload by transverse aortic constriction (TAC) was induced in 2-month-old, male mice with and without a FHC (R403Q) mutation in α-myosin heavy chain. A significantly greater number of FHC mice (n = 8) than wild-type (WT) mice (n = 5) died during the 9-week study period. TAC induced a significant increase in cardiac mass whether measured at 2 or 9 weeks post-TAC in both WT and FHC mice, albeit to a different extent. However, the temporal and morphological trajectory of ventricular remodeling was impacted by the FHC transgene. Both WT and FHC hearts responded to TAC with an early (2 weeks post-TAC) and significant augmentation of the relative wall thickness (RWT) indicative of concentric hypertrophy. By 9 weeks post-TAC, RWT decreased in WT hearts (eccentric hypertrophy) but remained elevated in FHC hearts. WT hearts following TAC demonstrated enhanced cardiac function as measured by the end-systolic pressure-volume relationship, pre-load recruitable stroke work (PRSW), and myocardial relaxation indicative of compensatory hypertrophy. Similarly, TAC induced differential histological and cellular remodeling; TAC reduced expression of the sarcoplasmic reticulum Ca(2+)-ATPase (2a) (SERCA2a; 2 and 9 weeks) and phospholamban (PLN; 2 weeks) but increased PLN phosphorylation (2 weeks) and β-myosin heavy chain (β-MyHC; 9 weeks) in WT hearts. FHC-TAC hearts showed increased β-MyHC (2 and 9 weeks) and a late (9 weeks) decrease in PLN expression concomitant with a significant increase in PLN phosphorylation. We conclude that FHC hearts respond to TAC induced pressure overload with increased premature death, severe concentric hypertrophy, and a differential ability to undergo morphological, functional, or cellular remodeling compared to WT hearts.
• Konhilas, J., McKee, L. A., Chen, H., Regan, J. A., Behunin, S. M., Walker, J. W., Walker, J. S., & Konhilas, J. -. (2013). Sexually dimorphic myofilament function and cardiac troponin I phosphospecies distribution in hypertrophic cardiomyopathy mice. Archives of biochemistry and biophysics, 535(1).
  More info

  The pathological progression of hypertrophic cardiomyopathy (HCM) is sexually dimorphic such that male HCM mice develop phenotypic indicators of cardiac disease well before female HCM mice. Here, we hypothesized that alterations in myofilament function underlies, in part, this sex dimorphism in HCM disease development. Firstly, 10-12month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca(2+)-sensitive tension development in demembranated cardiac trabeculae excised from 10-12month female and male HCM mice. Whereas HCM did not impact Ca(2+)-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca(2+) than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca(2+)-sensitive tension development was due to changes in Ca(2+) handling and sarcomeric proteins, including expression of SR Ca(2+) ATPase (2a) (SERCA2a), β-myosin heavy chain (β-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and β-MyHC protein whereas male HCM hearts showed a similar elevation of β-MyHC protein but a reduced level of cardiac troponin T (cTnT) phosphorylation. We also measured the distribution of cardiac troponin I (cTnI) phosphospecies using phosphate-affinity SDS-PAGE. The distribution of cTnI phosphospecies depended on sex and HCM. In conclusion, female and male HCM mice display sex dimorphic myofilament function that is accompanied by a sex- and HCM-dependent distribution of sarcomeric proteins and cTnI phosphospecies.
• Konhilas, J., Perez, J. N., Chen, H., Regan, J. A., Emert, A., Constantopoulos, E., Lynn, M., & Konhilas, J. -. (2013). Effects of chemically induced ovarian failure on voluntary wheel-running exercise and cardiac adaptation in mice. Comparative medicine, 63(3).
  More info

  The role of exercise in decreasing the risk of cardiovascular disease in postmenopausal women has not been studied sufficiently. Accordingly, we investigated the effect of voluntary wheel-running and forced treadmill exercise on cardiac adaptation in mice treated with 4-vinylcyclohexine diepoxide (VCD), which selectively accelerates the loss of primary and primordial follicles and results in a state that closely mimics human menopause. Two-month-old female C57BL/6 mice injected with VCD (160 mg/kg) for 20 consecutive days underwent ovarian failure by 60 to 90 d after injection. Responses to voluntary wheel running and treadmill exercise did not differ between VCD- and vehicle-treated 7-mo-old C57BL/6 or outbred B6C3F1 mice. Moreover, adaptive cardiac hypertrophy, hypertrophic marker expression, and skeletal muscle characteristics after voluntary cage-wheel exercise did not differ between VCD- and vehicle-treated mice. Because 5' AMP-activated protein kinase (AMPK) is a key component for the maintenance of cardiac energy balance during exercise, we determined the effect of exercise and VCD-induced ovarian failure on the AMPK signaling axis in the heart. According to Western blotting, VCD treatment followed by voluntary cage-wheel exercise differently affected the upstream AMPK regulatory components AMPKα1 and AMPKα2. In addition, net downstream AMPK signaling was reduced after VCD treatment and exercise. Our data suggest that VCD did not affect exercise-induced cardiac hypertrophy but did alter cellular cardiac adaptation in a mouse model of menopause.
• Lipovka, Y., & Konhilas, J. P. (2013). Abstract 320: Roles of Estrogen, AMPK and Micro RNAs in the Progression of Cardiac Hypertrophy. Circulation Research, 113.
  More info

  In industrialized countries, the prevalence of congestive heart failure (CHF) is increasing steadily and has become one of the leading causes of hospitalization. In addition, the risk of cardiovascular disease increases in post-menopausal women. Yet, the association between estrogen and the risk of CHF has not been adequately studied. Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. Our on-going studies indicate differential AMPK regulation through two miR species (miR195 and miR451) in a mouse model harboring a missense mutation (R403Q) in alpha-myosin heavy chain (αMHC) causing hypertrophic cardiomyopathy (HCM). Using bioinformatic algorithms (TargetScanMouse, 5.2), we were able to predict miR candidates that potentially target the AMPK axis. In addition, Altered expression of miRs that target AMPK axis was found in phenylephrine induced hypertrophic neonatal rat cardiomyocytes (NRCM). However, Estradiol treatment of NRCM blocked the hypertrophic changes induced by phenylephrine treatment. It was known that the activation of AMPK pathway inhibits cardiomyocyte hypertrophy. Our data showed that AMPK pathway was activated by Estradiol treatment, which can be blocked by estrogen receptor (ER) β antagonist. Therefore, estradiol increase AMPK pathway activation which in turn attenuate phenylephrine induced increase in cardiomyocyte cell size. Further studies are need to further explore the role of estrogen in the regulation of miR expression in hypertrophic cardiomyocytes, and the role of the expression changes of miRs regulated by estrogen in the development of hypertrophic phenotype.
• Haines, C. D., Harvey, P. A., Luczak, E. D., Barthel, K. K., Konhilas, J. P., Watson, P. A., Stauffer, B. L., & Leinwand, L. A. (2012). Estrogenic compounds are not always cardioprotective and can be lethal in males with genetic heart disease. Endocrinology, 153(9), 4470-9.
  More info

  Hypertrophic cardiomyopathy (HCM) is more severe in male than female mice eating a soy-based diet. We sought to determine whether the detrimental effects are mediated by the phytoestrogens present in soy, the mechanism by which phytoestrogens act, and to test whether estrogen modulates the sexually dimorphic phenotype. A soy-free diet (casein based) supplemented with the predominant phytoestrogens in soy, genistein and daidzein, recapitulated the fibrotic, proapoptotic and negative hemodynamic effects of soy in male hearts. As with the soy diet, the hearts of female HCM mice were not negatively affected by the phytoestrogen-containing diet. To determine the role of estrogen in the sex differences mediated by diet in HCM, gonadectomies were performed and estrogen was administered to male and female HCM mice on a casein- or phytoestrogen-supplemented diet. Somewhat surprisingly, estrogen was not protective in male or female mice with HCM and, in fact, was lethal in phytoestrogen-fed male mice with HCM. Because genistein is a potent tyrosine kinase inhibitor and tyrosine kinase inhibition has been associated with cardiotoxicity, we tested its effects in isolated adult cardiac myocytes. Genistein inhibited different tyrosine kinases depending on sex and, in combination with estrogen, resulted in apoptosis only in adult male cardiac myocytes. Finally, we show that phytoestrogens led to distinct programs of gene expression in hearts from males vs. females with HCM, suggesting mechanisms by which males are more sensitive to the detrimental effects of phytoestrogens and females are protected. These results implicate the phytoestrogen genistein in mediating cardiac pathology in males with HCM and, importantly, establish that estrogen is not protective in the setting of HCM.
• Konhilas, J. P., Behunin, S. M., & Lynch, R. M. (2012). Keeping the beat. Focus on "enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro". American journal of physiology. Cell physiology, 303(12), C1218-9.
• Konhilas, J. P., Hidalgo, C., Chen, H., & Behunin, S. M. (2012). Cardiac Troponin I Phosphorylation at ser149 by Protein Kinase A: A Potential Modulator of Myocardial Contractility. Biophysical Journal, 102(3), 358a. doi:10.1016/j.bpj.2011.11.1955
  More info

  In the heart, stimulation of β-adrenergic pathway and subsequent activation of protein kinase A (PKA) is known to increase myocardial contractility. The increase in contractility is, in part, due to target phosphorylation of troponin I (TnI). In this study, we sought to identify novel target sites for PKA that could potentially contribute to this increase in contractility. To induce phosphorylation of TnI, cardiac and fast skeletal muscle from 3-4 month old Sprague Dawley rats was mechanically disrupted and demembranated followed by incubation with the catalytic subunit of PKA (50U PKA/ 3mg tissue, 0-30 min). To identify target specific phosphorylation on fast skeletal (fsTnI) or cardiac (cTnI) TnI, western blot analysis with phospho-specific antibodies was performed. PKA treatment increased phosphorylation of cTnI at ser22/23, as expected, but also at ser149. Similarly, PKA treatment increased phosphorylation of fsTnI at ser117, which is the equivalent to ser149 in cTnI. Accordingly, fsTnI demonstrated no observable phosphorylation at ser22/23. Adenosine-monophosphate activated kinase (AMPK) has been shown to target ser149 of cTnI. Therefore, to validate PKA-dependent phosphorylation of cTnI at ser149, hearts were excised and perfused with AICAR, a known activator of AMPK. AICAR-perfused hearts demonstrated a time-dependent increase in phosphorylation of cTnI at ser149. These results demonstrate that PKA-dependent phosphorylation can target ser149 in cTnI and, equivalently, ser117 in fsTnI. The functional consequence of this target site phosphorylation and how it impacts contractility is currently under investigation.
• Konhilas, J., Chen, H., Untiveros, G. M., McKee, L. A., Perez, J., Li, J., Antin, P. B., & Konhilas, J. -. (2012). Micro-RNA-195 and -451 regulate the LKB1/AMPK signaling axis by targeting MO25. PloS one, 7(7).
  More info

  Recently, MicroRNAs (miR) and AMP-kinase (AMPK) have emerged as prominent players in the development of cardiac hypertrophy and heart failure. We hypothesized that components of the adenosine monophosphate-activated kinase (AMPK) pathway are targeted by miRs and alter AMPK signaling during pathological cardiac stress.
• Luczak, E. D., Luckey, S. W., Leinwand, L. A., Konhilas, J. P., & Haines, C. D. (2012). Abstract 276: Cyclin D2 Is a Critical Mediator of Exercise-Induced Cardiac Hypertrophy. Circulation Research, 111.
  More info

  Exercise training activates a number of hypertrophic signaling pathways that can be distinct from those activated by pathologic stimuli. However, there must be some overlap in those pathways underlying myocyte cell growth. Using a number of mouse genetic models, we investigated the role of several molecules implicated in pathologic cardiac hypertrophy for their cardiac responses to exercise. We used three-month-old female transgenic mice expressing the anti-hypertrophic molecules, cardiac-specific constitutively active glycogen synthase kinase-3β (caGSK-3β), an inhibitor of Ca 2+ -calmodulin-dependent protein kinase (CaMKII Inh), and doubly transgenic mice expressing both caGSK-3β and CaMKII inhibition (caGSK-3β/CaMKII Inh). We also studied the exercise responsiveness of mice expressing the pro-hypertrophic cardiac-specific activated (myr)Akt. MAPK/ERK kinase kinase-1 (MEKK1) has been shown to be essential for pathologic cardiac hypertrophy and we therefore studied the requirement of MEKK1 for exercise-induced cardiac growth. Cell cycle regulators such as cyclin D2 have been shown to be required for pathologic cardiac hypertrophy; therefore we studied cyclin D2 null mice. Mice were divided into sedentary and 21 days of voluntary exercise on a cage wheel. Across the seven different mouse models, exercise capacity was similar with regards to running duration, distance, and speed. Importantly, we analyzed the impact of exercise on cardiac hypertrophy by measuring heart weight-to-body weight (HW/BW) ratios of sedentary and exercised mice. While exercise had no impact on body weight, heart weight increased significantly in all mouse models except the cyclin D2 -/- mice. Overall there was a 3.5-fold range of percent increase in HW/BW ratios from the highest (caGSK-3β) to the lowest (cyclin D2 -/- ). In conclusion, genetic manipulation of these hypertrophic signaling pathways has little impact on exercise performance and only the loss of cyclin D2 attenuates exercise-induced cardiac growth. These data establish cyclin D2 as an important regulator of physiological hypertrophy and underscore the differences in pathologic and physiologic cardiac hypertrophy.
• Velden, J. V., Stienen, G. J., Slegtenhorst, M. V., Sequeira, V., Remedios, C. D., Regan, J. A., Michels, M., Konhilas, J. P., & Cate, F. J. (2012). Reduced Length-Dependent Activation in Human Cardiomyocytes Harboring the Troponin I Mutation R145W. Biophysical Journal, 102(3), 158a. doi:10.1016/j.bpj.2011.11.860
  More info

  Background: One of the mutated sarcomeric proteins in familial hypertrophic cardiomyopathy (FHC) is Troponin I (cTnI), a down-stream target of the β-adrenergic receptor pathway. Activation of this pathway leads to protein kinase A (PKA)-mediated phosphorylation of cTnI and sarcomere desensitization to Ca2+ to improve muscle relaxation. Moreover, cTnI phosphorylation was shown to enhance sarcomere length-dependent activation (i.e. Frank-Starling mechanism). We investigated if the responsiveness to PKA and to an increase in sarcomere length is impaired by mutant cTnI in human FHC.Methods: A comparison was made between cardiac samples from two FHC patients harboring the R145W mutation in the TNNI3 gene (TNNI3mut) and non-failing donor hearts. Isometric force was measured in Triton-permeabilized cardiomyocytes at different [Ca2+] and sarcomere lengths of 1.8 and 2.2 μm before and after treatment with exogenous PKA.Results: At a sarcomere length of 2.2 μm, TNNI3mut cells (n=30) showed lower maximal force (26.3±4.9 kN/m2) and similar passive force (2.1±0.2 kN/m2) compared to donor cells (n=48; 36.4±2.3 and 2.4±0.3 kN/m2, respectively). In addition, a significantly higher Ca2+-sensitivity (EC50)(2.69±0.16 μM) and a decreased steepness (2.17±0.10) of the force-Ca2+ relation were found in TNNI3mut compared to donor (3.09±0.09 μM and 3.75±0.12, respectively). The increase in Ca2+-sensitivity (i.e. length-dependent activation; ΔEC50) upon an increase in sarcomere length was significantly smaller in TNNI3mut (0.29±0.15) compared to donor (0.76±0.06) cells. Treatment with PKA decreased myofilament Ca2+-sensitivity in TNNI3mut to values observed in donor, but did not correct the reduced length-dependent activation.Conclusions: Our data show preserved responsiveness to PKA in FHC harboring the R145W mutation, but a blunted length-dependent activation, which was not corrected by PKA. Reduced maximal force and perturbed Frank-Starling mechanism, both at baseline and after PKA, may impair contractile performance in human FHC with mutant cTnI.
• Walker, J. S., Regan, J. A., Mckee, L. A., Leinwand, L. A., Konhilas, J. P., & Chen, H. (2012). Target Specific Phosphorylation of Cardiac Troponin I and Sex Dimorphic Myofilament Function in R403Q Mice. Biophysical Journal, 102(3), 555a-556a. doi:10.1016/j.bpj.2011.11.3027
  More info

  Male mice expressing an autosomal dominant mutation in alpha-myosin heavy chain (R403Q) develop hypertrophic cardiomyopathy characterized by progressive left-ventricular dilation and cardiac dysfunction, whereas females do not. We hypothesize that this sexual dimorphism exists on multiple levels, including cellular metabolism regulation, post-translational modulation of contractile proteins and mechanical functions of contractile fibers. Hearts from wild-type (WT) and R403Q male and female animals with established disease were analyzed for myosin heavy chain (MyHC) isoform expression, total troponin I (TnI) phosphorylation, site-specific TnI phosphorylation, AMPKα expression, and AMPK activity. Ca2+-sensitive tension development was measured in demembranated cardiac trabeculae. R403Q male and female mice demonstrated progressive cardiac hypertrophy beginning at 4 months of age. β-MyHC expression increased in male and female R403Q mice. Total phosphorylation of TnI expression was independent of sex and R403Q mutation. No difference was found in phospho-TnI-Ser22/23 in R403Q animals relative to WT controls, however R403Q females had increased expression of phospho-TnI-Ser150 relative to WT counterparts. R403Q males showed increased expression of AMPKα, however had decreased AMPK activity relative to WT counterparts. R403Q females had reduced AMPKα expression but unchanged AMPK activity. The R403Q mutation does not affect Ca2+-sensitive tension development in demembranated cardiac trabeculae from males; female R403Q cardiac trabeculae were more sensitive to Ca2+ than WT controls. In conclusion, R403Q mice display hypertrophy with increased expression of hypertrophy marker β-MyHC. There exists a sexually dimorphic pattern of TnI phosphorylation at Ser150, although global TnI phosphorylation was unchanged. There exists a sexually dimorphic pattern of AMPK expression and activity, suggesting sexual dimorphic regulation of cellular metabolism and energetics. Increased calcium sensitivity may provide sufficient contractile support in R403Q females maintaining a compensated state of cardiac function, a characteristic not exhibited in males.
• Luczak, E. D., Barthel, K. K., Stauffer, B. L., Konhilas, J. P., Cheung, T. H., & Leinwand, L. A. (2011). Remodeling the cardiac transcriptional landscape with diet. Physiological genomics, 43(12), 772-80.
  More info

  The perception that soy food products and dietary supplements will have beneficial effects on cardiovascular health has led to a massive consumer market. However, we have previously noted that diet profoundly affects disease progression in a genetic model of hypertrophic cardiomyopathy (HCM). In this model, a soy-based diet negatively impacts cardiac function in male mice. Given the frequent connection between functional changes and transcriptional changes, we investigated the effect of diet (soy- vs. milk-based) on cardiac gene expression and how it is affected by the additional factors of sex and disease. We found that gene expression in the heart is altered more by diet than by sex or an inherited disease. We also found that the healthy male heart may be sensitized to dietary perturbations of gene expression in that it displays a gene expression profile more similar to diseased male and female hearts than to healthy female hearts. These observations may in part account for documented divergence in HCM phenotypes between males and females and between diets.
• Mark-Kappeler, C. J., Sen, N., Lukefahr, A., McKee, L., Sipes, I. G., Konhilas, J., & Hoyer, P. B. (2011). Inhibition of ovarian KIT phosphorylation by the ovotoxicant 4-vinylcyclohexene diepoxide in rats. Biology of reproduction, 85(4), 755-62.
  More info

  In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 μM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 μg/ml) or ACK4 (80 μg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.
• Untiveros, G. M., Perez, J. N., Mckee, L. A., Konhilas, J. P., & Chen, H. (2011). Abstract P196: The Impact of MicroRNA195 on the Lkb1/AMPK Signaling Axis and Hypertrophic Cardiomyopathy. Circulation Research, 109.
  More info

  Objectives: MicroRNAs (miRs) have been identified as chief post-transcriptional regulators of cardiac disease progression. In addition, a critical role of the adenosine monophosphate-activated kinase (AMPK) pathway in the development of myocardial hypertrophy has been revealed. Yet, regulation of the AMPK pathway by miRs in the heart has not been addressed. We hypothesized that components of the AMPK pathway are targeted by miRs and alter AMPK signaling in a mouse model of hypertrophic cardiomyopathy (HCM). Methods and results: Using real-time PCR, a candidate miR screen that included 22 miRs implicated in pathological cardiac disease and/or metabolic dysregulation was performed on hearts from 60-, 120-, and 240-day-old transgenic HCM male mice harboring an R403Q mutation in the myosin heavy gene. Among early (60 day) elevated miRs were miR-195 and -451. Both miR-195 and -451 have conserved target sites in the 3′ UTR of CAB39 (MO25), a central component of the MO25/STRAD/LKB1 complex that acts as an upstream kinase for AMPK and its subsequent activation. We further confirmed the elevation of miR-195 and -451 by Northern blotting. Next, we demonstrated specific expression and a similar distribution pattern of miR-195 and -451 in cardiomyocytes of R403Q HCM hearts by in situ hybridization. To determine whether the conserved sites in MO25 3′ UTR acted as functional targets, either the miR-195 or miR-451 target sequence was cloned into a luciferase expression vector. MiR-195 but not miR-451 suppressed luciferase activity compared to the missense sequence control vector in C2C12 cells. In addition, over-expression of miR195 in C2C12 cells knocked down MO25 expression levels and downstream AMPK signaling (phosphorylation of Acetyl CoA carboxylase). Finally, parallel changes were measured in 60 day R403Q HCM male hearts that included reduced MO25 expression and lowered phosphorlation of AMPK and Acetyl CoA carboxylase. Conclusion: Our findings indicate that miR-195 targets the LKB1/AMPK signaling axis and suggest a functional role for miR-195 elevation in R403Q HCM disease progression.
• Konhilas, J. P. (2010). Sex Dimorphic Myofilament Function and AMPK Expression in R403Q Hearts. Biophysical Journal, 98(3), 716a. doi:10.1016/j.bpj.2009.12.3926
  More info

  Male mice expressing an autosomal dominant mutation in alpha-myosin heavy chain (R403Q) develop hypertrophic cardiomyopathy characterized by progressive left-ventricular dilation and cardiac dysfunction whereas females do not. We wished to determine whether these sex dimorphisms were due to underlying differences in myofilament contractile function. Therefore, we determined the sensitivity of the myofilaments to Ca2+ in demembranated cardiac trabeculae (CT) from wild-type (WT) and R403Q male and female mice (10-12 months of age). We demonstrate that the R403Q mutation did not affect Ca2+-sensitive tension development in CT from males. While Ca2+-sensitivity was greater in both male WT and R403Q CT compared to WT females, they were less sensitive to Ca2+ than CT from female R403Q hearts. We also determined rates of tension redevelopment (ktr) following a release-restretch protocol in CT from WT and R403Q male and female hearts at the same age. CT from R403Q male hearts exhibited an enhanced ktr compared to WT males. The ktr in WT female CT was similar to WT males. The ktr in R403Q female CT measured between WT and R403Q males. We hypothesized that the sex dimorphisms in myofialment function reflect an increase in the energetic cost of contraction when expressing the R403Q mutation. Therefore, we measured levels of Adenosine monophosphate-activated kinase (AMPK), a central sensor of the cellular energy state. Total AMPK protein levels were significantly increased in 10-12 month male R403Q hearts compared to WT controls. Female R403Q hearts showed the opposite: total AMPKα expression was lower compared to WT controls. We conclude that (1) the increased Ca2+-sensitivity may provide sufficient contractile support in female R403Q hearts maintaining a compensated state, and (2) the increased AMPK expression in male R403Q hearts is indicative of an increased energetic demand caused by the mutation.
• Konhilas, J. P., Boucek, D. M., Horn, T. R., Johnson, G. L., & Leinwand, L. A. (2010). The role of MEKK1 in hypertrophic cardiomyopathy. International heart journal, 51(4), 277-84.
  More info

  MEKK1 is a ubiquitously expressed mitogen activated protein kinase that is involved in tissue remodeling in a variety of settings including carotid artery blood flow cessation, wound healing, and breast adenocarcinoma intravasation. Here, we have tested the function of MEKK1 in genetic hypertrophic cardiomyopathy (HCM). MEKK1 was genetically deleted in C57Bl6/J mice expressing a mutant alpha-myosin heavy chain (HCM-MEKK1(-/-)). The absence of MEKK1 in HCM resulted in a more pronounced hypertrophy when compared to HCM mice with the MEKK1 gene intact without further increases in atrial natriuretic factor and beta-myosin heavy chain (MyHC) expression and fibrosis. Since MEKK1 is required for the induction of several tissue proteases, we tested the hypothesis that cardiac enlargement of HCM- MEKK1(-/-) mice was due to altered expression of urokinase-type plasminogen activator (uPA), JunB, matrix-metalloproteinase (MMP), and tissue inhibitors of MMPs (TIMPs). Because of its role in preventing apoptosis, we also tested the loss of MEKK1 on apoptotic mediators Bcl-2, cytochrome C, caspase-9, and caspase-3. uPA expression was decreased while JunB, MMP-9, caspase-9, and caspase-3 activities were elevated in HCM- MEKK1(-/-) hearts when compared to MEKK1(-/-), wild-type (WT), and HCM mice. Bcl-2 and Cyt C expression was elevated only in HCM mice. We conclude that the absence of MEKK1 induces a more pronounced cardiac hypertrophy to HCM through altered expression of proteases implicated in cardiac remodeling and increased apoptosis.
• Konhilas, J., & Konhilas, J. -. (2010). What we know and do not know about sex and cardiac disease. Journal of biomedicine & biotechnology, 2010.
  More info

  Cardiovascular disease (CVD) remains the single leading cause of death in both men and women. A large proportion of the population with CVD will die with a diagnosis of congestive heart failure (CHF). It is becoming increasingly recognized that sex differences exist in the etiology, development, and outcome of CHF. For example, compared to male counterparts, women that present with CHF are typically older and have systolic cardiac function that is not impaired. Despite a growing body of literature addressing the underlying mechanisms of sex dimorphisms in cardiac disease, there remain significant inconsistencies reported in these studies. Given that the development of CHF results from the complex integration of genetic and nongenetic cues, it is not surprising that the elucidation and subsequent identification of molecular mechanisms remains unclear. In this review, key aspects of sex differences in CVD and CHF will be highlighted with an emphasis on some of the unanswered questions regarding these differences. The contention is presented that it becomes critical to reference cellular mechanisms within the context of each sex to better understand these sex dimorphisms.
• Rosedale, R., Westman, E. C., & Konhilas, J. P. (2009). Clinical Experience of a Diet Designed to Reduce Aging. The journal of applied research, 9(4), 159-165.
  More info

  OBJECTIVE: Aging is associated with elevated levels of glucose, insulin, and triglycerides. Our objective was to assess the effect of a nutritional program designed to reduce these correlates of aging. DESIGN: This is a retrospective chart review of patients attending an outpatient metabolic management program including a high-fat, adequate-protein, low-carbohydrate diet, nutritional supplementation and periodic individual visits. Outcomes measured at baseline and follow-up included body weight, fasting serum glucose, insulin, leptin, lipids, and thyroid hormone. RESULTS: Thirty-one patients were identified with complete information. The mean age of patients was 57.6 ± 2.4 consisting of 53% female and 47% male patients. The average duration between follow up visits was 91.5 ± 8.5 days. Of the parameters measured at the follow-up visit, body weight, serum leptin, insulin, fasting glucose, triglyceride, and free T(3) significantly decreased by 8.1 ± 0.8%, 48.2 ± 3.8%, 40.1 ± 4.7%, 7.6 ± 2.1%, 28.3 ± 5.7%, and 10.8 ± 1.8%, respectively. Furthermore, the triglyceride/high density lipoprotein ratio decreased from 5.1 ± 1.7 to 2.6 ± 0.5. CONCLUSIONS: In the context of an outpatient medical clinic, a high-fat, adequate-protein, low-carbohydrate diet with nutritional supplementation led to improvements in serum factors related to the aging process. Further research regarding this dietary approach and its relationship to aging is in order.
• Jensen, D. R., Maclean, P. S., Knaub, L. A., Leinwand, L. A., Konhilas, J. P., Konhilas, J. P., Leinwand, L. A., Knaub, L. A., MacLean, P. S., Jensen, D. R., Eckel, R. H., & Eckel, R. H. (2008). Increased thermoregulation in cold-exposed transgenic mice overexpressing lipoprotein lipase in skeletal muscle: an avian phenotype?. Journal of lipid research, 49(4), 870-9. doi:10.1194/jlr.m700519-jlr200
  More info

  LPL is an enzyme involved in the breakdown and uptake of lipoprotein triglycerides. In the present study, we examined how the transgenic (Tg) overexpression of human LPL in mouse skeletal muscle affected tolerance to cold temperatures, cold-induced thermogenesis, and fuel utilization during this response. Tg mice and their nontransgenic controls were placed in an environmental chamber and housed in metabolic chambers that monitored oxygen consumption and carbon dioxide production with calorimetry. When exposed to 4 degrees C, an attenuation in the decline in body temperature in Tg mice was accompanied by an increased metabolic rate (15%; P < 0.001) and a reduction in respiratory quotient (P < 0.05). Activity levels, the expression of uncoupling proteins in brown fat and muscle, and lean mass failed to explain the enhanced cold tolerance and thermogenesis in Tg mice. The more oxidative type IIa fibers were favored over the more glycolytic type IIb fibers (P < 0.001) in the gastrocnemius and quadriceps muscles of Tg mice. These data suggest that Tg overexpression of LPL in skeletal muscle increases cold tolerance by enhancing the capacity for fat oxidation, producing an avian-like phenotype in which skeletal muscle contributes significantly to the thermogenic response to cold temperatures.
• Konhilas, J. P. (2008). What makes a dead cell attractive?. Journal of applied physiology (Bethesda, Md. : 1985), 104(3), 573-4. doi:10.1152/japplphysiol.01376.2007
  More info

  congestive heart failure represents the end stage of various cardiac disease etiologies, including hypertension, myocardial infarction/ischemia from atherosclerosis, viral myocarditis, valvular insufficiency, or mutations in genes encoding sarcomeric proteins ([1][1], [10][2], [11][3]). These events
• Konhilas, J. P., & Leinwand, L. A. (2007). The effects of biological sex and diet on the development of heart failure. Circulation, 116(23), 2747-59.
• Watson, P. A., Reusch, J. E., McCune, S. A., Leinwand, L. A., Luckey, S. W., Konhilas, J. P., Brown, D. A., Chicco, A. J., Sparagna, G. C., Long, C. S., & Moore, R. L. (2007). Restoration of CREB function is linked to completion and stabilization of adaptive cardiac hypertrophy in response to exercise. American journal of physiology. Heart and circulatory physiology, 293(1), H246-59.
  More info

  Potential regulation of two factors linked to physiological outcomes with left ventricular (LV) hypertrophy, resistance to apoptosis, and matching of metabolic capacity, by the transcription factor cyclic-nucleotide regulatory element binding protein (CREB), was examined in the two models of physiological LV hypertrophy: involuntary treadmill running of female Sprague-Dawley rats and voluntary exercise wheel running in female C57Bl/6 mice. Comparative studies were performed in the models of pathological LV hypertrophy and failure: the spontaneously hypertension heart failure (SHHF) rat and the hypertrophic cardiomyopathy (HCM) transgenic mouse, a model of familial idiopathic cardiomyopathy. Activating CREB serine-133 phosphorylation was decreased early in remodeling in response to both physiological (decreased 50-80%) and pathological (decreased 60-80%) hypertrophic stimuli. Restoration of LV CREB phosphorylation occurred concurrent with completion of physiological hypertrophy (94% of sedentary control), but remained decreased (by 90%) during pathological hypertrophy. In all models of hypertrophy, CREB phosphorylation/activation demonstrated strong positive correlations with 1) expression of the anti-apoptotic protein bcl-2 (a CREB-dependent gene) and subsequent reductions in the activation of caspase 9 and caspase 3; 2) expression of peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1; a major regulator of mitochondrial content and respiratory capacity), and 3) LV mitochondrial respiratory rates and mitochondrial protein content. Exercise-induced increases in LV mitochondrial respiratory capacity were commensurate with increases observed in LV mass, as previously reported in the literature. Exercise training of SHHF rats and HCM mice in LV failure improved cardiac phenotype, increased CREB activation (31 and 118%, respectively), increased bcl-2 content, improved apoptotic status, and enhanced PGC-1 content and mitochondrial gene expression. Adenovirus-mediated expression of constitutively active CREB in neonatal rat cardiac recapitulated exercise-induced upregulation of PGC-1 content and mitochondrial oxidative gene expression. These data support a model wherein CREB contributes to physiological hypertrophy by enhancing expression of genes important for efficient oxidative capacity and resistance to apoptosis.
• Konhilas, J. P., & Leinwand, L. A. (2006). Partnering up for cardiac hypertrophy. Circulation research, 98(8), 985-7.
• Stauffer, B. L., Luczak, E. D., Leinwand, L. A., & Konhilas, J. P. (2006). Soy Diet Worsens Heart Disease in Mice. Obstetrical & Gynecological Survey, 61(5), 326-328. doi:10.1097/01.ogx.0000216528.66156.49
  More info

  A soy-rich diet is viewed as being beneficial to health, especially the cardiovascular system. Although Americans spend billions on soy foods and dietary supplements, systematic studies on how genetic characteristics might relate to different diets and how gender might shape this interaction are lacking. The soy-based diet given to lab rodents reportedly prevents cancer and lowers cholesterol levels, but it may also increase androgen levels and decrease thyroid peroxidase. Many effects of a soy diet are ascribed to isoflavones or phytoestrogens. The investigators examined the effect of diet on a genetic mouse model of hypertrophic cardiomyopathy (HCM). Cardiac contractile function is preserved in female mice, which continues to increase their cardiac mass. Male mice, in contrast, develop thin ventricular walls and have poor cardiac contractility. The HCM mouse expresses a mutant myosin heavy chain transgene in the heart. The role of diet was examined by comparing HCM mice with WT (wild type) littermate control mice of both sexes. A standard soy-based diet was compared with a casein-based diet, which in some cases was supplemented with daidzein and genistein, the most prominent isoflavones in soy. The HCM mice had higher ratios of ventricular weight to tibial length than did sex-matched WT control mice fed the same diet. Male animals had larger hearts than females. HCM mice had significantly more hypertrophy on the casein diet than when taking a soy diet. Male HCM animals fed a casein-based diet had greater cardiac hypertrophy than those fed a soy diet. In contrast, diet did not influence cardiac hypertrophy in female HCM animals. Cardiac growth was less in both male and female HCM animals eating the standard soy diet. Adding phytoestrogens to the casein-based diet did not entirely reproduce this phenotype. Mutated male mice developed heart failure on a soy diet but, when fed a casein diet, did not develop severe dilated cardiomyopathy. Left ventricular size and contractile function were preserved in these animals. In addition, the casein diet prevented numerous pathologic changes in male mice, including fibrosis, induction of p-myosin heavy chain, inactivation of glycogen synthase kinase 3β, and markedly elevated caspase-3. The investigators conclude that the dilated cardiac phenotype in male HCM mice fed a soy diet represents augmented growth and increased programmed cell death. Decompensation is associated with a change from a balanced to an unbalanced relationship between cell growth and cell death. These mice exhibit more physiological and less pathologic cardiac growth when given a soy-free diet. Hopefully, further research will suggest novel dietary changes that will prevent the development or progression of hypertrophic cardiac disorders. Such research will focus on identifying specific genetic modifiers that influence the heart's response to diet.
• Stauffer, B. L., Luczak, E. D., Leinwand, L. A., & Konhilas, J. P. (2006). Soy diet worsens heart disease in mice.. The Journal of clinical investigation, 116(1), 209-16. doi:10.1172/jci24676
  More info

  We report that dietary modification from a soy-based diet to a casein-based diet radically improves disease indicators and cardiac function in a transgenic mouse model of hypertrophic cardiomyopathy. On a soy diet, males with a mutation in the alpha-myosin heavy chain gene progress to dilation and heart failure. However, males fed a casein diet no longer deteriorate to severe, dilated cardiomyopathy. Remarkably, their LV size and contractile function are preserved. Further, this diet prevents a number of pathologic indicators in males, including fibrosis, induction of beta-myosin heavy chain, inactivation of glycogen synthase kinase 3beta (GSK3beta), and caspase-3 activation.
• Watson, P. A., Stauffer, B. L., Rosenberg, P. B., Maass, A., Luckey, S. W., Leinwand, L. A., Konhilas, J. P., Horn, T. R., & Boucek, D. M. (2006). Exercise can prevent and reverse the severity of hypertrophic cardiomyopathy.. Circulation research, 98(4), 540-8. doi:10.1161/01.res.0000205766.97556.00
  More info

  Hypertrophic cardiomyopathy (HCM) is the most common form of sudden death in young competitive athletes. However, exercise has also been shown to be beneficial in the setting of other cardiac diseases. We examined the ability of voluntary exercise to prevent or reverse the phenotypes of a murine model of HCM harboring a mutant myosin heavy chain (MyHC). No differences in voluntary cage wheel performance between nontransgenic (NTG) and HCM male mice were seen. Exercise prevented fibrosis, myocyte disarray, and induction of "hypertrophic" markers including NFAT activity when initiated before established HCM pathology. If initiated in older HCM animals with documented disease, exercise reversed myocyte disarray (but not fibrosis) and "hypertrophic" marker induction. In addition, exercise returned the increased levels of phosphorylated GSK-3beta to those of NTG and decreased levels of phosphorylated CREB in HCM mice to normal levels. Exercise in HCM mice also favorably impacted components of the apoptotic signaling pathway, including Bcl-2 (an inhibitor of apoptosis) and procaspase-9 (an effector of apoptosis) expression, and caspase-3 activity. Remarkably, there were no differences in mortality between exercised NTG and HCM mice. Thus, not only was exercise not harmful but also it was able to prevent and even reverse established cardiac disease phenotypes in this HCM model.
• Widegren, U., Paul, A. C., Leinwand, L. A., Konhilas, J. P., Cleary, A. S., & Allen, D. L. (2005). Loaded wheel running and muscle adaptation in the mouse.. American journal of physiology. Heart and circulatory physiology, 289(1), H455-65. doi:10.1152/ajpheart.00085.2005
  More info

  Voluntary cage wheel exercise has been used extensively to determine the physiological adaptation of cardiac and skeletal muscle in mice. In this study, we tested the effect of different loading conditions on voluntary cage wheel performance and muscle adaptation. Male C57Bl/6 mice were exposed to a cage wheel with no-resistance (NR), low-resistance (LR), or high-resistance (HR) loads for 7 wk. Power output was elevated (3-fold) under increased loading (LR and HR) conditions compared with unloaded (NR) exercise training. Only unloaded (NR) exercise induced an increase in heart mass, whereas only loaded (LR and HR) exercise training induced an increase in skeletal (soleus) muscle mass. Moreover, unloaded and loaded exercise training had a differential impact on the cross-sectional area of muscle fibers, depending on the type of myosin heavy chain expressed by each fiber. The biochemical adaptation of the heart was characterized by a decrease in genes associated with pathological (but not physiological) cardiac hypertrophy and a decrease in calcineurin expression in all exercise groups. In addition, transcriptional activity of myocyte enhancer factor-2 (MEF-2) was significantly decreased in the hearts of the LR group as determined by a MEF-2-dependent transgene driving the expression of beta-galactosidase. Phosphorylation of glycogen synthase kinase-3beta, protein kinase B (Akt), and p70 S6 kinase was increased only in the hearts of the NR group, consistent with the significant increase in cardiac mass. In conclusion, unloaded and loaded cage wheel exercise have a differential impact on cage wheel performance and muscle (cardiac and skeletal) adaptation.
• Stauffer, B. L., Olson, E. N., Maass, A. H., Luckey, S. W., Leinwand, L. A., & Konhilas, J. P. (2004). Sex modifies exercise and cardiac adaptation in mice.. American journal of physiology. Heart and circulatory physiology, 287(6), H2768-76. doi:10.1152/ajpheart.00292.2004
  More info

  How an individual's sex and genetic background modify cardiac adaptation to increased workload is a topic of great interest. We systematically evaluated morphological and physiological cardiac adaptation in response to voluntary and forced exercise. We found that sex/gender is a dominant factor in exercise performance (in two exercise paradigms and two mouse strains) and that females of one of these strains have greater capacity to increase their cardiac mass in response to similar amounts of exercise. To explore the biochemical mechanisms for these differences, we examined signaling pathways previously implicated in cardiac hypertrophy. Ca2+/calmodulin-dependent protein kinase (CaMK) activity was significantly greater in males compared with females and increased after voluntary cage-wheel exposure in both sexes, but the proportional increase in CaMK activity was twofold higher in females compared with males. Phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was evident after 7 days of cage-wheel exposure in both sexes and remained elevated in females only by 21 days of exercise. Despite moderate increases in myocyte enhancer factor-2 (a downstream effector of CaMK) transcriptional activity and phosphorylation of Akt with exercise, there were no sex differences. Mitogen-activated protein kinase signaling components (p38 mitogen-activated protein kinase and extracellular regulated kinase 1/2) were not different between male and female mice and were not affected by exercise. We conclude that females have increased exercise capacity and increased hypertrophic response to exercise. We have also identified sex-specific differences in hypertrophic signaling within the cardiac myocyte that may contribute to sexual dimorphism in exercise and cardiac adaptation to exercise.
• Wolska, B. M., Tombe, P. P., Solaro, R. J., Martin, A. F., Konhilas, J. P., Jweied, E. E., & Irving, T. C. (2003). Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing.. The Journal of physiology, 547(Pt 3), 951-61. doi:10.1113/jphysiol.2002.038117
  More info

  Cyclic AMP-dependent protein kinase (PKA) targets contractile proteins, troponin-I (TnI) and myosin binding protein C (MyBP-C) in the heart and induces a decrease in myofilament Ca2+ sensitivity. Yet, the effect of sarcomere length (SL) change on Ca2+ sensitivity (length-dependent activation: LDA) following PKA-dependent phosphorylation is not clear. To clarify the role of PKA-dependent phosphorylation of TnI and MyBP-C on LDA in the heart, we examined LDA in skinned myocytes from a non-transgenic (NTG) and a transgenic murine model in which the native cardiac isoform (cTnI) was completely replaced by the slow skeletal isoform of TnI (ssTnI-TG) lacking the phosphorylation sites for PKA, while retaining PKA sites on MyBP-C. In NTG myocytes, PKA treatment decreased Ca2+ sensitivity at each SL, but enhanced the impact of SL change on Ca2+ sensitivity. Despite a greater sensitivity to Ca2+ and a reduction in LDA, neither Ca2+ responsiveness nor LDA was affected by PKA treatment in ssTnI-TG myocytes. To determine whether the above observations could be explained by the lateral separation between thick and thin filaments, as suggested by others, we measured interfilament spacing by X-ray diffraction as a function of SL in skinned cardiac trabeculae in the passive state from both NTG and ssTnI-TG models before and following treatment with PKA. Phosphorylation by PKA increased lattice spacing at every SL in NTG trabeculae. However, the relationship between SL and myofilament lattice spacing in ssTnI-TG was markedly shifted downward to an overall decreased myofilament lattice spacing following PKA treatment. We conclude: (1) PKA-dependent phosphorylation enhances length-dependent activation in NTG hearts; (2) replacement of native TnI with ssTnI increases Ca2+ sensitivity of tension but reduces length-dependent activation; (3) MyBP-C phosphorylation by PKA does not alter calcium responsiveness and induces a decrease in myofilament lattice spacing at all sarcomere lengths and (4) length-dependent activation in the heart cannot be entirely explained by alterations in myofilament lattice spacing.
• Stauffer, B. L., Maass, A., Leinwand, L. A., & Konhilas, J. P. (2002). From sarcomeric mutations to heart disease: understanding familial hypertrophic cardiomyopathy.. Cold Spring Harbor symposia on quantitative biology, 67(0), 409-15. doi:10.1101/sqb.2002.67.409
• Tombe, P. P., Konhilas, J. P., & Dobesh, D. P. (2002). Cooperative activation in cardiac muscle: impact of sarcomere length.. American journal of physiology. Heart and circulatory physiology, 282(3), H1055-62. doi:10.1152/ajpheart.00667.2001
  More info

  This study was undertaken to determine the impact of sarcomere length (SL) on the level of cooperative activation of the cardiac myofilament at physiological [Mg2+]. Active force development was measured in skinned rat cardiac trabeculae as a function of free [Ca2+] at five SLs (1.85-2.25 microm; 1 mM free [Mg2+]; 15 degrees C). Only muscle preparations with minimal force rundown during the entire protocol were included in the analysis (average 7.2 +/- 1.7%). Median SL was measured by on-line computer video micrometry and controlled within 0.01 microm. Care was taken to ensure a sufficient number of data points in the steep portion of the [Ca2+]-force relationship at every SL to allow for accurate fit of the data to a modified Hill equation. Multiple linear regression analysis of the fit parameters revealed that both maximum, Ca2+-saturated force and Ca2+ sensitivity were a significant function of SL (P < 0.001), whereas the level of cooperativity did not depend on SL (P = 0.2). Further analysis of the [Ca2+]-force relationships revealed a marked asymmetry that, also, was not affected by SL (P = 0.2-0.6). Finally, we found that the level of cooperativity in isolated skinned myocardium was comparable to that reported for intact, nonskinned myocardium. Our results suggest that an increase in SL induces an increase in the Ca2+ responsiveness of the cardiac sarcomere without affecting the level of cooperativity.
• Tombe, P. P., Konhilas, J. P., & Irving, T. C. (2002). Frank-Starling law of the heart and the cellular mechanisms of length-dependent activation.. Pflugers Archiv : European journal of physiology, 445(3), 305-10. doi:10.1007/s00424-002-0902-1
• Tombe, P. P., Konhilas, J. P., & Irving, T. C. (2002). Length-dependent activation in three striated muscle types of the rat.. The Journal of physiology, 544(Pt 1), 225-36. doi:10.1113/jphysiol.2002.024505
  More info

  The process whereby sarcomere length modulates the sensitivity of the myofilaments to Ca(2+) is termed length-dependent activation. Length-dependent activation is a property of all striated muscles, yet the relative extent of length-dependent activation between skeletal muscle and cardiac muscle is unclear. Although length-dependent activation may be greater in fast skeletal muscle (FSM) than in slow skeletal muscle (SSM), there has not been a well controlled comparison of length-dependent activation between skeletal muscle and cardiac muscle (CM). Accordingly, we measured sarcomere length-dependent properties in skinned soleus (SSM), psoas (FSM) and ventricular trabeculae (CM) of the rat under carefully controlled conditions. The free Ca(2+)-force relationship was determined at sarcomere lengths (SL) of 1.95 microm, 2.10 microm and 2.25 microm and fitted to a modified Hill equation. FSM and SSM were more sensitive to Ca(2+) than CM. Length-dependent activation was ordered as CM > FSM > SSM. Cooperativity as measured by the Hill coefficient of the Ca(2+)-force relationship was not significantly different between CM and FSM, both of which exhibited greater cooperativity than SSM. SL did not significantly alter this parameter in each muscle type. To establish whether the observed differences can be explained by alterations in interfilament spacing, we measured myofilament lattice spacing (LS) by synchrotron X-ray diffraction in relaxed, skinned muscle preparations. LS was inversely proportional to SL for each muscle type. The slope of the SL-LS relationship, however, was not significantly different between striated muscle types. We conclude that (1) length-dependent activation differs among the three types of striated muscle and (2) these differences in the length-dependent properties among the striated muscle types may not solely be explained by the differences in the response of interfilament spacing to changes in muscle length in relaxed, skinned isolated muscle preparations.
• Tombe, P. P., Konhilas, J. P., & Irving, T. C. (2002). Myofilament calcium sensitivity in skinned rat cardiac trabeculae: role of interfilament spacing.. Circulation research, 90(1), 59-65. doi:10.1161/hh0102.102269
  More info

  The increase in myofilament Ca(2+) responsiveness on an increase in sarcomere length (SL) is, in part, the cellular basis for Frank-Starling's law of the heart. It has been suggested that a decrease in myofilament lattice spacing (LS) in response to an increase in SL underlies this phenomenon. This hypothesis is supported by previous studies in which reduced muscle width induced by osmotic compression was associated with an increase in Ca(2+) sensitivity, mimicking those changes observed with an increase in SL. To evaluate this hypothesis, we directly measured LS by synchrotron x-ray diffraction as function of SL in skinned rat cardiac trabeculae bathed in 0% to 6% dextran solutions (MW 413 000). We found that EC(50), [Ca(2+)] at which force is half-maximal, at SL between 1.95 and 2.25 microm did not vary in proportion to LS when 3% or 6% dextran solutions were applied. We also found that moderate compression (1% dextran) of skinned trabeculae at SL=2.02 microm reduced LS (LS=42.29+/-0.14 nm) to match that of uncompressed fibers at a long SL (SL=2.19 microm; LS=42.28+/-0.15 nm). Whereas increasing SL from 2.02 to 2.19 microm significantly increased Ca(2+) sensitivity as indexed by the EC(50) parameter (2.87+/-0.11 micromol/L to 2.52+/-0.12 micromol/L), similar reduction in myofilament lattice spacing achieved by compression with 1% dextran did not alter Ca(2+) sensitivity (2.87+/-0.10 micromol/L) at the short SL. We conclude that alterations in myofilament lattice spacing may not be the mechanism that underlies the sarcomere length-induced alteration of calcium sensitivity in skinned myocardium.
• Wieczorek, D. F., Solaro, R. J., Pieples, K., Lorenz, J. N., Labitzke, E., Konhilas, J. P., Jagatheesan, G., Irving, T. C., Grupp, I. L., Detombe, P. P., Boivin, G. P., & Arteaga, G. M. (2002). Tropomyosin 3 expression leads to hypercontractility and attenuates myofilament length-dependent Ca(2+) activation.. American journal of physiology. Heart and circulatory physiology, 283(4), H1344-53. doi:10.1152/ajpheart.00351.2002
  More info

  Tropomyosin (TM), an integral component of the thin filament, is encoded by three striated muscle isoforms: alpha-TM, beta-TM, and TPM 3. Although the alpha-TM and beta-TM isoforms are well characterized, less is known about the function of the TPM 3 isoform, which is predominantly found in the slow-twitch musculature of mammals. To determine its functional significance, we ectopically expressed this isoform in the hearts of transgenic mice. We generated six transgenic mouse lines that produce varying levels of TPM 3 message with ectopic TPM 3 protein accounting for 40-60% of the total striated muscle tropomyosin. The transgenic mice have normal life spans and exhibit no morphological abnormalities in their sarcomeres or hearts. However, there are significant functional alterations in cardiac performance. Physiological assessment of these mice by using closed-chest analyses and a work-performing model reveals a hyperdynamic effect on systolic and diastolic function. Analysis of detergent-extracted fiber bundles demonstrates a decreased sensitivity to Ca(2+) in force generation and a decrease in length-dependent Ca(2+) activation with no detectable change in interfilament spacing as determined by using X-ray diffraction. Our data are the first to demonstrate that TM isoforms can affect sarcomeric performance by decreasing sensitivity to Ca(2+) and influencing the length-dependent Ca(2+) activation.
• Wolska, B. M., Vijayan, K., Tombe, P. P., Solaro, R. J., Phillips, R. M., Naya, T., Martin, A. F., Leiden, J. M., Konhilas, J. P., Kim, R., & Arteaga, G. M. (2001). Expression of slow skeletal troponin I in adult transgenic mouse heart muscle reduces the force decline observed during acidic conditions.. The Journal of physiology, 536(Pt 3), 863-70. doi:10.1111/j.1469-7793.2001.00863.x
  More info

  1. Acidosis in cardiac muscle is associated with a decrease in developed force. We hypothesized that slow skeletal troponin I (ssTnI), which is expressed in neonatal hearts, is responsible for the observed decreased response to acidic conditions. To test this hypothesis directly, we used adult transgenic (TG) mice that express ssTnI in the heart. Cardiac TnI (cTnI) was completely replaced by ssTnI either with a FLAG epitope introduced into the N-terminus (TG-ssTnI) or without the epitope (TG-ssTnI) in these mice. TG mice that express cTnI were also generated as a control TG line (TG-cTnI). Non-transgenic (NTG) littermates were used as controls. 2. We measured the force-calcium relationship in all four groups at pH 7.0 and pH 6.5 in detergent-extracted fibre bundles prepared from left ventricular papillary muscles. The force-calcium relationship was identical in fibre bundles from NTG and TG-cTnI mouse hearts, therefore NTG mice served as controls for TG-ssTnIand TG-ssTnI mice. Compared to NTG controls, the force generated by fibre bundles from TG mice expressing ssTnI was more sensitive to Ca(2+). The shift in EC(50) (the concentration of Ca(2+) at which half-maximal force is generated) caused by acidic pH was significantly smaller in fibre bundles isolated from TG hearts compared to those from NTG hearts. However, there was no difference in the force-calcium relationship between hearts from the TG-ssTnIand TG-ssTnI groups. 3. We also isolated papillary muscles from the right ventricle of NTG and TG mouse hearts expressing ssTnI and measured isometric force at extracellular pH 7.33 and pH 6.75. At acidic pH, after an initial decline, twitch force recovered to 60 +/- 3 % (n = 7) in NTG papillary muscles, 98 +/- 2 % (n = 5) in muscles from TG-ssTnIand 96 +/- 3 % (n = 7) in muscles from TG-ssTnI hearts. Our results indicate that TnI isoform composition plays a crucial role in the determination of myocardial force sensitivity to acidosis.
• Tombe, P. P., Perry, D., Konhilas, J. P., Irving, T. C., & Fischetti, R. F. (2000). Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium.. American journal of physiology. Heart and circulatory physiology, 279(5), H2568-73. doi:10.1152/ajpheart.2000.279.5.h2568
  More info

  The Frank-Starling relationship of the heart has, as its molecular basis, an increase in the activation of myofibrils by calcium as the sarcomere length increases. It has been suggested that this phenomenon may be due to myofilaments moving closer together at longer lengths, thereby enhancing the probability of favorable acto-myosin interaction, resulting in increased calcium sensitivity. Accordingly, we have developed an apparatus so as to obtain accurate measurements of myocardial interfilament spacing (by synchrotron X-ray diffraction) as a function of sarcomere length (by video microscopy) over the working range of the heart, using skinned as well as intact rat trabeculas as model systems. In both these systems, lattice spacing decreased significantly as sarcomere length was increased. Furthermore, lattice spacing in the intact muscle was significantly smaller than that in the skinned muscle at all sarcomere lengths studied. These observations are consistent with the hypothesis that lattice spacing underlies length-dependent activation in the myocardium.

Proceedings Publications

• Konhilas, J. (2020). Decision letter: Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload. In Elife review.
• Wolfkiel, C. J., Konhilas, J. P., & Holt, W. W. (1995). Regional pulmonary blood flow measurement in humans with electron-beam computed tomography. In Medical Imaging 1995: Physiology and Function from Multidimensional Images, 2433, 15-25.
  More info

  Electron beam computed tomography (EBCT) is a potentially useful modality to quantitate regional pulmonary flow (RPF) with minimal invasiveness, in part because it has good spatial and temporal resolution. The present studies used a single compartment model of indicator transport and EBCT to measure regional tissue flow in the lungs of human subjects. The model postulates that flow is proportional to maximal enhancement and assumes complete tissue accumulation of indicator before significant indicator washout (WO). EBCT flow studies were retrospectively analyzed with respect to RPF in 10 adult patients who had undergone clinically indicated or research cardiovascular studies. Time density curves from the left atrial (LA) cavity and one-third segments of left (LL) and right (RL) lungs (A: anterior, M: middle, and P: posterior segments) were used to calculate RPF. Washout was determined as the percent of the LA curve at the time of peak parenchymal opacification using gamma curve fits to both tissue data and the LA curve data. Mean +/- standard deviation RPF in ml/min/ml was 0.8 +/- 0.4, 1.1 +/- 0.4, and 1.3 +/- 0.4 for A, M, and P respectively for one-third regions in the left lung. Similar results were found in the right lung. No difference in RPF was found when images were measured either by including the largest of visible parenchymal vessels or when such vessels were excluded. Flow in A of LL and RL was less than that in M or P. Average WO was about 10%, with a range of 0-41% of the LA curve area. There was no significant difference between one-third segment WO using pairwise comparison on the left and right sides when tested separately. RPF values were greater in the posterior vs anterior regions of these supine patients. In conclusion, EBCT can detect gravity related flow differences in the human lung. EBCT has potential for clinical assessment of absolute regional pulmonary flow determination in animals and man.

Poster Presentations

• Fricks, J., Salcedo, V., Pier, M., Langlais, P. R., Konhilas, J. P., & Hoyer-Kimura, C. (2022, October). Estrogen Status in Menopause and Sex Differences  within the Gut Promote Functionally Distinct Changes. Arizona Physiological Society.
• Hay, M., Hay, M., Doyle, K., Doyle, K., Ossanna, N., Ossanna, N., Falk, T., Falk, T., Bartlett, M., Bartlett, M., Polt, R. L., Polt, R. L., Mansour, H. M., Mansour, H. M., Konhilas, J. P., Konhilas, J. P., Hoyer-Kimura, C., & Hoyer-Kimura, C. (2021, March/Spring). Novel Therapeutic and Inflammatory Biomarkers for Vascular Contributions to Cognitive Impairment and Dementia.. AD/PD 2021 15th International Conference on Alzheimer’s and Parkinson’s Diseases Virtual ConferenceAD/PD.
  More info

  Oral e-Poster presentation
• Chung, A., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M. I., Figueroa, A. G., Day, W. A., Konhilas, J. P., Mckay, B. S., Nguyen, T., & Doyle, K. (2018, June). Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis and mediates secondary neurodegeneration in an osteopontin dependent mechanism. Keystone Symposia Conference: New Frontiers in Neuroinflammation: What Happens when CNS and Periphery Meet?. Keystone, Colorado: Keystone Symposia Conference.
• Chung, A., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M. I., Figueroa, A. G., Day, W. A., Konhilas, J. P., Mckay, B. S., Nguyen, T., & Doyle, K. (2018, November). Liquefaction of the brain following stroke shares characteristics with atherosclerosis. Society for Neuroscience Conference. San Diego, California: Society for Neuroscience.
• Lopez-Pier, M., Cannon, D., Langlais, P. R., & Konhilas, J. P. (2018, October/Fall). AMP activated Protein Kinase and Estrogen-depednent mechanisms underlying increased susceptibility to CVD during Menopause. University of Arizona Biomedical Engineering Student Poster Session.