HESHAM SADEK

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• Ahmed, M. S., Nguyen, N. U., Nakada, Y., Hsu, C. C., Farag, A., Lam, N. T., Wang, P., Thet, S., Menendez-Montes, I., Elhelaly, W. M., Lou, X., Secco, I., Tomczyk, M., Zentilin, L., Pei, J., Cui, M., Dos Santos, M., Liu, X., Liu, Y., , Zaha, D., et al. (2024). Identification of FDA-approved drugs that induce heart regeneration in mammals. Nature cardiovascular research, 3(3), 372-388.
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  Targeting Meis1 and Hoxb13 transcriptional activity could be a viable therapeutic strategy for heart regeneration. In this study, we performd an in silico screening to identify FDA-approved drugs that can inhibit Meis1 and Hoxb13 transcriptional activity based on the resolved crystal structure of Meis1 and Hoxb13 bound to DNA. Paromomycin (Paro) and neomycin (Neo) induced proliferation of neonatal rat ventricular myocytes in vitro and displayed dose-dependent inhibition of Meis1 and Hoxb13 transcriptional activity by luciferase assay and disruption of DNA binding by electromobility shift assay. X-ray crystal structure revealed that both Paro and Neo bind to Meis1 near the Hoxb13-interacting domain. Administration of Paro-Neo combination in adult mice and in pigs after cardiac ischemia/reperfusion injury induced cardiomyocyte proliferation, improved left ventricular systolic function and decreased scar formation. Collectively, we identified FDA-approved drugs with therapeutic potential for induction of heart regeneration in mammals.
• Ali, S. R., & Sadek, H. A. (2024). Cardiomyocyte DNA Damage Predicts Functional Recovery in Heart Failure Patients. JACC. Heart failure, 12(4), 662-664.
• Ali, S. R., Nguyen, N. U., Menendez-Montes, I., Hsu, C. C., Elhelaly, W., Lam, N. T., Li, S., Elnwasany, A., Nakada, Y., Thet, S., Foo, R. S., & Sadek, H. A. (2024). Hypoxia-induced stabilization of HIF2A promotes cardiomyocyte proliferation by attenuating DNA damage. The journal of cardiovascular aging, 4(1).
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  Gradual exposure to a chronic hypoxic environment leads to cardiomyocyte proliferation and improved cardiac function in mouse models through a reduction in oxidative DNA damage. However, the upstream transcriptional events that link chronic hypoxia to DNA damage have remained obscure.
• Choi, Y. G., Ma, X., Das, S., Sierra-Pagan, J. E., Larson, T., Gong, W., Sadek, H. A., Zhang, J. J., Garry, M. G., & Garry, D. J. (2024). ETV2 transcriptionally activates Rig1 gene expression and promotes reprogramming of the endothelial lineage. Scientific reports, 14(1), 28688.
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  ETV2 is an essential transcription factor as Etv2 null murine embryos lack all vasculature, blood and are lethal early during embryogenesis. Previous studies have established that ETV2 functions as a pioneer factor and directly reprograms fibroblasts to endothelial cells. However, the underlying molecular mechanisms regulating this reprogramming process remain incompletely defined. In the present study, we examined the ETV2-RIG1 cascade as regulators that govern ETV2-mediated reprogramming. Mouse embryonic fibroblasts (MEFs) harboring an inducible ETV2 expression system were used to overexpress ETV2 and reprogram these somatic cells to the endothelial lineage. Single-cell RNA-seq from reprogrammed fibroblasts defined the induction of the transcriptional network involved in Rig1-like receptor signaling pathways. Studies using ChIP-seq, electrophoretic mobility shift assays, and transcriptional assays demonstrated that ETV2 was a direct upstream activator of Rig1 gene expression. We further demonstrated that the knockdown of Rig1 and separately, Nfκb1 using shRNA significantly reduced the efficiency of endothelial cell reprogramming. These results highlight that ETV2 reprograms fibroblasts to endothelial cells by directly activating RIG1. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of revascularization strategies for the treatment of ischemic tissues such as ischemic heart disease.
• Derks, W., Rode, J., Collin, S., Rost, F., Heinke, P., Hariharan, A., Pickel, L., Simonova, I., Lázár, E., Graham, E., Jashari, R., Andrä, M., Jeppsson, A., Salehpour, M., Alkass, K., Druid, H., Kyriakopoulos, C. P., Taleb, I., Shankar, T. S., , Selzman, C. H., et al. (2024). A Latent Cardiomyocyte Regeneration Potential in Human Heart Disease. Circulation.
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  Cardiomyocytes in the adult human heart show a regenerative capacity, with an annual renewal rate of ≈0.5%. Whether this regenerative capacity of human cardiomyocytes is employed in heart failure has been controversial.
• Nguyen, T., Nakada, Y., Wu, Y., Zhao, J., Garry, D. J., Sadek, H., & Zhang, J. (2024). Cell-Cycle-Specific Autoencoding Improves Cluster Analysis of Cycling Cardiomyocytes. Stem cells (Dayton, Ohio), 42(5), 445-459.
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  Our previous analyses of cardiomyocyte single-nucleus RNA sequencing (snRNAseq) data from the hearts of fetal pigs and pigs that underwent apical resection surgery on postnatal day (P) 1 (ARP1), myocardial infarction (MI) surgery on P28 (MIP28), both ARP1 and MIP28 (ARP1MIP28), or controls (no surgical procedure or CTL) identified 10 cardiomyocyte subpopulations (clusters), one of which appeared to be primed to proliferate in response to MI. However, the clusters composed of primarily proliferating cardiomyocytes still contained noncycling cells, and we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Here, we improved the precision of our assessments by conducting similar analyses with snRNAseq data for only the 1646 genes included under the Gene Ontology term "cell cycle."
• Nguyen, T., Rosa-Garrido, M., Sadek, H., Garry, D. J., & Zhang, J. J. (2024). Promoting cardiomyocyte proliferation for myocardial regeneration in large mammals. Journal of molecular and cellular cardiology, 188, 52-60.
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  From molecular and cellular perspectives, heart failure is caused by the loss of cardiomyocytes-the fundamental contractile units of the heart. Because mammalian cardiomyocytes exit the cell cycle shortly after birth, the cardiomyocyte damage induced by myocardial infarction (MI) typically leads to dilatation of the left ventricle (LV) and often progresses to heart failure. However, recent findings indicate that the hearts of neonatal pigs completely regenerated the cardiomyocytes that were lost to MI when the injury occurred on postnatal day 1 (P1). This recovery was accompanied by increases in the expression of markers for cell-cycle activity in cardiomyocytes. These results suggest that the repair process was driven by cardiomyocyte proliferation. This review summarizes findings from recent studies that found evidence of cardiomyocyte proliferation in 1) the uninjured hearts of newborn pigs on P1, 2) neonatal pig hearts after myocardial injury on P1, and 3) the hearts of pigs that underwent apical resection surgery (AR) on P1 followed by MI on postnatal day 28 (P28). Analyses of cardiomyocyte single-nucleus RNA sequencing data collected from the hearts of animals in these three experimental groups, their corresponding control groups, and fetal pigs suggested that although the check-point regulators and other molecules that direct cardiomyocyte cell-cycle progression and proliferation in fetal, newborn, and postnatal pigs were identical, the mechanisms that activated cardiomyocyte proliferation in response to injury may differ from those that regulate cardiomyocyte proliferation during development.
• Ahmed, M. S., Farag, A. B., Boys, I. N., Wang, P., Menendez-Montes, I., Nguyen, N. U., Eitson, J. L., Ohlson, M. B., Fan, W., McDougal, M. B., Mar, K., Thet, S., Ortiz, F., Kim, S. Y., Solmonson, A., Williams, N. S., Lemoff, A., DeBerardinis, R. J., Schoggins, J. W., & Sadek, H. A. (2023). FDA approved drugs with antiviral activity against SARS-CoV-2: From structure-based repurposing to host-specific mechanisms. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 162, 114614.
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  The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5'-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.
• Garry, D. J., Zhang, J. J., Larson, T. A., Sadek, H. A., & Garry, M. G. (2023). Networks that Govern Cardiomyocyte Proliferation to Facilitate Repair of the Injured Mammalian Heart. Methodist DeBakey cardiovascular journal, 19(5), 16-25.
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  Cardiovascular diseases are the number one cause of death worldwide and in the United States (US). Cardiovascular diseases frequently progress to end-stage heart failure, and curative therapies are extremely limited. Intense interest has focused on deciphering the cascades and networks that govern cardiomyocyte proliferation and regeneration of the injured heart. For example, studies have shown that lower organisms such as the adult newt and adult zebrafish have the capacity to completely regenerate their injured heart with restoration of function. Similarly, the neonatal mouse and pig are also able to completely regenerate injured myocardium due to cardiomyocyte proliferation from preexisting cardiomyocytes. Using these animal models and transcriptome analyses, efforts have focused on the definition of factors and signaling pathways that can reactivate and induce cardiomyocyte proliferation in the adult mammalian injured heart. These studies and discoveries have the potential to define novel therapies to promote cardiomyocyte proliferation and repair of the injured, mammalian heart.
• Hönemann, J. N., Gerlach, D., Hoffmann, F., Kramer, T., Weis, H., Hellweg, C. E., Konda, B., Zaha, V. G., Sadek, H. A., van Herwarden, A. E., Olthaar, A. J., Reuter, H., Baldus, S., Levine, B. D., Jordan, J., Tank, J., & Limper, U. (2023). Hypoxia and Cardiac Function in Patients With Prior Myocardial Infarction. Circulation research, 132(9), 1165-1167.
• Abdisalaam, S., Mukherjee, S., Bhattacharya, S., Kumari, S., Sinha, D., Ortega, J., Li, G. M., Sadek, H. A., Krishnan, S., & Asaithamby, A. (2022). NBS1-CtIP-mediated DNA end resection suppresses cGAS binding to micronuclei. Nucleic acids research, 50(5), 2681-2699.
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  Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor-in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor-functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head-associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1's interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.
• Galal, M. W., Ahmed, M., Shao, Y., Xing, C., Ali, W., Baly, A. E., Elfiky, A., Amer, K., Schoggins, J., Sadek, H. A., & Gobara, Z. N. (2022). The Use of Mebendazole in COVID-19 Patients: An Observational Retrospective Single Center Study. Advances in virology, 2022, 3014686.
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  An screen identified mebendazole with potential antiviral activity that could be a repurposed drug against SARS-CoV-2. Mebendazole is a well-tolerated and cheap antihelminthic agent that is readily available worldwide and thus could be a therapeutic tool in the fight against COVID-19.
• Jain, M. K., De Lemos, J. A., McGuire, D. K., Ayers, C., Eitson, J. L., Sanchez, C. L., Kamel, D., Meisner, J. A., Thomas, E. V., Hegde, A. A., Mocherla, S., Strebe, J. K., Li, X., Williams, N. S., Xing, C., Ahmed, M. S., Wang, P., Sadek, H. A., & Schoggins, J. W. (2022). Atovaquone for treatment of COVID-19: A prospective randomized, double-blind, placebo-controlled clinical trial. Frontiers in pharmacology, 13, 1020123.
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  An screen was performed to identify FDA approved drugs that inhibit SARS-CoV-2 main protease (M), followed by viral replication assays, and pharmacokinetic studies in mice. These studies identified atovaquone as a promising candidate for inhibiting viral replication. A 2-center, randomized, double-blind, placebo-controlled trial was performed among patients hospitalized with COVID-19 infection. Enrolled patients were randomized 2:1 to atovaquone 1500 mg BID matched placebo. Patients received standard of care treatment including remdesivir, dexamethasone, or convalescent plasma as deemed necessary by the treating team. Saliva was collected at baseline and twice per day for up to 10 days for RNA extraction for SARS-CoV-2 viral load measurement by quantitative reverse-transcriptase PCR. The primary outcome was the between group difference in log-transformed viral load (copies/mL) using a generalized linear mixed-effect models of repeated measures from all samples. Of the 61 patients enrolled; 41 received atovaquone and 19 received placebo. Overall, the population was predominately male (63%) and Hispanic (70%), with a mean age of 51 years, enrolled a mean of 5 days from symptom onset. The log viral load was 5.25 copies/mL . 4.79 copies/mL at baseline in the atovaquone . placebo group. Change in viral load did not differ over time between the atovaquone plus standard of care arm the placebo plus standard of care arm. Pharmacokinetic (PK) studies of atovaquone plasma concentration demonstrated a wide variation in atovaquone levels, with an inverse correlation between BMI and atovaquone levels, (Rho -0.45, = 0.02). In post hoc analysis, an inverse correlation was observed between atovaquone levels and viral load (Rho -0.54, = 0.005). In this prospective, randomized, placebo-controlled trial, atovaquone did not demonstrate evidence of enhanced SARS-CoV-2 viral clearance compared with placebo. However, based on the observed inverse correlation between atovaquone levels and viral load, additional PK-guided studies may be warranted to examine the antiviral effect of atovaquone in COVID-19 patients.
• Lam, N. T., Nguyen, N. U., Ahmed, M. S., Hsu, C. C., Rios Coronado, P. E., Li, S., Menendez-Montes, I., Thet, S., Elhelaly, W. M., Xiao, F., Wang, X., Williams, N. S., Canseco, D. C., Red-Horse, K., Rothermel, B. A., & Sadek, H. A. (2022). Targeting calcineurin induces cardiomyocyte proliferation in adult mice. Nature cardiovascular research, 1(7), 679-688.
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  The mammalian neonatal heart can regenerate for 1 week after birth, after which, the majority of cardiomyocytes exit the cell cycle. Recent studies demonstrated that calcineurin mediates cell-cycle arrest of postnatal cardiomyocytes, partly through induction of nuclear translocation of the transcription factor Hoxb13 (a cofactor of Meis1). Here we show that inducible cardiomyocyte-specific deletion of calcineurin B1 in adult cardiomyocytes markedly decreases cardiomyocyte size and promotes mitotic entry, resulting in increased total cardiomyocyte number and improved left ventricular (LV) systolic function after myocardial infarction (MI). Similarly, pharmacological inhibition of calcineurin activity using FK506 promotes cardiomyocyte proliferation in vivo and increases cardiomyocyte number; however, FK506 administration after MI in mice failed to improve LV systolic function, possibly due to inhibition of vasculogenesis and blunting of the post-MI inflammatory response. Collectively, our results demonstrate that loss of calcineurin activity in adult cardiomyocytes promotes cell cycle entry; however, the effects of the calcineurin inhibitor FK506 on other cell types preclude a significant improvement of LV systolic function after MI.
• Menendez-Montes, I., & Sadek, H. A. (2022). WNT links metabolism and cell cycle in postnatal cardiomyocytes. The journal of cardiovascular aging, 2(2).
• Pana, T. A., Savla, J., Kepinski, I., Fairbourn, A., Afzal, A., Mammen, P., Drazner, M., Subramaniam, R. M., Xing, C., Morton, K. A., Drakos, S. G., Zaha, V. G., & Sadek, H. A. (2022). Bidirectional Changes in Myocardial F-Fluorodeoxyglucose Uptake After Human Ventricular Unloading. Circulation, 145(2), 151-154.
• Solmonson, A., Faubert, B., Gu, W., Rao, A., Cowdin, M. A., Menendez-Montes, I., Kelekar, S., Rogers, T. J., Pan, C., Guevara, G., Tarangelo, A., Zacharias, L. G., Martin-Sandoval, M. S., Do, D., Pachnis, P., Dumesnil, D., Mathews, T. P., Tasdogan, A., Pham, A., , Cai, L., et al. (2022). Compartmentalized metabolism supports midgestation mammalian development. Nature, 604(7905), 349-353.
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  Mammalian embryogenesis requires rapid growth and proper metabolic regulation. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development. Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.
• Zhu, W., Sun, J., Bishop, S. P., Sadek, H., & Zhang, J. (2022). Turning back the clock: A concise viewpoint of cardiomyocyte cell cycle activation for myocardial regeneration and repair. Journal of molecular and cellular cardiology, 170, 15-21.
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  Patients with acute myocardial infarction (MI) could progress to end-stage congestive heart failure, which is one of the most significant problems in public health. From the molecular and cellular perspective, heart failure often results from the loss of cardiomyocytes-the fundamental contractile unit of the heart-and the damage caused by myocardial injury in adult mammals cannot be repaired, in part because mammalian cardiomyocytes undergo cell-cycle arrest during the early perinatal period. However, recent studies in the hearts of neonatal small and large mammals suggest that the onset of cardiomyocyte cell-cycle arrest can be reversed, which may lead to the development of entirely new strategies for the treatment of heart failure. In this Viewpoint, we summarize these and other provocative findings about the cellular and molecular mechanisms that regulate cardiomyocyte proliferation and how they may be targeted to turn back the clock of cardiomyocyte cell-cycle arrest and improve recovery from cardiac injury and disease.
• Ahmed, M. S., Wang, P., Nguyen, N. U., Nakada, Y., Menendez-Montes, I., Ismail, M., Bachoo, R., Henkemeyer, M., Sadek, H. A., & Kandil, E. S. (2021). Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain. Proceedings of the National Academy of Sciences of the United States of America, 118(10).
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  Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.
• Menendez-Montes, I., Abdisalaam, S., Xiao, F., Lam, N. T., Mukherjee, S., Szweda, L. I., Asaithamby, A., & Sadek, H. A. (2021). Mitochondrial fatty acid utilization increases chromatin oxidative stress in cardiomyocytes. Proceedings of the National Academy of Sciences of the United States of America, 118(34).
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  The inability of adult mammalian cardiomyocytes to proliferate underpins the development of heart failure following myocardial injury. Although the newborn mammalian heart can spontaneously regenerate for a short period of time after birth, this ability is lost within the first week after birth in mice, partly due to increased mitochondrial reactive oxygen species (ROS) production which results in oxidative DNA damage and activation of DNA damage response. This increase in ROS levels coincides with a postnatal switch from anaerobic glycolysis to fatty acid (FA) oxidation by cardiac mitochondria. However, to date, a direct link between mitochondrial substrate utilization and oxidative DNA damage is lacking. Here, we generated ROS-sensitive fluorescent sensors targeted to different subnuclear compartments (chromatin, heterochromatin, telomeres, and nuclear lamin) in neonatal rat ventricular cardiomyocytes, which allowed us to determine the spatial localization of ROS in cardiomyocyte nuclei upon manipulation of mitochondrial respiration. Our results demonstrate that FA utilization by the mitochondria induces a significant increase in ROS detection at the chromatin level compared to other nuclear compartments. These results indicate that mitochondrial metabolic perturbations directly alter the nuclear redox status and that the chromatin appears to be particularly sensitive to the prooxidant effect of FA utilization by the mitochondria.
• Messerschmidt, V. L., Chintapula, U., Kuriakose, A. E., Laboy, S., Truong, T. T., Kydd, L. A., Jaworski, J., Pan, Z., Sadek, H., Nguyen, K. T., & Lee, J. (2021). Corrigendum: Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules. Frontiers in cardiovascular medicine, 8, 785910.
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  [This corrects the article DOI: 10.3389/fcvm.2021.707897.].
• Nakada, Y., & Sadek, H. A. (2021). Experimental Hypoxia as a Model for Cardiac Regeneration in Mice. Methods in molecular biology (Clifton, N.J.), 2158, 337-344.
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  Experimental hypoxia has been used for decades to examine the adaptive response to low-oxygen environments. Various models have been studied, including flies, worms, fish, rodents, and humans. Our lab has recently used this technology to examine the effect of environmental hypoxia on mammalian heart regeneration. In this chapter, we describe studies of systemic hypoxia in mice. We found that systemic hypoxia can blunt oxidative DNA damage and induce cardiomyocyte proliferation. While our primary interests are focused on cardiovascular research, these hypoxia protocols are applicable to any other organ system.
• Abdisalaam, S., Bhattacharya, S., Mukherjee, S., Sinha, D., Srinivasan, K., Zhu, M., Akbay, E. A., Sadek, H. A., Shay, J. W., & Asaithamby, A. (2020). Dysfunctional telomeres trigger cellular senescence mediated by cyclic GMP-AMP synthase. The Journal of biological chemistry, 295(32), 11144-11160.
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  Defective DNA damage response (DDR) signaling is a common mechanism that initiates and maintains the cellular senescence phenotype. Dysfunctional telomeres activate DDR signaling, genomic instability, and cellular senescence, but the links among these events remains unclear. Here, using an array of biochemical and imaging techniques, including a highly regulatable CRISPR/Cas9 strategy to induce DNA double strand breaks specifically in the telomeres, ChIP, telomere immunofluorescence, fluorescence hybridization (FISH), micronuclei imaging, and the telomere shortest length assay (TeSLA), we show that chromosome mis-segregation due to imperfect DDR signaling in response to dysfunctional telomeres creates a preponderance of chromatin fragments in the cytosol, which leads to a premature senescence phenotype. We found that this phenomenon is caused not by telomere shortening, but by cyclic GMP-AMP synthase (cGAS) recognizing cytosolic chromatin fragments and then activating the stimulator of interferon genes (STING) cytosolic DNA-sensing pathway and downstream interferon signaling. Significantly, genetic and pharmacological manipulation of cGAS not only attenuated immune signaling, but also prevented premature cellular senescence in response to dysfunctional telomeres. The findings of our study uncover a cellular intrinsic mechanism involving the cGAS-mediated cytosolic self-DNA-sensing pathway that initiates premature senescence independently of telomere shortening.
• Ahmed, M. S., & Sadek, H. A. (2020). Hypoxia Induces Cardiomyocyte Proliferation in Humans. JACC. Basic to translational science, 5(5), 461-462.
• Ali, S. R., Menendez-Montes, I., Warshaw, J., Xiao, F., & Sadek, H. A. (2020). Homotypic Fusion Generates Multinucleated Cardiomyocytes in the Murine Heart. Circulation, 141(23), 1940-1942.
• Ali, S. R., Nguyen, D., Wang, B., Jiang, S., & Sadek, H. A. (2020). Deep Learning Identifies Cardiomyocyte Nuclei With High Precision. Circulation research, 127(5), 696-698.
• Cardoso, A. C., Pereira, A. H., & Sadek, H. A. (2020). Mechanisms of Neonatal Heart Regeneration. Current cardiology reports, 22(5), 33.
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  This review provides an overview of the molecular mechanisms underpinning the cardiac regenerative capacity during the neonatal period and the potential targets for developing novel therapies to restore myocardial loss.
• Li, S., Nguyen, N. U., Xiao, F., Menendez-Montes, I., Nakada, Y., Tan, W. L., Anene-Nzelu, C. G., Foo, R. S., Thet, S., Cardoso, A. C., Wang, P., Elhelaly, W. M., Lam, N. T., Pereira, A. H., Hill, J. A., & Sadek, H. A. (2020). Mechanism of Eccentric Cardiomyocyte Hypertrophy Secondary to Severe Mitral Regurgitation. Circulation, 141(22), 1787-1799.
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  Primary valvular heart disease is a prevalent cause of morbidity and mortality in both industrialized and developing countries. Although the primary consequence of valvular heart disease is myocardial dysfunction, treatment of valvular heart diseases centers around valve repair or replacement rather than prevention or reversal of myocardial dysfunction. This is particularly evident in primary mitral regurgitation (MR), which invariably results in eccentric hypertrophy and left ventricular (LV) failure in the absence of timely valve repair or replacement. The mechanism of LV dysfunction in primary severe MR is entirely unknown.
• Sadek, H. A., & Porrello, E. R. (2020). Neonatal heart regeneration: Moving from phenomenology to regenerative medicine. The Journal of thoracic and cardiovascular surgery, 159(6), 2451-2455.
• Sadek, H., & Olson, E. N. (2020). Toward the Goal of Human Heart Regeneration. Cell stem cell, 26(1), 7-16.
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  Heart regeneration, a relatively new field of biology, is one of the most active and controversial areas of biomedical research. The potential impact of successful human heart regeneration therapeutics cannot be overstated, given the magnitude and prognosis of heart failure. However, the regenerative process is highly complex, and premature claims of successful heart regeneration have both fueled interest and created controversy. The field as a whole is now in the process of course correction, and a clearer picture is beginning to emerge. Despite the challenges, fundamental principles in developmental biology have provided a framework for hypothesis-driven approaches toward the ultimate goal of adult heart regeneration and repair. In this review, we discuss the current state of the field and outline the potential paths forward toward regenerating the human myocardium.
• Elhelaly, W. M., Cardoso, A. C., Pereira, A. H., Elnawasany, A., Ebrahimi, S., Nakada, Y., & Sadek, H. A. (2019). C-Kit Cells Do Not Significantly Contribute to Cardiomyogenesis During Neonatal Heart Regeneration. Circulation, 139(4), 559-561.
• Nakada, Y., Nhi Nguyen, N. U., Xiao, F., Savla, J. J., Lam, N. T., Abdisalaam, S., Bhattacharya, S., Mukherjee, S., Asaithamby, A., Gillette, T. G., Hill, J. A., Sadek, H. A., Nakada, Y., Nhi Nguyen, N. U., Xiao, F., Savla, J. J., Lam, N. T., Abdisalaam, S., Bhattacharya, S., , Mukherjee, S., et al. (2019). DNA Damage Response Mediates Pressure Overload-Induced Cardiomyocyte Hypertrophy. Circulation, 139(9), 1237-1239.
• Villalobos, E., Criollo, A., Schiattarella, G. G., Altamirano, F., French, K. M., May, H. I., Jiang, N., Nguyen, N. U., Romero, D., Roa, J. C., García, L., Diaz-Araya, G., Morselli, E., Ferdous, A., Conway, S. J., Sadek, H. A., Gillette, T. G., Lavandero, S., & Hill, J. A. (2019). Fibroblast Primary Cilia Are Required for Cardiac Fibrosis. Circulation, 139(20), 2342-2357.
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  The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling.
• Lam, N. T., & Sadek, H. A. (2018). Neonatal Heart Regeneration: Comprehensive Literature Review. Circulation, 138(4), 412-423.
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  The adult mammalian heart is incapable of meaningful functional recovery after injury, and thus promoting heart regeneration is 1 of the most important therapeutic targets in cardiovascular medicine. In contrast to the adult mammalian heart, the neonatal mammalian heart is capable of regeneration after various types of injury. Since the first report in 2011, a number of groups have reported their findings on neonatal heart regeneration. The current review provides a comprehensive analysis of heart regeneration studies in neonatal mammals conducted to date, outlines lessons learned, and poses unanswered questions.
• Rotter, D., Peiris, H., Grinsfelder, D. B., Martin, A. M., Burchfield, J., Parra, V., Hull, C., Morales, C. R., Jessup, C. F., Matusica, D., Parks, B. W., Lusis, A. J., Nguyen, N. U., Oh, M., Iyoke, I., Jakkampudi, T., McMillan, D. R., Sadek, H. A., Watt, M. J., , Gupta, R. K., et al. (2018). Regulator of Calcineurin 1 helps coordinate whole-body metabolism and thermogenesis. EMBO reports, 19(12).
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  Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.
• Amofa, D., Hulin, A., Nakada, Y., Sadek, H. A., & Yutzey, K. E. (2017). Hypoxia promotes primitive glycosaminoglycan-rich extracellular matrix composition in developing heart valves. American journal of physiology. Heart and circulatory physiology, 313(6), H1143-H1154.
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  During postnatal heart valve development, glycosaminoglycan (GAG)-rich valve primordia transform into stratified valve leaflets composed of GAGs, fibrillar collagen, and elastin layers accompanied by decreased cell proliferation as well as thinning and elongation. The neonatal period is characterized by the transition from a uterine environment to atmospheric O, but the role of changing O levels in valve extracellular matrix (ECM) composition or morphogenesis is not well characterized. Here, we show that tissue hypoxia decreases in mouse aortic valves in the days after birth, concomitant with ECM remodeling and cell cycle arrest of valve interstitial cells. The effects of hypoxia on late embryonic valve ECM composition, Sox9 expression, and cell proliferation were examined in chicken embryo aortic valve organ cultures. Maintenance of late embryonic chicken aortic valve organ cultures in a hypoxic environment promotes GAG expression, Sox9 nuclear localization, and indicators of hyaluronan remodeling but does not affect fibrillar collagen content or cell proliferation. Chronic hypoxia also promotes GAG accumulation in murine adult heart valves in vivo. Together, these results support a role for hypoxia in maintaining a primitive GAG-rich matrix in developing heart valves before birth and also in the induction of hyaluronan remodeling in adults. Tissue hypoxia decreases in mouse aortic valves after birth, and exposure to hypoxia promotes glycosaminoglycan accumulation in cultured chicken embryo valves and adult murine heart valves. Thus, hypoxia maintains a primitive extracellular matrix during heart valve development and promotes extracellular matrix remodeling in adult mice, as occurs in myxomatous disease.
• Eschenhagen, T., Bolli, R., Braun, T., Field, L. J., Fleischmann, B. K., Frisén, J., Giacca, M., Hare, J. M., Houser, S., Lee, R. T., Marbán, E., Martin, J. F., Molkentin, J. D., Murry, C. E., Riley, P. R., Ruiz-Lozano, P., Sadek, H. A., Sussman, M. A., & Hill, J. A. (2017). Cardiomyocyte Regeneration: A Consensus Statement. Circulation, 136(7), 680-686.
• Hill, J. A., Ardehali, R., Clarke, K. T., Del Zoppo, G. J., Eckhardt, L. L., Griendling, K. K., Libby, P., Roden, D. M., Sadek, H. A., Seidman, C. E., Vaughan, D. E., & , A. H. (2017). Fundamental Cardiovascular Research: Returns on Societal Investment: A Scientific Statement From the American Heart Association. Circulation research, 121(3), e2-e8.
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  Recent decades have witnessed robust successes in conquering the acutely lethal manifestations of heart and vascular diseases. Many patients who previously would have died now survive. Lifesaving successes like these provide a tremendous and easily recognized benefit to individuals and society. Although cardiovascular mortality has declined, the devastating impact of chronic heart disease and comorbidities on quality of life and healthcare resources continues unabated. Future strides, extending those made in recent decades, will require continued research into mechanisms underlying disease prevention, pathogenesis, progression, and therapeutic intervention. However, severe financial constraints currently jeopardize these efforts. To chart a path for the future, this report analyzes the challenges and opportunities we face in continuing the battle against cardiovascular disease and highlights the return on societal investment afforded by fundamental cardiovascular research.
• Lázár, E., Sadek, H. A., & Bergmann, O. (2017). Cardiomyocyte renewal in the human heart: insights from the fall-out. European heart journal, 38(30), 2333-2342.
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  The capacity of the mammalian heart to regenerate cardiomyocytes has been debated over the last decades. However, limitations in existing techniques to track and identify nascent cardiomyocytes have often led to inconsistent results. Radiocarbon (14C) birth dating, in combination with other quantitative strategies, allows to establish the number and age of human cardiomyocytes, making it possible to describe their age distribution and turnover dynamics. Accurate estimates of cardiomyocyte generation in the adult heart can provide the foundation for novel regenerative strategies that aim to stimulate cardiomyocyte renewal in various cardiac pathologies.
• Elhelaly, W. M., Lam, N. T., Hamza, M., Xia, S., & Sadek, H. A. (2016). Redox Regulation of Heart Regeneration: An Evolutionary Tradeoff. Frontiers in cell and developmental biology, 4, 137.
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  Heart failure is a costly and deadly disease, affecting over 23 million patients worldwide, half of which die within 5 years of diagnosis. The pathophysiological basis of heart failure is the inability of the adult heart to regenerate lost or damaged myocardium. Although limited myocyte turnover does occur in the adult heart, it is insufficient for restoration of contractile function (Nadal-Ginard, 2001; Laflamme et al., 2002; Quaini et al., 2002; Hsieh et al., 2007; Bergmann et al., 2009, 2012). In contrast to lower vertebrates (Poss et al., 2002; Poss, 2007; Jopling et al., 2010; Kikuchi et al., 2010; Chablais et al., 2011; González-Rosa et al., 2011; Heallen et al., 2011), adult mammalian heart cardiomyogenesis following injury is very limited (Nadal-Ginard, 2001; Laflamme et al., 2002; Quaini et al., 2002; Bergmann et al., 2009, 2012) and is insufficient to restore normal cardiac function. Studies in the late 90s elegantly mapped the DNA synthesis and cell cycle dynamics of the mammalian heart during development and following birth (Soonpaa et al., 1996; Soonpaa and Field, 1997, 1998), where they showed that DNA synthesis drops significantly around birth with low-level DNA synthesis few days after birth. Around P5 to P7, cardiomyocytes undergo a final round of DNA synthesis without cytokinesis, and the majority become binucleated and exit the cell cycle permanently. Therefore, due to the similarities between the immature mammalian heart and lower vertebrates (Poss, 2007; Walsh et al., 2010), it became important to determine whether they have similar regenerative abilities. Recently, we demonstrated that removal of up to 15% of the apex of the left ventricle of postnatal day 1 (P1) mice results in complete regeneration within 3 weeks without any measurable fibrosis and cardiac dysfunction (Porrello et al., 2011). This response is characterized by robust cardiomyocyte proliferation with gradual restoration of normal cardiac morphology. In addition to the histological evidence of proliferating myocytes, genetic fate-mapping studies confirmed that the majority of newly formed cardiomyocytes are derived from proliferation of preexisting cardiomyocytes (Porrello et al., 2011). More recently, we established an ischemic injury model where the left anterior descending coronary artery was ligated in P1 neonates (Porrello et al., 2013). The injury response was similar to the resection model, with robust cardiomyocyte proliferation throughout the myocardium, as well as restoration of normal morphology by 21 days. However, this regenerative capacity is lost by P7, after which injury results in the typical cardiomyocyte hypertrophy and scar-formation characteristic of the adult mammalian heart. Not surprisingly, the loss of this regenerative capacity coincides with binucleation and cell cycle exit of cardiomyocytes (Soonpaa et al., 1996; Walsh et al., 2010). An important approach toward a deeper understanding the loss of cardiac regenerative capacity in mammals is to first consider , and not only , this happens. Regeneration of the early postnatal heart following resection or ischemic infarction involves replacement of lost myocardium and vasculature with restoration of normal myocardial thickness and architecture, with long-term normalization of systolic function. Why would the heart permanently forego such a remarkable regenerative program shortly after birth? The answer may lie in within the fundamental principal of evolutionary tradeoff.
• Kimura, W., Xiao, F., Canseco, D. C., Muralidhar, S., Thet, S., Zhang, H. M., Abderrahman, Y., Chen, R., Garcia, J. A., Shelton, J. M., Richardson, J. A., Ashour, A. M., Asaithamby, A., Liang, H., Xing, C., Lu, Z., Cheng Zhang, C., & Sadek, H. A. (2016). Corrigendum: Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart. Nature, 532(7598), 268.
• Mani, R. S., Amin, M. A., Li, X., Kalyana-Sundaram, S., Veeneman, B. A., Wang, L., Ghosh, A., Aslam, A., Ramanand, S. G., Rabquer, B. J., Kimura, W., Tran, M., Cao, X., Roychowdhury, S., Dhanasekaran, S. M., Palanisamy, N., Sadek, H. A., Kapur, P., Koch, A. E., & Chinnaiyan, A. M. (2016). Inflammation-Induced Oxidative Stress Mediates Gene Fusion Formation in Prostate Cancer. Cell reports, 17(10), 2620-2631.
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  Approximately 50% of prostate cancers are associated with gene fusions of the androgen-regulated gene TMPRSS2 to the oncogenic erythroblast transformation-specific (ETS) transcription factor ERG. The three-dimensional proximity of TMPRSS2 and ERG genes, in combination with DNA breaks, facilitates the formation of TMPRSS2-ERG gene fusions. However, the origins of DNA breaks that underlie gene fusion formation in prostate cancers are far from clear. We demonstrate a role for inflammation-induced oxidative stress in the formation of DNA breaks leading to recurrent TMPRSS2-ERG gene fusions. The transcriptional status and epigenetic features of the target genes influence this effect. Importantly, inflammation-induced de novo genomic rearrangements are blocked by homologous recombination (HR) and promoted by non-homologous end-joining (NHEJ) pathways. In conjunction with the association of proliferative inflammatory atrophy (PIA) with human prostate cancer, our results support a working model in which recurrent genomic rearrangements induced by inflammatory stimuli lead to the development of prostate cancer.
• Canseco, D. C., Kimura, W., Garg, S., Mukherjee, S., Bhattacharya, S., Abdisalaam, S., Das, S., Asaithamby, A., Mammen, P. P., & Sadek, H. A. (2015). Human ventricular unloading induces cardiomyocyte proliferation. Journal of the American College of Cardiology, 65(9), 892-900.
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  The adult mammalian heart is incapable of meaningful regeneration after substantial cardiomyocyte loss, primarily due to the inability of adult cardiomyocytes to divide. Our group recently showed that mitochondria-mediated oxidative DNA damage is an important regulator of postnatal cardiomyocyte cell cycle arrest. However, it is not known whether mechanical load also plays a role in this process. We reasoned that the postnatal physiological increase in mechanical load contributes to the increase in mitochondrial content, with subsequent activation of DNA damage response (DDR) and permanent cell cycle arrest of cardiomyocytes.
• Kimura, W., Xiao, F., Canseco, D. C., Muralidhar, S., Thet, S., Zhang, H. M., Abderrahman, Y., Chen, R., Garcia, J. A., Shelton, J. M., Richardson, J. A., Ashour, A. M., Asaithamby, A., Liang, H., Xing, C., Lu, Z., Zhang, C. C., & Sadek, H. A. (2015). Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart. Nature, 523(7559), 226-30.
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  Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts, mediated primarily by proliferation of pre-existing cardiomyocytes. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.
• Kocabas, F., Xie, L., Xie, J., Yu, Z., DeBerardinis, R. J., Kimura, W., Thet, S., Elshamy, A. F., Abouellail, H., Muralidhar, S., Liu, X., Chen, C., Sadek, H. A., Zhang, C. C., & Zheng, J. (2015). Hypoxic metabolism in human hematopoietic stem cells. Cell & bioscience, 5, 39.
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  Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment, known as niche in the endosteal regions of the bone marrow. This stem cell niche with low oxygen tension requires HSCs to adopt a unique metabolic profile. We have recently demonstrated that mouse long-term hematopoietic stem cells (LT-HSCs) utilize glycolysis instead of mitochondrial oxidative phosphorylation as their main energy source. However, the metabolic phenotype of human hematopoietic progenitor and stem cells (HPSCs) remains unknown.
• Nakada, Y., Kimura, W., & Sadek, H. A. (2015). Defining the Limit of Embryonic Heart Regeneration. Circulation, 132(2), 77-8.
• Rimmelé, P., Liang, R., Bigarella, C. L., Kocabas, F., Xie, J., Serasinghe, M. N., Chipuk, J., Sadek, H., Zhang, C. C., & Ghaffari, S. (2015). Mitochondrial metabolism in hematopoietic stem cells requires functional FOXO3. EMBO reports, 16(9), 1164-76.
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  Hematopoietic stem cells (HSC) are primarily dormant but have the potential to become highly active on demand to reconstitute blood. This requires a swift metabolic switch from glycolysis to mitochondrial oxidative phosphorylation. Maintenance of low levels of reactive oxygen species (ROS), a by-product of mitochondrial metabolism, is also necessary for sustaining HSC dormancy. Little is known about mechanisms that integrate energy metabolism with hematopoietic stem cell homeostasis. Here, we identify the transcription factor FOXO3 as a new regulator of metabolic adaptation of HSC. ROS are elevated in Foxo3(-/-) HSC that are defective in their activity. We show that Foxo3(-/-) HSC are impaired in mitochondrial metabolism independent of ROS levels. These defects are associated with altered expression of mitochondrial/metabolic genes in Foxo3(-/-) hematopoietic stem and progenitor cells (HSPC). We further show that defects of Foxo3(-/-) HSC long-term repopulation activity are independent of ROS or mTOR signaling. Our results point to FOXO3 as a potential node that couples mitochondrial metabolism with HSC homeostasis. These findings have critical implications for mechanisms that promote malignant transformation and aging of blood stem and progenitor cells.
• Sen, S., & Sadek, H. A. (2015). Neonatal heart regeneration: mounting support and need for technical standards. Journal of the American Heart Association, 4(1), e001727.
• Aurora, A. B., Porrello, E. R., Tan, W., Mahmoud, A. I., Hill, J. A., Bassel-Duby, R., Sadek, H. A., & Olson, E. N. (2014). Macrophages are required for neonatal heart regeneration. The Journal of clinical investigation, 124(3), 1382-92.
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  Myocardial infarction (MI) leads to cardiomyocyte death, which triggers an immune response that clears debris and restores tissue integrity. In the adult heart, the immune system facilitates scar formation, which repairs the damaged myocardium but compromises cardiac function. In neonatal mice, the heart can regenerate fully without scarring following MI; however, this regenerative capacity is lost by P7. The signals that govern neonatal heart regeneration are unknown. By comparing the immune response to MI in mice at P1 and P14, we identified differences in the magnitude and kinetics of monocyte and macrophage responses to injury. Using a cell-depletion model, we determined that heart regeneration and neoangiogenesis following MI depends on neonatal macrophages. Neonates depleted of macrophages were unable to regenerate myocardia and formed fibrotic scars, resulting in reduced cardiac function and angiogenesis. Immunophenotyping and gene expression profiling of cardiac macrophages from regenerating and nonregenerating hearts indicated that regenerative macrophages have a unique polarization phenotype and secrete numerous soluble factors that may facilitate the formation of new myocardium. Our findings suggest that macrophages provide necessary signals to drive angiogenesis and regeneration of the neonatal mouse heart. Modulating inflammation may provide a key therapeutic strategy to support heart regeneration.
• Kimura, W., Muralidhar, S., Canseco, D. C., Puente, B., Zhang, C. C., Xiao, F., Abderrahman, Y. H., & Sadek, H. A. (2014). Redox signaling in cardiac renewal. Antioxidants & redox signaling, 21(11), 1660-73.
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  Utilizing oxygen (O2) through mitochondrial oxidative phosphorylation enables organisms to generate adenosine triphosphate (ATP) with a higher efficiency than glycolysis, but it results in increased reactive oxygen species production from mitochondria, which can result in stem cell dysfunction and senescence.
• Kocabas, F., Zheng, J., Zhang, C., & Sadek, H. A. (2014). Metabolic characterization of hematopoietic stem cells. Methods in molecular biology (Clifton, N.J.), 1185, 155-64.
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  An important feature of stem cells is their maintenance in their respective hypoxic niche. Survival in this low-oxygen microenvironment requires significant metabolic adaptation. We demonstrated that mouse HSCs utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. We have adapted various tools for characterization of the metabolic properties of hematopoietic stem cells (HSCs). These techniques include flow cytometric profiling of HSCs based on mitochondrial potential and NADH fluorescence as well as measurement of ATP content, oxygen consumption rate, and glycolytic flux in purified HSCs.
• Mahmoud, A. I., Canseco, D., Xiao, F., & Sadek, H. A. (2014). Cardiomyocyte cell cycle: Meis-ing something?. Cell cycle (Georgetown, Tex.), 13(7), 1057-8.
• Mahmoud, A. I., Porrello, E. R., Kimura, W., Olson, E. N., & Sadek, H. A. (2014). Surgical models for cardiac regeneration in neonatal mice. Nature protocols, 9(2), 305-11.
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  Although amphibian and fish models of heart regeneration have existed for decades, a mammalian equivalent has long remained elusive. Our discovery of a brief postnatal window for heart regeneration in neonatal mice has led to the establishment of surgical models for cardiac regenerative studies in mammals for the first time. This protocol describes a 10-min surgical procedure to induce cardiac injury in 1-d-old neonatal mice. This allows for the analysis of cardiac regeneration after surgical amputation of the left ventricle (LV) (apical resection) and coronary artery occlusion (myocardial infarction (MI)). A comparative analysis of neonatal and adult responses to myocardial injury should enable identification of the key differences between regenerative and nonregenerative responses to cardiac injury. This protocol can also be adapted to the growing repertoire of genetic models available in the mouse, and it provides a valuable tool for unlocking the molecular mechanisms that guide mammalian heart regeneration during early postnatal life.
• Puente, B. N., Kimura, W., Muralidhar, S. A., Moon, J., Amatruda, J. F., Phelps, K. L., Grinsfelder, D., Rothermel, B. A., Chen, R., Garcia, J. A., Santos, C. X., Thet, S., Mori, E., Kinter, M. T., Rindler, P. M., Zacchigna, S., Mukherjee, S., Chen, D. J., Mahmoud, A. I., , Giacca, M., et al. (2014). The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell, 157(3), 565-79.
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  The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.
• Sadek, H. A., Martin, J. F., Takeuchi, J. K., Leor, J., Nie, Y., Giacca, M., & Lee, R. T. (2014). Multi-investigator letter on reproducibility of neonatal heart regeneration following apical resection. Stem cell reports, 3(1), 1.
• Zhang, C. C., & Sadek, H. A. (2014). Hypoxia and metabolic properties of hematopoietic stem cells. Antioxidants & redox signaling, 20(12), 1891-901.
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  The effect of redox signaling on hematopoietic stem cell (HSC) function is not clearly understood.
• Zheng, J., Lu, Z., Kocabas, F., Böttcher, R. T., Costell, M., Kang, X., Liu, X., Deberardinis, R. J., Wang, Q., Chen, G. Q., Sadek, H., & Zhang, C. C. (2014). Profilin 1 is essential for retention and metabolism of mouse hematopoietic stem cells in bone marrow. Blood, 123(7), 992-1001.
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  How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we demonstrated that the deletion of the cytoskeleton-modulating protein profilin 1 (pfn1) in hematopoietic stem cell (HSCs) led to bone marrow failure, loss of quiescence, and mobilization and apoptosis of HSCs in vivo. A switch from glycolysis to mitochondrial respiration with increased reactive oxygen species (ROS) level was also observed in HSCs on pfn1 deletion. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that the metabolism is mechanistically linked to the cell cycle quiescence of stem cells. The actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Our study provided evidence that pfn1 at least partially acts through the axis of pfn1/Gα13/EGR1 to regulate stem cell retention and metabolism in the bone marrow.
• Mahmoud, A. I., Kocabas, F., Muralidhar, S. A., Kimura, W., Koura, A. S., Thet, S., Porrello, E. R., & Sadek, H. A. (2013). Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature, 497(7448), 249-253.
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  The neonatal mammalian heart is capable of substantial regeneration following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by postnatal day 7 and the mechanisms of cardiomyocyte cell cycle arrest remain unclear. The homeodomain transcription factor Meis1 is required for normal cardiac development but its role in cardiomyocytes is unknown. Here we identify Meis1 as a critical regulator of the cardiomyocyte cell cycle. Meis1 deletion in mouse cardiomyocytes was sufficient for extension of the postnatal proliferative window of cardiomyocytes, and for re-activation of cardiomyocyte mitosis in the adult heart with no deleterious effect on cardiac function. In contrast, overexpression of Meis1 in cardiomyocytes decreased neonatal myocyte proliferation and inhibited neonatal heart regeneration. Finally, we show that Meis1 is required for transcriptional activation of the synergistic CDK inhibitors p15, p16 and p21. These results identify Meis1 as a critical transcriptional regulator of cardiomyocyte proliferation and a potential therapeutic target for heart regeneration.
• Muralidhar, S. A., Mahmoud, A. I., Canseco, D., Xiao, F., & Sadek, H. A. (2013). Harnessing the power of dividing cardiomyocytes. Global cardiology science & practice, 2013(3), 212-21.
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  Lower vertebrates, such as newt and zebrafish, retain a robust cardiac regenerative capacity following injury. Recently, our group demonstrated that neonatal mammalian hearts have a remarkable regenerative potential in the first few days after birth. Although adult mammals lack this regenerative potential, it is now clear that there is measurable cardiomyocyte turnover that occurs in the adult mammalian heart. In both neonatal and adult mammals, proliferation of pre-existing cardiomyocytes appears to be the underlying mechanism of myocyte turnover. This review will highlight the advances and landmark studies that opened new frontiers in cardiac regeneration.
• Porrello, E. R., Mahmoud, A. I., Simpson, E., Johnson, B. A., Grinsfelder, D., Canseco, D., Mammen, P. P., Rothermel, B. A., Olson, E. N., & Sadek, H. A. (2013). Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proceedings of the National Academy of Sciences of the United States of America, 110(1), 187-92.
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  We recently identified a brief time period during postnatal development when the mammalian heart retains significant regenerative potential after amputation of the ventricular apex. However, one major unresolved question is whether the neonatal mouse heart can also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. Here, we induced ischemic myocardial infarction (MI) in 1-d-old mice and found that this results in extensive myocardial necrosis and systolic dysfunction. Remarkably, the neonatal heart mounted a robust regenerative response, through proliferation of preexisting cardiomyocytes, resulting in full functional recovery within 21 d. Moreover, we show that the miR-15 family of microRNAs modulates neonatal heart regeneration through inhibition of postnatal cardiomyocyte proliferation. Finally, we demonstrate that inhibition of the miR-15 family from an early postnatal age until adulthood increases myocyte proliferation in the adult heart and improves left ventricular systolic function after adult MI. We conclude that the neonatal mammalian heart can regenerate after myocardial infarction through proliferation of preexisting cardiomyocytes and that the miR-15 family contributes to postnatal loss of cardiac regenerative capacity.
• Xin, M., Kim, Y., Sutherland, L. B., Murakami, M., Qi, X., McAnally, J., Porrello, E. R., Mahmoud, A. I., Tan, W., Shelton, J. M., Richardson, J. A., Sadek, H. A., Bassel-Duby, R., & Olson, E. N. (2013). Hippo pathway effector Yap promotes cardiac regeneration. Proceedings of the National Academy of Sciences of the United States of America, 110(34), 13839-44.
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  The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process.
• Aurora, A. B., Mahmoud, A. I., Luo, X., Johnson, B. A., van Rooij, E., Matsuzaki, S., Humphries, K. M., Hill, J. A., Bassel-Duby, R., Sadek, H. A., & Olson, E. N. (2012). MicroRNA-214 protects the mouse heart from ischemic injury by controlling Ca²⁺ overload and cell death. The Journal of clinical investigation, 122(4), 1222-32.
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  Early reperfusion of ischemic cardiac tissue remains the most effective intervention for improving clinical outcome following myocardial infarction. However, abnormal increases in intracellular Ca²⁺ during myocardial reperfusion can cause cardiomyocyte death and consequent loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Therapeutic modulation of Ca²⁺ handling provides some cardioprotection against the paradoxical effects of restoring blood flow to the heart, highlighting the significance of Ca²⁺ overload to IR injury. Cardiac IR is also accompanied by dynamic changes in the expression of microRNAs (miRNAs); for example, miR-214 is upregulated during ischemic injury and heart failure, but its potential role in these processes is unknown. Here, we show that genetic deletion of miR-214 in mice causes loss of cardiac contractility, increased apoptosis, and excessive fibrosis in response to IR injury. The cardioprotective roles of miR-214 during IR injury were attributed to repression of the mRNA encoding sodium/calcium exchanger 1 (Ncx1), a key regulator of Ca²⁺ influx; and to repression of several downstream effectors of Ca²⁺ signaling that mediate cell death. These findings reveal a pivotal role for miR-214 as a regulator of cardiomyocyte Ca²⁺ homeostasis and survival during cardiac injury.
• Kimura, W., & Sadek, H. A. (2012). The cardiac hypoxic niche: emerging role of hypoxic microenvironment in cardiac progenitors. Cardiovascular diagnosis and therapy, 2(4), 278-89.
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  Resident stem cells persist throughout the entire lifetime of an organism where they replenishing damaged cells. Numerous types of resident stem cells are housed in a low-oxygen tension (hypoxic) microenvironment, or niches, which seem to be critical for survival and maintenance of stem cells. Recently our group has identified the adult mammalian epicardium and subepicardium as a hypoxic niche for cardiac progenitor cells. Similar to hematopoietic stem cells (LT-HSCs), progenitor cells in the hypoxic epicardial niche utilize cytoplasmic glycolysis instead of mitochondrial oxidative phosphorylation, where hypoxia inducible factor 1α (Hif-1α) maintains them in glycolytic undifferentiated state. In this review we summarize the relationship between hypoxic signaling and stem cell function, and discuss potential roles of several cardiac stem/progenitor cells in cardiac homeostasis and regeneration.
• Kocabas, F., Zheng, J., Thet, S., Copeland, N. G., Jenkins, N. A., DeBerardinis, R. J., Zhang, C., & Sadek, H. A. (2012). Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells. Blood, 120(25), 4963-72.
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  The role of Meis1 in leukemia is well established, but its role in hematopoietic stem cells (HSCs) remains poorly understood. Previously, we showed that HSCs use glycolytic metabolism to meet their energy demands. However, the mechanism of regulation of HSC metabolism, and the importance of maintaining this distinct metabolic phenotype on HSC function has not been determined. More importantly, the primary function of Meis1 in HSCs remains unknown. Here, we examined the effect of loss of Meis1 on HSC function and metabolism. Inducible Meis1 deletion in adult mouse HSCs resulted in loss of HSC quiescence, and failure of bone marrow repopulation after transplantation. While we previously showed that Meis1 regulates Hif-1α transcription in vitro, we demonstrate here that loss of Meis1 results in down-regulation of both Hif-1α and Hif-2α in HSCs. This resulted in a shift to mitochondrial metabolism, increased reactive oxygen species production, and apoptosis of HSCs. Finally, we demonstrate that the effect of Meis1 knockout on HSCs is entirely mediated through reactive oxygen species where treatment of the Meis1 knockout mice with the scavenger N-acetylcystein restored HSC quiescence and rescued HSC function. These results uncover an important transcriptional network that regulates metabolism, oxidant defense, and maintenance of HSCs.
• Zang, Q. S., Sadek, H., Maass, D. L., Martinez, B., Ma, L., Kilgore, J. A., Williams, N. S., Frantz, D. E., Wigginton, J. G., Nwariaku, F. E., Wolf, S. E., & Minei, J. P. (2012). Specific inhibition of mitochondrial oxidative stress suppresses inflammation and improves cardiac function in a rat pneumonia-related sepsis model. American journal of physiology. Heart and circulatory physiology, 302(9), H1847-59.
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  Using a mitochondria-targeted vitamin E (Mito-Vit-E) in a rat pneumonia-related sepsis model, we examined the role of mitochondrial reactive oxygen species in sepsis-mediated myocardial inflammation and subsequent cardiac contractile dysfunction. Sepsis was produced in adult male Sprague-Dawley rats via intratracheal injection of S. pneumonia (4 × 10(6) colony formation units per rat). A single dose of Mito-Vit-E, vitamin E, or control vehicle, at 21.5 μmol/kg, was administered 30 min postinoculation. Blood was collected, and heart tissue was harvested at various time points. Mito-Vit-E in vivo distribution was confirmed by mass spectrometry. In cardiac mitochondria, Mito-Vit-E improved total antioxidant capacity and suppressed H(2)O(2) generation, whereas vitamin E offered little effect. In cytosol, both antioxidants decreased H(2)O(2) levels, but only vitamin E strengthened antioxidant capacity. Mito-Vit-E protected mitochondrial structure and function in the heart during sepsis, demonstrated by reduction in lipid and protein oxidation, preservation of mitochondrial membrane integrity, and recovery of respiratory function. While both Mito-Vit-E and vitamin E suppressed sepsis-induced peripheral and myocardial production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), Mito-Vit-E exhibited significantly higher efficacy (P < 0.05). Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induced myeloperoxidase accumulation in myocardium, suggesting its effect on neutrophil infiltration. Echocardiography analysis indicated that Mito-Vit-E ameliorated cardiac contractility of sepsis animals, shown by improved fractional shortening and ejection fraction. Together, our data suggest that targeted scavenging of mitochondrial reactive oxygen species protects mitochondrial function, attenuates tissue-level inflammation, and improves whole organ activities in the heart during sepsis.
• Simsek, T., Kocabas, F., Zheng, J., Deberardinis, R. J., Mahmoud, A. I., Olson, E. N., Schneider, J. W., Zhang, C. C., & Sadek, H. A. (2010). The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell stem cell, 7(3), 380-90.
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  Bone marrow transplantation is the primary therapy for numerous hematopoietic disorders. The efficiency of bone marrow transplantation depends on the function of long-term hematopoietic stem cells (LT-HSCs), which is markedly influenced by their hypoxic niche. Survival in this low-oxygen microenvironment requires significant metabolic adaptation. Here, we show that LT-HSCs utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. We used flow cytometry to identify a unique low mitochondrial activity/glycolysis-dependent subpopulation that houses the majority of hematopoietic progenitors and LT-HSCs. Finally, we demonstrate that Meis1 and Hif-1alpha are markedly enriched in LT-HSCs and that Meis1 regulates HSC metabolism through transcriptional activation of Hif-1alpha. These findings reveal an important transcriptional network that regulates HSC metabolism.
• Small, E. M., Thatcher, J. E., Sutherland, L. B., Kinoshita, H., Gerard, R. D., Richardson, J. A., Dimaio, J. M., Sadek, H., Kuwahara, K., & Olson, E. N. (2010). Myocardin-related transcription factor-a controls myofibroblast activation and fibrosis in response to myocardial infarction. Circulation research, 107(2), 294-304.
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  Myocardial infarction (MI) results in loss of cardiac myocytes in the ischemic zone of the heart, followed by fibrosis and scar formation, which diminish cardiac contractility and impede angiogenesis and repair. Myofibroblasts, a specialized cell type that switches from a fibroblast-like state to a contractile, smooth muscle-like state, are believed to be primarily responsible for fibrosis of the injured heart and other tissues, although the transcriptional mediators of fibrosis and myofibroblast activation remain poorly defined. Myocardin-related transcription factors (MRTFs) are serum response factor (SRF) cofactors that promote a smooth muscle phenotype and are emerging as components of stress-responsive signaling.
• Sadek, H. A., Martin, C. M., Latif, S. S., Garry, M. G., & Garry, D. J. (2009). Bone-marrow-derived side population cells for myocardial regeneration. Journal of cardiovascular translational research, 2(2), 173-81.
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  Bone-marrow-derived stem cells have displayed the potential for myocardial regeneration in animal models as well as in clinical trials. Unfractionated bone marrow mononuclear cell (MNC) population is a heterogeneous group of cells known to include a number of stem cell populations. Cells in the side population (SP) fraction have a high capacity for differentiation into multiple lineages. In the current study, we investigated the role of murine and human bone-marrow-derived side population cells in myocardial regeneration. In these studies, we show that mouse bone-marrow-derived SP cells expressed the contractile protein, alpha-actinin, following culture with neonatal cardiomyocytes and after delivery into the myocardium following injury. Moreover, the number of green-fluorescent-protein-positive cells, of bone marrow side population origin, increased progressively within the injured myocardium over 90 days. Transcriptome analysis of these bone marrow cells reveals a pattern of expression consistent with immature cardiomyocytes. Additionally, the differentiation capacity of human granulocyte colony-stimulating factor stimulated peripheral blood stem cells were assessed following injection into injured rat myocardium. Bone marrow mononuclear cell and side population cells were both readily identified within the rat myocardium 1 month following injection. These human cells expressed human-specific cardiac troponin I as determined by immunohistochemistry as well as numerous cardiac transcripts as determined by polymerase chain reaction. Both human bone marrow mononuclear cells and human side population cells augmented cardiac systolic function following a modest drop in function as a result of cryoinjury. The augmentation of cardiac function following injection of side population cells occurred earlier than with bone marrow mononuclear cells despite the fact that the number of side population cells used was one tenth that of bone marrow mononuclear cells (9 x 10(5) cells per heart in the MNC group compared to 9 x 10(4) per heart in the SP group). These results support the hypotheses that rodent and human-bone-marrow derived side population cells are capable of acquiring a cardiac fate and that human bone-marrow-derived side population cells are superior to unfractionated bone marrow mononuclear cells in augmenting left ventricular systolic function following cryoinjury.
• Martin, C. M., Ferdous, A., Gallardo, T., Humphries, C., Sadek, H., Caprioli, A., Garcia, J. A., Szweda, L. I., Garry, M. G., & Garry, D. J. (2008). Hypoxia-inducible factor-2alpha transactivates Abcg2 and promotes cytoprotection in cardiac side population cells. Circulation research, 102(9), 1075-81.
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  Stem and progenitor cell populations occupy a specialized niche and are consequently exposed to hypoxic as well as oxidative stresses. We have previously established that the multidrug resistance protein Abcg2 is the molecular determinant of the side population (SP) progenitor cell population. We observed that the cardiac SP cells increase in number more than 3-fold within 3 days of injury. Transcriptome analysis of the SP cells isolated from the injured adult murine heart reveals increased expression of cytoprotective transcripts. Overexpression of Abcg2 results in an increased ability to consume hydrogen peroxide and is associated with increased levels of alpha-glutathione reductase protein expression. Importantly, overexpression of Abcg2 also conferred a cell survival benefit following exposure to hydrogen peroxide. To further examine the molecular regulation of the Abcg2 gene, we demonstrated that hypoxia-inducible factor (HIF)-2alpha binds an evolutionary conserved HIF-2alpha response element in the murine Abcg2 promoter. Transcriptional assays reveal a dose-dependent activation of Abcg2 expression by HIF-2alpha. These results support the hypothesis that Abcg2 is a direct downstream target of HIF-2alpha which functions with other factors to initiate a cytoprotective program for this progenitor SP cell population that resides in the adult heart.
• Sadek, H., Hannack, B., Choe, E., Wang, J., Latif, S., Garry, M. G., Garry, D. J., Longgood, J., Frantz, D. E., Olson, E. N., Hsieh, J., & Schneider, J. W. (2008). Cardiogenic small molecules that enhance myocardial repair by stem cells. Proceedings of the National Academy of Sciences of the United States of America, 105(16), 6063-8.
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  The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5, using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs.
• Sadek, H., Latif, S., Collins, R., Garry, M. G., & Garry, D. J. (2008). Use of ferumoxides for stem cell labeling. Regenerative medicine, 3(6), 807-16.
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  Although numerous clinical trials have shown promising results with regards to the cardiac regenerative capacity of different types of stem cells, there remains virtually no evidence of the fate of stem cells in these human studies, primarily owing to safety concerns associated with the use of cell-labeling strategies.
• Sadek, H. A., & Hoit, B. D. (2007). Real time embolization of a mitral valve vegetation. Echocardiography (Mount Kisco, N.Y.), 24(7), 768-9.
• Sadek, H., Gilkeson, R. C., Hoit, B. D., & Brozovich, F. V. (2006). Images in cardiovascular medicine. Case of anomalous right superior vena cava. Circulation, 114(15), e532-3.
• Nulton-Persson, A. C., Szweda, L. I., & Sadek, H. A. (2004). Inhibition of cardiac mitochondrial respiration by salicylic acid and acetylsalicylate. Journal of cardiovascular pharmacology, 44(5), 591-5.
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  Acetylsalicylate, the active ingredient in aspirin, has been shown to be beneficial in the treatment and prevention of cardiovascular disease. Because of the increasing frequency with which salicylates are used, it is important to more fully characterize extra- and intracellular processes that are altered by these compounds. Evidence is provided that treatment of isolated cardiac mitochondria with salicylic acid and to a lesser extent acetylsalicylate resulted in an increase in the rate of uncoupled respiration. In contrast, both compounds inhibited ADP-dependent NADH-linked (state 3) respiration to similar degrees. Under the conditions of our experiments, loss in state 3 respiration resulted from inhibition of the Krebs cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH). Kinetic analysis indicates that salicylic acid acts as a competitive inhibitor at the alpha-ketoglutarate binding site. In contrast, acetylsalicylate inhibited the enzyme in a noncompetitive fashion consistent with interaction with the alpha-ketoglutarate binding site followed by enzyme-catalyzed acetylation. The effects of salicylic acid and acetylsalicylate on cardiac mitochondrial function may contribute to the known cardioprotective effects of therapeutic doses of aspirin, as well as to the toxicity associated with salicylate overdose.
• Sadek, H. A., Szweda, P. A., & Szweda, L. I. (2004). Modulation of mitochondrial complex I activity by reversible Ca2+ and NADH mediated superoxide anion dependent inhibition. Biochemistry, 43(26), 8494-502.
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  Complex I, a key component of the mitochondrial respiratory chain, exhibits diminished activity as a result of cardiac ischemia/reperfusion. Cardiac ischemia/reperfusion is associated with increases in the levels of mitochondrial Ca(2+) and pro-oxidants. In the current in vitro study, we sought evidence for a mechanistic link between Ca(2+), pro-oxidants, and inhibition of complex I utilizing mitochondria isolated from rat heart. Our results indicate that addition of Ca(2+) to solubilized mitochondria results in loss in complex I activity. Ca(2+) induced a maximum decrease in complex I activity of approximately 35% at low micromolar concentrations over a narrow physiologically relevant pH range. Loss in activity required reducing equivalents in the form of NADH and was not reversed upon addition of EGTA. The antioxidants N-acetylcysteine and superoxide dismutase, but not catalase, prevented inhibition, indicating the involvement of superoxide anion (O2(*-)) in the inactivation process. Importantly, the sulfhydryl reducing agent DTT was capable of fully restoring complex I activity implicating the formation of sulfenic acid and/or disulfide derivatives of cysteine in the inactivation process. Finally, complex I can reactivate endogenously upon Ca(2+) removal if NADH is present and the enzyme is allowed to turnover catalytically. Thus, the present study provides a mechanistic link between three alterations known to occur during cardiac ischemia/reperfusion, mitochondrial Ca(2+) accumulation, free radical production, and complex I inhibition. The reversibility of these processes suggests redox regulation of Ca(2+) handling.
• Sadek, H. A., Humphries, K. M., Szweda, P. A., & Szweda, L. I. (2002). Selective inactivation of redox-sensitive mitochondrial enzymes during cardiac reperfusion. Archives of biochemistry and biophysics, 406(2), 222-8.
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  Reperfusion of ischemic myocardial tissue results in an increase in mitochondrial free radical production and declines in respiratory activity. The effects of ischemia and reperfusion on the activities of Krebs cycle enzymes, as well as enzymes involved in electron transport, were evaluated to provide insight into whether free radical events are likely to affect enzymatic and mitochondrial function(s). An in vivo rat model was utilized in which ischemia is induced by ligating the left anterior descending coronary artery. Reperfusion, initiated by release of the ligature, resulted in a significant decline in NADH-linked ADP-dependent mitochondrial respiration as assessed in isolated cardiac mitochondria. Assays of respiratory chain complexes revealed reduction in the activities of complex I and, to a lesser extent, complex IV exclusively during reperfusion, with no alterations in the activities of complexes II and III. Moreover, Krebs cycle enzymes alpha-ketoglutarate dehydrogenase and aconitase were susceptible to reperfusion-induced inactivation with no decline in the activities of other Krebs cycle enzymes. The decline in alpha-ketoglutarate dehydrogenase activity during reperfusion was associated with a loss in native lipoic acid on the E2 subunit, suggesting oxidative inactivation. Inhibition of complex I in vitro promotes free radical generation. alpha-Ketoglutarate dehydrogenase and aconitase are uniquely susceptible to in vitro oxidative inactivation. Thus, our results suggest a scenario in which inhibition of complex I promotes free radical production leading to oxidative inactivation of alpha-ketoglutarate dehydrogenase and aconitase.