Jared Churko

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• Falana, S. L., Kazmouz, S. G., Iwanski, J. B., Sarvagalla, S., Bas, B. E., Juneman, E., Moukabary, T., Ma, N., Gundry, R. L., Rohani, L., Hanson, P., Laksman, Z., James, C. A., Calkin, H., & Churko, J. M. (2025). Modelling arrhythmogenic cardiomyopathy fattyfibro pathology with PKP2-deficient epicardial cells derived from human iPSCs. Communications Biology, 8(Issue 1). doi:10.1038/s42003-025-08921-z
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  Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease marked by progressive fattyfibro replacement of the ventricular myocardium, life-threatening arrhythmias, and sudden cardiac death. To dissect epicardial contributions to ACM pathogenesis, we generated iPSC lines from patients carrying plakophilin 2 (PKP2) 1849C > T or PKP2 2013delC mutations, their CRISPR/Cas9–corrected isogenic controls, and a PKP2 knockout line. Epicardial cells (hPSC-EPCs) differentiated from mutant and knockout backgrounds exhibit enhanced epithelial-to-mesenchymal transition characteristics, increased lipid accumulation, and a pronounced fibrotic phenotype. RNA-seq performed on ACM hPSC-EPCs reveals dysregulation of Wnt, interferon, and Rho GTPase signaling, including an upregulation of insulin growth factor 2 (IGF2) and a key adipogenic transcription factor, CEBPA. Subsequent treatment of control and PKP2KO hPSC-EPCs with recombinant IGF2 enhances CEBPA expression, suggesting that insulin growth factor signaling contributes to ACM fattyfibro remodeling.
• Falana, S. L., Kazmouz, S. G., Iwanski, J. B., Sarvagalla, S., Bas, B. E., Juneman, E., Moukabary, T., Ma, N., Gundry, R. L., Rohani, L., Hanson, P., Laksman, Z., James, C. A., Calkin, H., & Churko, J. M. (2025). Modelling arrhythmogenic cardiomyopathy fattyfibro pathology with PKP2-deficient epicardial cells derived from human iPSCs. Communications biology, 8(1), 1502.
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  Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease marked by progressive fattyfibro replacement of the ventricular myocardium, life-threatening arrhythmias, and sudden cardiac death. To dissect epicardial contributions to ACM pathogenesis, we generated iPSC lines from patients carrying plakophilin 2 (PKP2) 1849C > T or PKP2 2013delC mutations, their CRISPR/Cas9-corrected isogenic controls, and a PKP2 knockout line. Epicardial cells (hPSC-EPCs) differentiated from mutant and knockout backgrounds exhibit enhanced epithelial-to-mesenchymal transition characteristics, increased lipid accumulation, and a pronounced fibrotic phenotype. RNA-seq performed on ACM hPSC-EPCs reveals dysregulation of Wnt, interferon, and Rho GTPase signaling, including an upregulation of insulin growth factor 2 (IGF2) and a key adipogenic transcription factor, CEBPA. Subsequent treatment of control and PKP2KO hPSC-EPCs with recombinant IGF2 enhances CEBPA expression, suggesting that insulin growth factor signaling contributes to ACM fattyfibro remodeling.
• Huang, K., Ashraf, M., Rohani, L., Luo, Y., Sacayanan, A., Huang, H., Haegert, A., Volik, S., Sar, F., LeBihan, S., Liew, J., Backx, P. H., Roberts, J. D., Tibbits, G. F., Churko, J. M., Sanatani, S., Collins, C., Brunham, L. R., & Laksman, Z. (2025). Atrial Fibrillation Related Titin Truncation Is Associated With Atrial Myopathy in Patient-Derived Induced Pluripotent Stem Cell Disease Models. Circulation. Genomic and precision medicine, 18(1), e004412.
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  Protein-truncating mutations in the titin gene are associated with increased risk of atrial fibrillation. However, little is known about the underlying pathophysiology.
• Huang, K., Ashraf, M., Rohani, L., Luo, Y., Sacayanan, A., Huang, H., Haegert, A., Volik, S., Sar, F., Lebihan, S., Liew, J., Backx, P. H., Roberts, J. D., Tibbits, G. F., Churko, J. M., Sanatani, S., Collins, C., Brunham, L. R., & Laksman, Z. (2025). Atrial Fibrillation Related Titin Truncation Is Associated with Atrial Myopathy in Patient-Derived Induced Pluripotent Stem Cell Disease Models. Circulation: Genomic and Precision Medicine, 18(Issue 1). doi:10.1161/circgen.123.004412
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  BACKGROUND: Protein-truncating mutations in the titin gene are associated with increased risk of atrial fibrillation. However, little is known about the underlying pathophysiology. METHODS: We identified a heterozygous titin truncating variant (TTNtv) in a patient with unexplained early onset atrial fibrillation and normal ventricular function. We generated patient-specific atrial- and ventricular-like induced pluripotent stem cell-derived cardiomyocytes and engineered heart tissue to evaluate the impact of the TTNtv on electrophysiology, sarcomere structure, contractility, and gene expression. RESULTS: We demonstrate that the TTNtv increases susceptibility to pacing-induced arrhythmia, promotes sarcomere disorganization, and reduces contractile force in atrial induced pluripotent stem cell-derived cardiomyocytes compared with their CRISPR/Cas9-corrected isogenic controls. In ventricular induced pluripotent stem cell-derived cardiomyocytes, this variant was associated with abnormal electrophysiology and sarcomere organization without a reduction in contractile force compared with their isogenic controls. RNA-sequencing revealed an upregulation of cell adhesion and extracellular matrix genes in the presence of the TTNtv for both atrial and ventricular engineered heart tissues. CONCLUSIONS: In a patient with unexplained atrial fibrillation, induced pluripotent stem cell-derived cardiomyocytes with a TTNtv showed structural and electrophysiological abnormalities in both atrial and ventricular models, while only atrial engineered heart tissues demonstrated reduced contractility. The observed chamber-specific effect suggests that structural disorganization and reduced contractile function may be associated with atrial myopathy in the presence of truncated titin.
• Iwanski, J. B., Lawal, O. S., Kwon, W. T., Vazquez, I., & Churko, J. M. (2025). Minimal Component, Protein-Free, and Cost-effective Human Pluripotent Stem Cell Cardiomyocyte Differentiation. Current Protocols, 5(Issue 2). doi:10.1002/cpz1.70099
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  Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become a powerful source for the in vitro modeling of cardiac diseases and various other essential applications, including cardiotoxicity screening and regenerative cell replacement therapies. Although many differentiation protocols have been developed to generate cardiomyocytes from human pluripotent stem cells, these protocols are costly and complex, requiring expensive and often unnecessary components (e.g., B27 medium supplement). In addition, the use of animal-derived growth factors limits their use for regenerative medicine purposes. To address these issues, herein, we have developed an efficient, cost-effective, and protein-free hPSC-CM protocol using only two components: DMEM/F12 basal medium and l-ascorbic acid 2-phosphate. By eliminating xenobiotic and complex components, the efficiency of directed differentiations is increased, the variability between cardiac differentiations is decreased, and the scalability of cell production is enhanced. Adaptation of this efficient, low-cost, and user-friendly cardiac differentiation protocol will enrich the utility and applicability of hPSC-CMs in drug discovery, cell therapies, tissue engineering, disease modeling, precision medicine, and cardiac regenerative medicine. © 2025 Wiley Periodicals LLC. Basic Protocol 1: hPSC cell culture. Basic Protocol 2: hPSC-CM differentiation. Basic Protocol 3: Characterization of hPSC-CMs by immunofluorescence (IF) imaging.
• Iwanski, J. B., Lawal, O. S., Kwon, W. T., Vazquez, I., & Churko, J. M. (2025). Minimal Component, Protein-Free, and Cost-effective Human Pluripotent Stem Cell Cardiomyocyte Differentiation. Current protocols, 5(2), e70099.
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  Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become a powerful source for the in vitro modeling of cardiac diseases and various other essential applications, including cardiotoxicity screening and regenerative cell replacement therapies. Although many differentiation protocols have been developed to generate cardiomyocytes from human pluripotent stem cells, these protocols are costly and complex, requiring expensive and often unnecessary components (e.g., B27 medium supplement). In addition, the use of animal-derived growth factors limits their use for regenerative medicine purposes. To address these issues, herein, we have developed an efficient, cost-effective, and protein-free hPSC-CM protocol using only two components: DMEM/F12 basal medium and l-ascorbic acid 2-phosphate. By eliminating xenobiotic and complex components, the efficiency of directed differentiations is increased, the variability between cardiac differentiations is decreased, and the scalability of cell production is enhanced. Adaptation of this efficient, low-cost, and user-friendly cardiac differentiation protocol will enrich the utility and applicability of hPSC-CMs in drug discovery, cell therapies, tissue engineering, disease modeling, precision medicine, and cardiac regenerative medicine. © 2025 Wiley Periodicals LLC. Basic Protocol 1: hPSC cell culture Basic Protocol 2: hPSC-CM differentiation Basic Protocol 3: Characterization of hPSC-CMs by immunofluorescence (IF) imaging.
• Subramanian, S. P., Wojtkiewicz, M., Yu, F., Castro, C., Schuette, E. N., Rodriguez-Paar, J., Churko, J., Renavikar, P., Anderson, D., Mahr, C., & Gundry, R. L. (2025). Integrated Multiomics Reveals Alterations in Paucimannose and Complex Type N-Glycans in Cardiac Tissue of Patients with COVID-19. Molecular & cellular proteomics : MCP, 24(4), 100929.
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  Coronavirus infectious disease of 2019 (COVID-19) can lead to cardiac complications, yet the molecular mechanisms driving these effects remain unclear. Protein glycosylation is crucial for viral replication, immune response, and organ function and has been found to change in the lungs and liver of patients with COVID-19. However, how COVID-19 impacts cardiac protein glycosylation has not been defined. Our study combined single nuclei transcriptomics, mass spectrometry (MS)-based glycomics, and lectin-based tissue imaging to investigate alterations in N-glycosylation in the human heart post-COVID-19. We identified significant expression differences in glycogenes involved in N-glycan biosynthesis and MS analysis revealed a reduction in high mannose and isomers of paucimannose structures post-infection, with changes in paucimannose directly correlating with COVID-19 independent of comorbidities. Our observations suggest that COVID-19 primes cardiac tissues to alter the glycome at all levels, namely, metabolism, nucleotide sugar transport, and glycosyltransferase activity. Given the role of N-glycosylation in cardiac function, this study provides a basis for understanding the molecular events leading to cardiac damage post-COVID-19 and informing future therapeutic strategies to treat cardiac complications resulting from coronavirus infections.
• Subramanian, S. P., Wojtkiewicz, M., Yu, F., Castro, C., Schuette, E. N., Rodriguez-Paar, J., Churko, J., Renavikar, P., Anderson, D., Mahr, C., & Gundry, R. L. (2025). Integrated Multiomics Reveals Alterations in Paucimannose and Complex Type N-Glycans in Cardiac Tissue of Patients with COVID-19. Molecular and Cellular Proteomics, 24(Issue 4). doi:10.1016/j.mcpro.2025.100929
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  Coronavirus infectious disease of 2019 (COVID-19) can lead to cardiac complications, yet the molecular mechanisms driving these effects remain unclear. Protein glycosylation is crucial for viral replication, immune response, and organ function and has been found to change in the lungs and liver of patients with COVID-19. However, how COVID-19 impacts cardiac protein glycosylation has not been defined. Our study combined single nuclei transcriptomics, mass spectrometry (MS)-based glycomics, and lectin-based tissue imaging to investigate alterations in N-glycosylation in the human heart post-COVID-19. We identified significant expression differences in glycogenes involved in N-glycan biosynthesis and MS analysis revealed a reduction in high mannose and isomers of paucimannose structures post-infection, with changes in paucimannose directly correlating with COVID-19 independent of comorbidities. Our observations suggest that COVID-19 primes cardiac tissues to alter the glycome at all levels, namely, metabolism, nucleotide sugar transport, and glycosyltransferase activity. Given the role of N-glycosylation in cardiac function, this study provides a basis for understanding the molecular events leading to cardiac damage post-COVID-19 and informing future therapeutic strategies to treat cardiac complications resulting from coronavirus infections.
• Thai, B. Q., Luff, S. A., Churko, J. M., Young, J. N., Sturgeon, C. M., & Bhattacharya, D. (2025). WNT signaling in human pluripotent stem cells promotes HDAC2-dependent epigenetic programs and development of retinoic acid-responsive mesoderm. bioRxiv : the preprint server for biology.
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  Human pluripotent stem cells (hPSCs) can be used as a scalable source of lymphocytes for adoptive cell therapies, contingent on the robust generation of definitive hematopoietic intermediates. Early WNT activation with CHIR99021 during mesoderm induction promoted the formation of KDR+ ALDH1A2+ mesodermal progenitors and subsequent generation of T cells in a retinoic acid (RA)-dependent manner. Integrated scRNA-seq and ATAC-seq defined a WNT-dependent developmental trajectory from hPSCs to KDR+ ALDH1A2+ mesoderm. Gene regulatory network modeling predicted HDAC2 and E-box transcription factors as regulators of RA-responsive mesodermal differentiation downstream of WNT. HDAC2 knockout impaired, while HDAC2 overexpression enhanced, KDR+ ALDH1A2+ progenitor formation. E-box factor manipulation had no discernible effect. An orthogonal chemical screen confirmed that HDAC2 inhibition suppressed KDR+ ALDH1A2+ mesodermal progenitors, whereas modulating histone methylation enhanced their formation. These findings reveal mechanisms by which WNT signaling promotes RA-responsive mesoderm and suggest methods to improve the generation of lymphocytes from hPSCs.
• Iwanski, J., Pappas, C., Mayfield, R., Farman, G., Ahrens-Nicklas, R., Churko, J., & Gregorio, C. (2024). Leiomodin 2 neonatal dilated cardiomyopathy mutation results in altered actin gene signatures and cardiomyocyte dysfunction. npj Regenerative Medicine, 9(1). doi:10.1038/s41536-024-00366-y
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  Neonatal dilated cardiomyopathy (DCM) is a poorly understood muscular disease of the heart. Several homozygous biallelic variants in LMOD2, the gene encoding the actin-binding protein Leiomodin 2, have been identified to result in severe DCM. Collectively, LMOD2-related cardiomyopathies present with cardiac dilation and decreased heart contractility, often resulting in neonatal death. Thus, it is evident that Lmod2 is essential to normal human cardiac muscle function. This study aimed to understand the underlying pathophysiology and signaling pathways related to the first reported LMOD2 variant (c.1193 G > A, p.Trp398*). Using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and a mouse model harboring the homologous mutation to the patient, we discovered dysregulated actin-thin filament lengths, altered contractility and calcium handling properties, as well as alterations in the serum response factor (SRF)-dependent signaling pathway. These findings reveal that LMOD2 may be regulating SRF activity in an actin-dependent manner and provide a potential new strategy for the development of biologically active molecules to target LMOD2-related cardiomyopathies.
• Berg Luecke, L., Waas, M., Littrell, J., Wojtkiewicz, M., Castro, C., Burkovetskaya, M., Schuette, E. N., Buchberger, A. R., Churko, J. M., Chalise, U., Waknitz, M., Konfrst, S., Teuben, R., Morrissette-McAlmon, J., Mahr, C., Anderson, D. R., Boheler, K. R., & Gundry, R. L. (2023). Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts. Nature Cardiovascular Research, 2(Issue 1). doi:10.1038/s44161-022-00200-y
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  Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.
• Chakrabarti, J., Mallick, S., Churko, J., Eschbacher, J., Little, A. S., Ji Yuen, K. C., & Zavros, Y. (2023). OR20-05 Generation Of CDH23-mutated Pituitary Neuroendocrine Tumor Organoids From Induced Pluripotent Stem Cells To Model Cushing’s Disease Pathogenesis. Journal of the Endocrine Society, 7(Supplement_1). doi:10.1210/jendso/bvad114.1314
• Mallick, S., Chakrabarti, J., Eschbacher, J., Moraitis, A. G., Greenstein, A. E., Churko, J., Pond, K. W., Livolsi, A., Thorne, C. A., Little, A. S., Yuen, K. C., & Zavros, Y. (2023). Genetically engineered human pituitary corticotroph tumor organoids exhibit divergent responses to glucocorticoid receptor modulators. Translational Research, 256(Issue). doi:10.1016/j.trsl.2023.01.002
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  Cushing's disease (CD) is a serious endocrine disorder attributed to an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that that subsequently leads to chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSCUSP48 and iPSCUSP8 cultures, mifepristone induced a predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of Cushing's disease patients.
• 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(Issue 20). doi:10.14814/phy2.15838
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  Cardiac ischemic reperfusion injury (IRI) is paradoxically instigated by reestablishing blood-flow to ischemic myocardium typically from a myocardial infarction (MI). Although revascularization following MI remains the standard of care, effective strategies remain limited to prevent or attenuate IRI. We hypothesized that epicardial placement of human placental amnion/chorion (HPAC) grafts will protect against IRI. Using a clinically relevant model of IRI, swine were subjected to 45 min percutaneous ischemia followed with (MI + HPAC, n = 3) or without (MI only, n = 3) HPAC. Cardiac function was assessed by echocardiography, and regional punch biopsies were collected 14 days post-operatively. A deep phenotyping approach was implemented by using histological interrogation and incorporating global proteomics and transcriptomics in nonischemic, ischemic, and border zone biopsies. Our results established HPAC limited the extent of cardiac injury by 50% (11.0 ± 2.0% vs. 22.0 ± 3.0%, p = 0.039) and preserved ejection fraction in HPAC-treated swine (46.8 ± 2.7% vs. 35.8 ± 4.5%, p = 0.014). We present comprehensive transcriptome and proteome profiles of infarct (IZ), border (BZ), and remote (RZ) zone punch biopsies from swine myocardium during the proliferative cardiac repair phase 14 days post-MI. Both HPAC-treated and untreated tissues showed regional dynamic responses, whereas only HPAC-treated IZ revealed active immune and extracellular matrix remodeling. Decreased endoplasmic reticulum (ER)-dependent protein secretion and increased antiapoptotic and anti-inflammatory responses were measured in HPAC-treated biopsies. We provide quantitative evidence HPAC reduced cardiac injury from MI in a preclinical swine model, establishing a potential new therapeutic strategy for IRI. Minimizing the impact of MI remains a central clinical challenge. We present a new strategy to attenuate post-MI cardiac injury using HPAC in a swine model of IRI. Placement of HPAC membrane on the heart following MI minimizes ischemic damage, preserves cardiac function, and promotes anti-inflammatory signaling pathways.
• Chakrabarti, J., Pandey, R., Churko, J. M., Eschbacher, J., Mallick, S., Chen, Y., Hermes, B., Mallick, P., Stansfield, B. N., Pond, K. W., Thorne, C. A., Yuen, K. C., Little, A. S., & Zavros, Y. (2022). Development of Human Pituitary Neuroendocrine Tumor Organoids to Facilitate Effective Targeted Treatments of Cushing’s Disease. Cells, 11(Issue 21). doi:10.3390/cells11213344
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  (1) Background: Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, obesity, cognitive impairment, anxiety, depression, and death. The first-line treatment for CD is pituitary surgery. Current surgical remission rates reported in only 56% of patients depending on several criteria. The lack of specificity, poor tolerability, and low efficacy of the subsequent second-line medical therapies make CD a medical therapeutic challenge. One major limitation that hinders the development of specific medical therapies is the lack of relevant human model systems that recapitulate the cellular composition of PitNET microenvironment. (2) Methods: human pituitary tumor tissue was harvested during transsphenoidal surgery from CD patients to generate organoids (hPITOs). (3) Results: hPITOs generated from corticotroph, lactotroph, gonadotroph, and somatotroph tumors exhibited morphological diversity among the organoid lines between individual patients and amongst subtypes. The similarity in cell lineages between the organoid line and the patient’s tumor was validated by comparing the neuropathology report to the expression pattern of PitNET specific markers, using spectral flow cytometry and exome sequencing. A high-throughput drug screen demonstrated patient-specific drug responses of hPITOs amongst each tumor subtype. Generation of induced pluripotent stem cells (iPSCs) from a CD patient carrying germline mutation CDH23 exhibited dysregulated cell lineage commitment. (4) Conclusions: The human pituitary neuroendocrine tumor organoids represent a novel approach in how we model complex pathologies in CD patients, which will enable effective personalized medicine for these patients.
• Churko, J. M., Frankman, Z., & Salem, T. (2022). Tissue Engineering Techniques for Induced Pluripotent Stem Cell Derived Three-Dimensional Cardiac Constructs. Tissue Engineering Part B: Reviews, 28(4), 891-911. doi:10.1089/ten.teb.2021.0088
• Pivniouk, V., Pivniouk, O., DeVries, A., Uhrlaub, J. L., Michael, A., Pivniouk, D., VanLinden, S. R., Conway, M. Y., Hahn, S., Malone, S. P., Ezeh, P., Churko, J. M., Anderson, D., Kraft, M., Nikolich-Zugich, J., & Vercelli, D. (2022). The OM-85 bacterial lysate inhibits SARS-CoV-2 infection of epithelial cells by downregulating SARS-CoV-2 receptor expression. Journal of Allergy and Clinical Immunology, 149(Issue 3). doi:10.1016/j.jaci.2021.11.019
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  Background: Treatments for coronavirus disease 2019, which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are urgently needed but remain limited. SARS-CoV-2 infects cells through interactions of its spike (S) protein with angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) on host cells. Multiple cells and organs are targeted, particularly airway epithelial cells. OM-85, a standardized lysate of human airway bacteria with strong immunomodulating properties and an impeccable safety profile, is widely used to prevent recurrent respiratory infections. We found that airway OM-85 administration inhibits Ace2 and Tmprss2 transcription in the mouse lung, suggesting that OM-85 might hinder SARS-CoV-2/host cell interactions. Objectives: We sought to investigate whether and how OM-85 treatment protects nonhuman primate and human epithelial cells against SARS-CoV-2. Methods: ACE2 and TMPRSS2 mRNA and protein expression, cell binding of SARS-CoV-2 S1 protein, cell entry of SARS-CoV-2 S protein–pseudotyped lentiviral particles, and SARS-CoV-2 cell infection were measured in kidney, lung, and intestinal epithelial cell lines, primary human bronchial epithelial cells, and ACE2-transfected HEK293T cells treated with OM-85 in vitro. Results: OM-85 significantly downregulated ACE2 and TMPRSS2 transcription and surface ACE2 protein expression in epithelial cell lines and primary bronchial epithelial cells. OM-85 also strongly inhibited SARS-CoV-2 S1 protein binding to, SARS-CoV-2 S protein–pseudotyped lentivirus entry into, and SARS-CoV-2 infection of epithelial cells. These effects of OM-85 appeared to depend on SARS-CoV-2 receptor downregulation. Conclusions: OM-85 inhibits SARS-CoV-2 epithelial cell infection in vitro by downregulating SARS-CoV-2 receptor expression. Further studies are warranted to assess whether OM-85 may prevent and/or reduce the severity of coronavirus disease 2019.
• Stansfield, B. N., Rangasamy, S., Ramsey, K., Khanna, M., & Churko, J. M. (2022). Generation of an iPSC line from a Pontocerebellar Hypoplasia 1B patient harboring a homozygous c.395 A > C mutation in EXOSC3 along with a family matched control. Stem Cell Research, 65(Issue). doi:10.1016/j.scr.2022.102944
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  Pontocerebellar Hypoplasia 1B (PCH1B) is a severe autosomal recessive neurological disorder that is associated with mutations in the exosome complex component RRP40 (EXOSC3) gene. We generated and characterized an iPSC line from an individual with PCH1B that harbors a recessive homozygous c.395 A > C mutation in EXOSC3 and a family matched control from the probands unaffected mother. Each iPSC line presents with normal morphology and karyotype and express high levels of pluripotent markers. UAZTi009-A and UAZTi011-A are capable of directed differentiation and can be used as a vital experimental tool to study the development of PCH1B.
• Vercelli, D., Nikolich-Zugich, J., Kraft, M., Anderson, D., Churko, J., Ezeh, P., Malone, S. P., Hahn, S., Conway, M. Y., VanLinden, S. R., Pivniouk, D., Michael, A., Uhrlaub, J. L., DeVries, A., Pivniouk, O., & Pivniouk, V. I. (2021). The OM-85 bacterial lysate inhibits SARS-CoV-2 infection of epithelial cells by downregulating SARS-CoV-2 receptor expression. The Journal of Allergy and Clinical Immunology. doi:10.1016/j.jaci.2021.11.019
• Yuen, M., Worgan, L., Iwanski, J., Pappas, C. T., Joshi, H., Churko, J. M., Arbuckle, S., Kirk, E. P., Zhu, Y., Roscioli, T., Gregorio, C. C., & Cooper, S. T. (2022). Neonatal-lethal dilated cardiomyopathy due to a homozygous LMOD2 donor splice-site variant. European Journal of Human Genetics, 30(Issue 4). doi:10.1038/s41431-022-01043-8
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  Dilated cardiomyopathy (DCM) is characterized by cardiac enlargement and impaired ventricular contractility leading to heart failure. A single report identified variants in leiomodin-2 (LMOD2) as a cause of neonatally-lethal DCM. Here, we describe two siblings with DCM who died shortly after birth due to heart failure. Exome sequencing identified a homozygous LMOD2 variant in both siblings, (GRCh38)chr7:g.123656237G > A; NM_207163.2:c.273 + 1G > A, ablating the donor 5′ splice-site of intron-1. Pre-mRNA splicing studies and western blot analysis on cDNA derived from proband cardiac tissue, MyoD-transduced proband skin fibroblasts and HEK293 cells transfected with LMOD2 gene constructs established variant-associated absence of canonically spliced LMOD2 mRNA and full-length LMOD2 protein. Immunostaining of proband heart tissue unveiled abnormally short actin-thin filaments. Our data are consistent with LMOD2 c.273 + 1G > A abolishing/reducing LMOD2 transcript expression by: (1) variant-associated perturbation in initiation of transcription due to ablation of the intron-1 donor; and/or (2) degradation of aberrant LMOD2 transcripts (resulting from use of alternative transcription start-sites or cryptic splice-sites) by nonsense-mediated decay. LMOD2 expression is critical for life and the absence of LMOD2 is associated with thin filament shortening and severe cardiac contractile dysfunction. This study describes the first splice-site variant in LMOD2 and confirms the role of LMOD2 variants in DCM.
• Churko, J. M., Chan, B. Y., Cho, W. J., Granzier, H., Keschrumrus, V., Poirier, M., Roczkowsky, A., Schulz, R., & Sergi, C. (2021). MMP inhibitors attenuate doxorubicin cardiotoxicity by preventing intracellular and extracellular matrix remodelling.. Cardiovascular research, 117(1), 188-200. doi:10.1093/cvr/cvaa017
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  Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix..Eight-week-old male C57BL/6J mice were treated with DXR weekly with or without MMP inhibitors doxycycline or ONO-4817 by daily oral gavage for 4 weeks. Echocardiography was used to determine cardiac function and left ventricular remodelling before and after treatment. MMP inhibitors ameliorated DXR-induced systolic and diastolic dysfunction by reducing the loss in left ventricular ejection fraction, fractional shortening, and E'/A'. MMP inhibitors attenuated adverse left ventricular remodelling, reduced cardiomyocyte dropout, and prevented myocardial fibrosis. DXR increased myocardial MMP-2 activity in part also by upregulating N-terminal truncated MMP-2. Immunogold transmission electron microscopy showed that DXR elevated MMP-2 levels within the sarcomere and mitochondria which were associated with myofilament lysis, mitochondrial degeneration, and T-tubule distention. DXR-induced myofilament lysis was associated with increased titin proteolysis in the heart which was prevented by ONO-4817. DXR also increased the level and activity of MMP-2 in human embryonic stem cell-derived cardiomyocytes, which was reduced by ONO-4817..MMP-2 activation is an early event in DXR cardiotoxicity and contributes to myofilament lysis by proteolyzing cardiac titin. Two orally available MMP inhibitors ameliorated DXR cardiotoxicity by attenuating intracellular and extracellular matrix remodelling, suggesting their use may be a potential prophylactic strategy to prevent heart injury during chemotherapy.
• 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(Issue 10). doi:10.1016/j.stemcr.2021.08.018
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  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.
• Beh, S. T., Frisch, C., Brafman, D. A., Churko, J., Walker, J. E., Serrano, G. E., Sue, L. I., Reiman, E. M., Beach, T. G., & Lue, L. F. (2020). Human autopsy-derived scalp fibroblast biobanking for age-related neurodegenerative disease research. Cells, 9(Issue 11). doi:10.3390/cells9112383
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  The Arizona Study of Aging and Neurodegenerative Disorders/Brain and Body Donation Program at Banner Sun Health Research Institute (BSHRI) is a longitudinal clinicopathological study with a current enrollment of more than 900 living subjects for aging and neurodegenerative disease research. Annual clinical assessments are done by cognitive and movement neurologists and neuropsychologists. Brain and body tissues are collected at a median postmortem interval of 3.0 h for neuropathological diagnosis and banking. Since 2018, the program has undertaken banking of scalp fibroblasts derived from neuropathologically characterized donors with Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative diseases. Here, we describe the procedure development and cell characteristics from 14 male and 15 female donors (mean ± SD of age: 83.6 ± 12.2). Fibroblasts from explant cultures were banked at passage 3. The results of mRNA analysis showed positive expression of fibroblast activation protein, vimentin, fibronectin, and THY1 cell surface antigen. We also demonstrated that the banked fibroblasts from a postmortem elderly donor were successfully reprogramed to human-induced pluripotent stem cells (hiPSCs). Taken together, we have demonstrated the successful establishment of a human autopsy-derived fibroblast banking program. The cryogenically preserved cells are available for request at the program website of the BSHRI.
• Jabart, E., Molho, J., Sin, K., Stansfield, B., Kazmouz, S. G., Ventro, D., Gardner, K., Wu, J. C., & Churko, J. M. (2020). Single-cell protein expression of hiPSC-derived cardiomyocytes using Single-Cell Westerns. Journal of Molecular and Cellular Cardiology, 149(Issue). doi:10.1016/j.yjmcc.2020.09.012
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  The ability to reprogram human somatic cells into human induced pluripotent stem cells (hiPSCs) has enabled researchers to generate cell types in vitro that have the potential to faithfully recapitulate patient-specific disease processes and phenotypes. hiPSC-derived cardiomyocytes (hiPSC-CMs) offer the promise of in vitro patient- and disease-specific models for drug testing and the discovery of novel therapeutic approaches for treating cardiovascular diseases. While methods to differentiate hiPSCs into cardiomyocytes have been demonstrated, the heterogeneity and immaturity of these differentiated populations have restricted their potential in reproducing human disease and the associated target cell phenotypes. These barriers may be overcome through comprehensive single-cell characterization to dissect the rich heterogeneity of hiPSC-CMs and to study the source of varying cell fates. In this study, we optimized and validated a new Single-Cell Western method to assess protein expression in hiPSC-CMs. To better understand distinct subpopulations generated from cardiomyocyte differentiations and to track populations at single-cell resolution over time, we measured and quantified the expression of cardiomyocyte subtype-specific proteins (MLC2V and MLC2A) using Single-Cell Westerns. By understanding their heterogeneity through single-cell protein expression and quantification, we may improve upon current cardiomyocyte differentiation protocols, generate hiPSC-CMs that are more representative of in vivo derived cardiomyocytes for disease modeling, and utilize hiPSC-CMs for regenerative medicine purposes. Single-Cell Westerns provide a robust platform for protein expression analysis at single-cell resolution.
• Lee, J., Termglinchan, V., Diecke, S., Itzhaki, I., Lam, C. K., Garg, P., Lau, E., Greenhaw, M., Seeger, T., Wu, H., Zhang, J. Z., Chen, X., Gil, I. P., Ameen, M., Sallam, K., Rhee, J. W., Churko, J. M., Chaudhary, R., Chour, T., , Wang, P. J., et al. (2019). Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy. Nature, 572(Issue 7769). doi:10.1038/s41586-019-1406-x
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  Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-β (PDGFRB) as a potential therapeutic target.
• Churko, J. M. (2018). Linking Clinical Parameters and Genotype in Dilated Cardiomyopathy. Circulation. Heart failure, 11(11), e005459.
• Churko, J. M. (2018). Linking Clinical Parameters and Genotype in Dilated Cardiomyopathy. Circulation. Heart failure, 11(Issue 11). doi:10.1161/circheartfailure.118.005459
• Churko, J. M., Garg, P., Treutlein, B., Venkatasubramanian, M., Wu, H., Lee, J., Wessells, Q. N., Chen, S. Y., Chen, W. Y., Chetal, K., Mantalas, G., Neff, N., Jabart, E., Sharma, A., Nolan, G. P., Salomonis, N., & Wu, J. C. (2018). Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis. Nature Communications, 9(Issue 1). doi:10.1038/s41467-018-07333-4
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  Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a powerful tool for human disease modeling and therapeutic testing. However, their use remains limited by their immaturity and heterogeneity. To characterize the source of this heterogeneity, we applied complementary single-cell RNA-seq and bulk RNA-seq technologies over time during hiPSC cardiac differentiation and in the adult heart. Using integrated transcriptomic and splicing analysis, more than half a dozen distinct single-cell populations were observed, several of which were coincident at a single time-point, day 30 of differentiation. To dissect the role of distinct cardiac transcriptional regulators associated with each cell population, we systematically tested the effect of a gain or loss of three transcription factors (NR2F2, TBX5, and HEY2), using CRISPR genome editing and ChIP-seq, in conjunction with patch clamp, calcium imaging, and CyTOF analysis. These targets, data, and integrative genomics analysis methods provide a powerful platform for understanding in vitro cellular heterogeneity.
• Churko, J. M., Garg, P., Treutlein, B., Venkatasubramanian, M., Wu, H., Lee, J., Wessells, Q. N., Chen, S. Y., Chen, W. Y., Chetal, K., Mantalas, G., Neff, N., Jabart, E., Sharma, A., Nolan, G. P., Salomonis, N., & Wu, J. C. (2018). Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis. Nature communications, 9(1), 4906.
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  Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a powerful tool for human disease modeling and therapeutic testing. However, their use remains limited by their immaturity and heterogeneity. To characterize the source of this heterogeneity, we applied complementary single-cell RNA-seq and bulk RNA-seq technologies over time during hiPSC cardiac differentiation and in the adult heart. Using integrated transcriptomic and splicing analysis, more than half a dozen distinct single-cell populations were observed, several of which were coincident at a single time-point, day 30 of differentiation. To dissect the role of distinct cardiac transcriptional regulators associated with each cell population, we systematically tested the effect of a gain or loss of three transcription factors (NR2F2, TBX5, and HEY2), using CRISPR genome editing and ChIP-seq, in conjunction with patch clamp, calcium imaging, and CyTOF analysis. These targets, data, and integrative genomics analysis methods provide a powerful platform for understanding in vitro cellular heterogeneity.
• Shao, N., Paik, D. T., Wu, H., Guo, H., Termglinchan, V., Churko, J. M., Kim, Y., Kitani, T., Zhao, M., Zhang, Y., Wilson, K. D., Karakikes, I., Snyder, M. P., Wu, J. C., & Lee, J. C. (2018). SETD7 Drives Cardiac Lineage Commitment through Stage-Specific Transcriptional Activation.. Cell stem cell, 22(3), 428-444.e5. doi:10.1016/j.stem.2018.02.005
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  Cardiac development requires coordinated and large-scale rearrangements of the epigenome. The roles and precise mechanisms through which specific epigenetic modifying enzymes control cardiac lineage specification, however, remain unclear. Here we show that the H3K4 methyltransferase SETD7 controls cardiac differentiation by reading H3K36 marks independently of its enzymatic activity. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that SETD7 targets distinct sets of genes to drive their stage-specific expression during cardiomyocyte differentiation. SETD7 associates with different co-factors at these stages, including SWI/SNF chromatin-remodeling factors during mesodermal formation and the transcription factor NKX2.5 in cardiac progenitors to drive their differentiation. Further analyses revealed that SETD7 binds methylated H3K36 in the bodies of its target genes to facilitate RNA polymerase II (Pol II)-dependent transcription. Moreover, abnormal SETD7 expression impairs functional attributes of terminally differentiated cardiomyocytes. Together, these results reveal how SETD7 acts at sequential steps in cardiac lineage commitment, and they provide insights into crosstalk between dynamic epigenetic marks and chromatin-modifying enzymes.
• Sharma, A., Zhang, Y., Buikema, J. W., Serpooshan, V., Chirikian, O., Kosaric, N., Churko, J. M., Dzilic, E., Shieh, A., Burridge, P. W., Wu, J. C., & Wu, S. M. (2018). Stage-specific Effects of Bioactive Lipids on Human iPSC Cardiac Differentiation and Cardiomyocyte Proliferation.. Scientific reports, 8(1), 6618. doi:10.1038/s41598-018-24954-3
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  Bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate diverse processes including cell proliferation, differentiation, and migration. However, their roles in cardiac differentiation and cardiomyocyte proliferation have not been explored. Using a 96-well differentiation platform for generating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) we found that S1P and LPA can independently enhance cardiomyocyte generation when administered at an early stage of differentiation. We showed that the combined S1P and LPA treatment of undifferentiated hiPSCs resulted in increased nuclear accumulation of β-catenin, the canonical Wnt signaling pathway mediator, and synergized with CHIR99021, a glycogen synthase kinase 3 beta inhibitor, to enhance mesodermal induction and subsequent cardiac differentiation. At later stages of cardiac differentiation, the addition of S1P and LPA resulted in cell cycle initiation in hiPSC-CMs, an effect mediated through increased ERK signaling. Although the addition of S1P and LPA alone was insufficient to induce cell division, it was able to enhance β-catenin-mediated hiPSC-CM proliferation. In summary, we demonstrated a developmental stage-specific effect of bioactive lipids to enhance hiPSC-CM differentiation and proliferation via modulating the effect of canonical Wnt/β-catenin and ERK signaling. These findings may improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.
• Wnorowski, A., Sharma, A., Chen, H., Wu, H., Shao, N., Churko, J. M., Matsa, E., Countryman, S., Wu, S. M., Lee, P. H., Wu, J. C., & Rubins, K. H. (2018). Abstract 379: Effects of Microgravity on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function. Circulation Research, 123(Suppl_1). doi:10.1161/res.123.suppl_1.379
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  With extended stays aboard the International Space Station (ISS) becoming commonplace as humanity prepares for exploration-class space missions, the need to better understand the effects of cardiac function during spaceflight is critical. However, primary human heart tissues, which would be useful for in vitro studies on heart function, are difficult to obtain and maintain. As a model system, we utilized cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) to study the effects of microgravity on human cardiac function and gene expression at the cellular level. We derived hiPSCs from three healthy volunteers and produced hiPSC-CMs using a high-efficiency hiPSC-CM differentiation protocol. We cultured hiPSC-CMs in a microgravity environment aboard the ISS for approximately one month, during which weekly media changes were conducted. We analyzed the gene expression, structure, and function of flight, post-flight, and ground control hiPSC-CM samples using RNA-sequencing, immunofluorescence, calcium imaging, and contractility assessment. Exposure to microgravity on the ISS resulted in decreased contractile velocity and calcium recycling in the hiPSC-CM, along with increased beating irregularities. RNA-sequencing analysis demonstrated that there were 2635 genes differentially expressed with p≤0.05 between flight, post-flight, and ground control samples. These included genes involved in metabolism, DNA/RNA modification, and molecular transport, indicating that these pathways may be permanently altered by long-term space flight . This study represents the first time that hiPSC technology has been used to study the effects of microgravity on human cardiac function and is the first to demonstrate that microgravity affects human heart function on the cellular level.
• Churko, J. M., Ameen, M., Gu, M., Venkatasubramanian, M., Diecke, S., Sallam, K., Im, H., Wang, G., Gold, J. D., Salomonis, N., Snyder, M. P., Wu, J. C., & Lee, J. C. (2017). Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods.. Nature biomedical engineering, 1(10), 826-837. doi:10.1038/s41551-017-0141-6
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  Many reprogramming methods can generate human induced pluripotent stem cells (hiPSCs) that closely resemble human embryonic stem cells (hESCs). This has led to assessments of how similar hiPSCs are to hESCs, by evaluating differences in gene expression, epigenetic marks and differentiation potential. However, all previous studies were performed using hiPSCs acquired from different laboratories, passage numbers, culturing conditions, genetic backgrounds and reprogramming methods, all of which may contribute to the reported differences. Here, by using high-throughput sequencing under standardized cell culturing conditions and passage number, we compare the epigenetic signatures (H3K4me3, H3K27me3 and HDAC2 ChIP-seq profiles) and transcriptome differences (by RNA-seq) of hiPSCs generated from the same primary fibroblast population by using six different reprogramming methods. We found that the reprogramming method impacts the resulting transcriptome and that all hiPSC lines could terminally differentiate, regardless of the reprogramming method. Moreover, by comparing the differences between the hiPSC and hESC lines, we observed a significant proportion of differentially expressed genes that could be attributed to polycomb repressive complex targets.
• Sallam, K., Haodi, W., Sayed, N., Rhee, J., Churko, J. M., Chen, I. Y., Wnorowski, A., Termglinchan, V., & Wu, J. C. (2017). Abstract 20145: Anemone Toxin II Unmasks Dilated Cardiomyopathy Phenotype in Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model. Circulation, 136.
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  Introduction: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have been used to as a cellular model of cardiac disorders including dilated cardiomyopathy (DCM) demonstrating excelle...
• Sharma, A., Burridge, P. W., Mckeithan, W. L., Serrano, R., Sayed, N., Churko, J. M., Kitani, T., Wu, H., Holmstrom, A., Matsa, E., Zhang, Y., Kumar, A., Fan, A. C., Alamo, J. C., Wu, S. M., Moslehi, J. J., Mercola, M., Wu, J. C., & Shukla, P. K. (2017). High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.. Science translational medicine, 9(377). doi:10.1126/scitranslmed.aaf2584
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  Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
• He, C., Hu, H., Wilson, K. D., Wu, H., Feng, J., Xia, S., Qu, K., Chang, H. Y., Wu, J. C., & Churko, J. M. (2016). Systematic Characterization of Long Noncoding RNAs Reveals the Contrasting Coordination of Cis- and Trans-Molecular Regulation in Human Fetal and Adult Hearts.. Circulation. Cardiovascular genetics, 9(2), 110-8. doi:10.1161/circgenetics.115.001264
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  The molecular regulation of heart development is regulated by cis- and trans-factors acting on the genome and epigenome. As a class of important regulatory RNAs, the role of long noncoding RNAs (lncRNAs) in human heart development is still poorly understood. Furthermore, factors that interact with lncRNAs in this process are not well characterized..Using RNA sequencing, we systematically define the contrasting lncRNA expression patterns between fetal and adult hearts. We report that lncRNAs upregulated in adult versus fetal heart have different sequence features and distributions. For example, the adult heart expresses more sense lncRNAs compared with fetal heart. We also report the coexpression of lncRNAs and neighboring coding genes that have important functions in heart development. Importantly, the regulation of lncRNA expression during fetal to adult heart development seems to be due, in part, to the coordination of specific developmental epigenetic modifications, such as H3K4me1 and H3k4me3. The expression of promoter-associated lncRNAs in adult and fetal hearts also seems to be related to these epigenetic states. Finally, transcription factor-binding analysis suggests that lncRNAs are directly regulating cardiac gene expression during development..We provide a systematic analysis of lncRNA control of heart development that gives clues to the roles that specific lncRNAs play in fetal and adult hearts.
• Kodo, K., Ong, S., Jahanbani, F., Termglinchan, V., Hirono, K., Inanloorahatloo, K., Ebert, A. D., Abilez, O. J., Churko, J. M., Karakikes, I., Jung, G., Ichida, F., Wu, S. M., Snyder, M. P., Bernstein, D., Wu, J. C., & Shukla, P. K. (2016). iPSC-derived cardiomyocytes reveal abnormal TGF-β signalling in left ventricular non-compaction cardiomyopathy.. Nature cell biology, 18(10), 1031-42. doi:10.1038/ncb3411
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  Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.
• Liang, P., Sallam, K., Wu, H., Li, Y., Itzhaki, I., Garg, P., Zhang, Y., Vermglinchan, V., Lan, F., Gu, M., Gong, T., Zhuge, Y., He, C., Ebert, A. D., Sanchez-freire, V., Hu, S., Sharma, A., Lam, C. K., Scheinman, M. M., , Bers, D. M., et al. (2016). Patient-Specific and Genome-Edited Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Brugada Syndrome.. Journal of the American College of Cardiology, 68(19), 2086-2096. doi:10.1016/j.jacc.2016.07.779
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  Brugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder..The objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)..This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs..BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression..Patient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.
• Matsa, E., Burridge, P. W., Yu, K., Ahrens, J. H., Termglinchan, V., Wu, H., Liu, C., Sayed, N., Churko, J. M., Shao, N., Woo, N. A., Chao, A. S., Gold, J. D., Karakikes, I., Snyder, M. P., Wu, J. C., & Shukla, P. K. (2016). Transcriptome Profiling of Patient-Specific Human iPSC-Cardiomyocytes Predicts Individual Drug Safety and Efficacy Responses In Vitro.. Cell stem cell, 19(3), 311-25. doi:10.1016/j.stem.2016.07.006
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  Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.
• Shukla, P. K., Churko, J. M., Matsa, E., Shao, N., Abilez, O. J., Wu, J. C., & Lam, C. K. (2016). Abstract 19048: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Hypokalemia Induced Exacerbation of Ventricular Arrhythmogenicity of Antiarrhythmic Drugs. Circulation.
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  Hypokalemia is an independent risk factor associated with an increased risk of arrhythmia and all-cause mortality in patients with cardiovascular disease and exacerbates the cardiac arrhythmia (i.e...
• Abilez, O. J., Bernstein, D., Churko, J. M., Ebert, A. D., Inanloorahatloo, K., Jahanbani, F., Jung, G., Karakikes, I., Kodo, K., Ong, S., Shukla, P., Snyder, M. P., Termglinchan, V., & Wu, J. C. (2015). Abstract 248: Aberrant TGFβ Signaling as an Etiology of Left Ventricular Non-compaction Cardiomyopathy. Circulation Research, 117(suppl_1). doi:10.1161/res.117.suppl_1.248
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  Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and has a unique phenotype with characteristically extensive hypertrabeculation of the left ventricle, similar to the embryonic left ventricle, suggesting a developmental defect of the embryonic myocardium. However, studying this disease has been challenging due to the lack of an animal model that can faithfully recapitulate the clinical phenotype of LVNC. To address this, we showed that patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from a family with LVNC history recapitulated a developmental defect consistent with the LVNC phenotype at the single-cell level. We then utilized hiPSC-CMs to show that increased transforming growth factor beta (TGFβ) signaling is one of the central mechanisms underlying the pathogenesis of LVNC. LVNC hiPSC-CMs demonstrated decreased proliferative capacity due to abnormal activation of TGFβ signaling (Figs A-B). Exome sequencing demonstrated a mutation in TBX20, which regulates TGFβ signaling through upregulation of ITGAV, contributing to the LVNC phenotype. Inhibition of abnormal TGFβ signaling or genetic correction of the TBX20 mutation (Figs C-D) using TALEN reversed the proliferation defects seen in LVNC hiPSC-CMs. Our results demonstrate that hiPSC-CMs are a useful tool for the exploration of novel mechanisms underlying poorly understood cardiomyopathies such as LVNC. Here we provide the first evidence of activation of TGFβ signaling as playing a role in the pathogenesis of LVNC.
• Altman, R. B., Chan, C., Churko, J. M., Fleischmann, D., Hong, W. X., Hu, S., Lee, W. H., Li, Y. F., Liang, G., Nguyen, P. K., Nguyen, I., Ong, S., Wang, J., & Wu, J. C. (2015). Erratum: Assessment of the radiation effects of cardiac CT angiography using protein and genetic biomarkers (JACC: Cardiovascular Imaging (2015) 8 (873-84)). Jacc-cardiovascular Imaging, 8(11), 1350. doi:10.1016/j.jcmg.2015.10.001
• Churko, J. M., Bers, D. M., Engler, A. J., Gold, J. D., Gu, M., Lan, F., Lee, J. C., Matsa, E., Sallam, K. I., Sharma, A., Vincent, L. G., Wang, Q., Wu, J. C., Wu, H., & Xiang, Y. K. (2015). Epigenetic Regulation of Phosphodiesterases 2A and 3A Underlies Compromised β-Adrenergic Signaling in an iPSC Model of Dilated Cardiomyopathy.. Cell stem cell, 17(1), 89-100. doi:10.1016/j.stem.2015.04.020
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  β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
• Diecke, S., Lu, J., Termglinchan, V., Kooreman, N. G., Burridge, P. W., Ebert, A. D., Churko, J. M., Sharma, A., Kay, M. A., Wu, J. C., & Lee, J. C. (2015). Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency.. Scientific reports, 5(1), 8081. doi:10.1038/srep08081
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  The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.
• Gu, M., Mordwinkin, N. M., Kooreman, N. G., Wu, H., Hu, S., Churko, J. M., Diecke, S., Burridge, P. W., He, C., Barron, F. E., Ong, S., Gold, J. D., Wu, J. C., & Lee, J. C. (2015). Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism.. European heart journal, 36(13), 806-16. doi:10.1093/eurheartj/ehu411
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  High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays..Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester..This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.
• Kodo, K., Ong, S., Jahanbani, F., Termglinchan, V., Keiichi, H., Inanloorahatloo, K., Ebert, A. D., Shukla, P., Abilez, O. J., Churko, J. M., Karakikes, I., Jung, G., Ichida, F., Snyder, M., Bernstein, D., & Wu, J. C. (2015). Abstract 12501: Abnormal Activation of TGFβ Signaling as a Pathogenesis of Left Ventricular Non-compaction Cardiomyopathy. Circulation, 132.
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  Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and has a unique phenotype with characteristically extensive hypertrabeculation of the left ventricle, similar to the embryonic left ventricle, suggesting a developmental defect of the embryonic myocardium. However, in-depth investigation of this disease has been challenging due to the lack of animal models that can faithfully recapitulate the clinical phenotype of LVNC. In this study, we demonstrated that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in cardiac transcription factor TBX20 recapitulated a developmental defect consistent with the LVNC phenotype at the single-cell level. We first performed genetic tests and identified two TBX20 mutations, Y317* and T262M from multiple members in a family with LVNC history and one isolated patient. We then utilized iPSC-CMs to show that increased transforming growth factor beta (TGFβ) signaling is one of the central mechanisms underlying the pathogenesis of LVNC caused by TBX20 mutation. LVNC iPSC-CMs demonstrated decreased proliferative capacity due to abnormal activation of TGFβ signaling (Figs. A-B). TBX20 regulated the expression of TGFβ signaling modifiers including ITGAV and the Smad cofactor, PRDM16, which is a known genetic cause of LVNC. Genome editing of PRDM16 in control iPSC-CMs led to proliferation defects as seen in LVNC iPSC-CMs. Inhibition of abnormal TGFβ signaling by TGFβ receptor-1 inhibitors or ITGAV inhibitor, or genetic correction of the TBX20 mutation using TALEN reversed the proliferation defects seen in LVNC iPSC-CMs (Figs. C-D). Our results demonstrate that iPSC-CMs are a useful tool for the exploration of novel mechanisms underlying poorly understood cardiomyopathies such as LVNC. Here we provide the first evidence of activation of TGFβ signaling as playing a role in the pathogenesis of LVNC.
• Nguyen, P. K., Lee, W. H., Li, Y. F., Hong, W. X., Hu, S., Chan, C., Liang, G., Nguyen, I., Ong, S., Wang, J., Altman, R. B., Fleischmann, D., Wu, J. C., & Churko, J. M. (2015). Assessment of the Radiation Effects of Cardiac CT Angiography Using Protein and Genetic Biomarkers.. JACC. Cardiovascular imaging, 8(8), 873-84. doi:10.1016/j.jcmg.2015.04.016
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  The purpose of this study was to evaluate whether radiation exposure from cardiac computed tomographic angiography (CTA) is associated with deoxyribonucleic acid (DNA) damage and whether damage leads to programmed cell death and activation of genes involved in apoptosis and DNA repair..Exposure to radiation from medical imaging has become a public health concern, but whether it causes significant cell damage remains unclear..We conducted a prospective cohort study in 67 patients undergoing cardiac CTA between January 2012 and December 2013 in 2 U.S. medical centers. Median blood radiation exposure was estimated using phantom dosimetry. Biomarkers of DNA damage and apoptosis were measured by flow cytometry, whole genome sequencing, and single cell polymerase chain reaction..The median dose length product was 1,535.3 mGy·cm (969.7 to 2,674.0 mGy·cm). The median radiation dose to the blood was 29.8 mSv (18.8 to 48.8 mSv). Median DNA damage increased 3.39% (1.29% to 8.04%, p < 0.0001) and median apoptosis increased 3.1-fold (interquartile range [IQR]: 1.4- to 5.1-fold, p < 0.0001) post-radiation. Whole genome sequencing revealed changes in the expression of 39 transcription factors involved in the regulation of apoptosis, cell cycle, and DNA repair. Genes involved in mediating apoptosis and DNA repair were significantly changed post-radiation, including DDB2 (1.9-fold [IQR: 1.5- to 3.0-fold], p < 0.001), XRCC4 (3.0-fold [IQR: 1.1- to 5.4-fold], p = 0.005), and BAX (1.6-fold [IQR: 0.9- to 2.6-fold], p < 0.001). Exposure to radiation was associated with DNA damage (odds ratio [OR]: 1.8 [1.2 to 2.6], p = 0.003). DNA damage was associated with apoptosis (OR: 1.9 [1.2 to 5.1], p < 0.0001) and gene activation (OR: 2.8 [1.2 to 6.2], p = 0.002)..Patients exposed to >7.5 mSv of radiation from cardiac CTA had evidence of DNA damage, which was associated with programmed cell death and activation of genes involved in apoptosis and DNA repair.
• Anversa, P., Churko, J., Czarna, A., Kim, J., Leri, A., Pereira, J. D., Rota, M., Sanada, F., & Wu, J. C. (2014). Abstract 235: Molecular Signature Of Cardiomyogenic c-kit-positive Bone Marrow Cells. Circulation Research, 115(suppl_1). doi:10.1161/res.115.suppl_1.235
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  Conflicting results have been reported concerning the ability of c-kit-positive bone marrow cells (c-kit-BMCs) to transdifferentiate into cardiomyocytes in vivo. We have raised the possibility that c-kit-BMCs may constitute a functionally heterogeneous pool, containing cells with different cardiomyogenic potential. To test this hypothesis, clonal analysis of individual c-kit-BMCs was introduced to track in vivo the progeny derived from a single founder cell. By employing viral gene-tagging, only a limited fraction of clonal c-kit-BMC was found to be able to generate cardiomyocytes in the acutely infarcted heart. On this basis, c-kit-BMC-derived clones were classified as myogenic or non-myogenic. The gene profile of these two categories of cells was studied by RNA-sequencing to determine whether myogenic c-kit-BMCs had a distinct molecular signature. Genes that showed a significant (p
• Asimaki, A., Kapoor, S., Plovie, E., Adams, E., Liu, Z., James, C. A., Judge, D. P., Calkins, H., Wu, J. C., Macrae, C. A., Kleber, A. G., Saffitz, J. E., Arndt, A. K., & Churko, J. M. (2014). Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy.. Science translational medicine, 6(240), 240ra74. doi:10.1126/scitranslmed.3008008
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  Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte-specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in I(Na) and I(K1) current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities.
• Asimaki, A., Plovie, E., Adams, E., Liu, Z., James, C. A., Judge, D. P., Calkins, H., Churko, J. M., Wu, J. C., Macrae, C. A., Kleber, A. G., Saffitz, J. E., Arndt, A. K., & Kapoor, S. (2014). Erratum: "Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy" (Science Translational Medicine (2014) 6:261 (261er6)). Science Translational Medicine, 6(261). doi:10.1126/scitranslmed.aaa1333
• Burridge, P. W., Matsa, E., Lin, Z. C., Churko, J. M., Ebert, A. D., Lan, F., Diecke, S., Mordwinkin, N. M., Plews, J. R., Abilez, O. J., Cui, B., Gold, J. D., Wu, J. C., Huber, B. C., & Shukla, P. K. (2014). Chemically defined generation of human cardiomyocytes.. Nature methods, 11(8), 855-60. doi:10.1038/nmeth.2999
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  Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.
• Chen, H. I., Poduri, A., Kivela, R., Saharinen, P., Mckay, A. S., Tian, X., Zhou, B., Wu, J. C., Alitalo, K., Red-horse, K., Churko, J. M., Numi, H. J., & Raftrey, B. C. (2014). VEGF-C and aortic cardiomyocytes guide coronary artery stem development.. The Journal of clinical investigation, 124(11), 4899-914. doi:10.1172/jci77483
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  Coronary arteries (CAs) stem from the aorta at 2 highly stereotyped locations, deviations from which can cause myocardial ischemia and death. CA stems form during embryogenesis when peritruncal blood vessels encircle the cardiac outflow tract and invade the aorta, but the underlying patterning mechanisms are poorly understood. Here, using murine models, we demonstrated that VEGF-C-deficient hearts have severely hypoplastic peritruncal vessels, resulting in delayed and abnormally positioned CA stems. We observed that VEGF-C is widely expressed in the outflow tract, while cardiomyocytes develop specifically within the aorta at stem sites where they surround maturing CAs in both mouse and human hearts. Mice heterozygous for islet 1 (Isl1) exhibited decreased aortic cardiomyocytes and abnormally low CA stems. In hearts with outflow tract rotation defects, misplaced stems were associated with shifted aortic cardiomyocytes, and myocardium induced ectopic connections with the pulmonary artery in culture. These data support a model in which CA stem development first requires VEGF-C to stimulate vessel growth around the outflow tract. Then, aortic cardiomyocytes facilitate interactions between peritruncal vessels and the aorta. Derangement of either step can lead to mispatterned CA stems. Studying this niche for cardiomyocyte development, and its relationship with CAs, has the potential to identify methods for stimulating vascular regrowth as a treatment for cardiovascular disease.
• Ebert, A. D., Kodo, K., Liang, P., Wu, H., Huber, B. C., Riegler, J., Lan, F., Diecke, S., Burridge, P. W., Gold, J. D., Mochly-rosen, D., Wu, J. C., Almeida, P. D., Churko, J. M., & Lee, J. C. (2014). Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system.. Science translational medicine, 6(255), 255ra130. doi:10.1126/scitranslmed.3009027
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  Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.
• Nguyen, P. K., Hu, S., Ong, S., Churko, J. M., Chan, C., Wang, J., Longaker, M. T., Hlatky, M. A., Fleischmann, D., Wu, J. C., Hong, W. X., & Lee, W. H. (2014). Abstract 17674: Cell Damage and Even Death Detected in Peripheral T-Lymphocytes in Patients Undergoing Cardiac Computed Tomographic Angiography. Circulation, 130.
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  Background: It is well known that radiation can cause cell damage and even cell death at high doses. Whether a similar albeit less pronounced effect exists at low dose remains unclear. Objective: The purpose of this study is to determine whether low dose radiation from cardiac computed tomographic angiography (CCTA) is associated with cellular damage. Methods: Blood was collected from 56 adult patients immediately before and after CCTA. Changes in protein phosphorylation and gene expression were analyzed by quantitative flow cytometry and single cell gene expression profiling, respectively. The presence of cell death was analyzed by flow cytometry in a subset of patients (n=25). Results: Activation of multiple proteins and genes were detected by quantitative flow cytometry and single cell expression profiling after exposure to ≥25 mSv of radiation in vitro. Similarly, blood collected from patients undergoing CCTA showed increased protein (i.e., H2AX, p53, and ATM) and gene (i.e., p53, BAX, MDM2, CDK4, ATF6, and DDB2) activation. A linear-dose response relationship was found in phosphorylated ATM, CDK4 and DDB2 in vivo. Sixty-eight percent (n=17/25; average % cell death 1.4%±0.9%; n=100,000 events) of patients had evidence of T-lymphocyte death after exposure. Patients with DNA damage (i.e. affected patients) as indicated by phosphorylation of H2AX, ATM and/or p53 had more cell death than those without DNA damage (3.7 vs. 1.09 fold, p=0.005) (Figure 1A). Patients with increased cell death were exposed to higher amounts of radiation (38.5 ± 18.4 mSv vs. 15.1 ± 13.4 mSv, p=0.004). With the exception of two patients, most patients who had significant cell death (i.e., >2 fold) had exposure to at least 20 mSv of radiation (Figure 1B). Conclusion: Radiation exposure from CCTA activates multiple proteins and genes involved in the DNA damage response pathway. Cell death was detected in over half of the patients although the magnitude was small. ![][1] [1]: /embed/graphic-1.gif
• Penuela, S., Kelly, J. J., Churko, J. M., Barr, K. J., Berger, A. C., & Laird, D. W. (2014). Panx1 regulates cellular properties of keratinocytes and dermal fibroblasts in skin development and wound healing.. The Journal of investigative dermatology, 134(7), 2026-2035. doi:10.1038/jid.2014.86
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  Pannexin1 (Panx1), a channel-forming glycoprotein is expressed in neonatal but not in aged mouse skin. Histological staining of Panx1 knockout (KO) mouse skin revealed a reduction in epidermal and dermal thickness and an increase in hypodermal adipose tissue. Following dorsal skin punch biopsies, mutant mice exhibited a significant delay in wound healing. Scratch wound and proliferation assays revealed that cultured keratinocytes from KO mice were more migratory, whereas dermal fibroblasts were more proliferative compared with controls. In addition, collagen gels populated with fibroblasts from KO mice exhibited significantly reduced contraction, comparable to WT fibroblasts treated with the Panx1 blocker, probenecid. KO fibroblasts did not increase α-smooth muscle actin expression in response to TGF-β, as is the case for differentiating WT myofibroblasts during wound contraction. We conclude that Panx1 controls cellular properties of keratinocytes and dermal fibroblasts during early stages of skin development and modulates wound repair upon injury.
• Sharma, A., Hamaguchi, R., Burridge, P. W., Rajarajan, K., Churko, J. M., Wu, H., Sallam, K. I., Matsa, E., Sturzu, A. C., Che, Y., Diecke, S., Liang, P., Red-horse, K., Carette, J. E., Wu, S. M., Wu, J. C., Ebert, A. D., & Marceau, C. D. (2014). Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.. Circulation research, 115(6), 556-66. doi:10.1161/circresaha.115.303810
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  Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection..This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy..hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonβ1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonβ1 treatment..This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
• Wu, H., Gu, M., Lan, F., Churko, J. M., Sanchez-freire, V., Matsa, E., Ebert, A. D., Sallam, K., Gold, J. D., Wu, J. C., & Lee, J. C. (2014). Abstract 20459: Epigenetic Activation of Phosphodiesterase Subtypes Lead to Compromised Beta-Adrenergic Signaling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes From Dilated Cardiomyopathy Patients. Circulation, 130.
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  Introduction: Familial dilated cardiomyopathy (DCM) has been modeled by human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, the mechanisms of compromised signaling transduction and contractile function in DCM iPSC-CMs are still not well understood. Methods and Results: Beating iPSC-CMs were generated from healthy individuals and DCM patients. RNA-seq and real-time PCR showed strict regulation of the main beta-adrenergic signaling proteins in iPSC-CMs during maturation. Confocal imaging of spontaneous calcium activity and hydrogel-based traction force microscopy (TFM) technology demonstrated beta-adrenergic stimulation induced both inotropic and chronotropic regulation in the contractility of iPSC-CMs. Following extended in vitro maturation of iPSC-CMs, we observed a transition in the beta-adrenergic receptor (beta-AR) subtype dependence from beta-2 AR dominance at early stage (day 30) to beta-1/2 AR co-existence at late stage (day 60). Comparison of the beta-adrenergic responsiveness between iPSC-CMs from DCM patients and their familial controls showed compromised beta-adrenergic signaling in DCM cells. Microarray data and expression profiling indicated up-regulated phosphodiesterases (PDE) 2A, 3A and 5A in DCM iPSC-CMs, which impaired cAMP generation and blunted the beta-adrenergic response. By blocking PDE2A, 3A or 5A, beta-adrenergic signaling reactivity and contractile function in DCM iPSC-CMs were both greatly improved. To further elucidate underlying mechanism of PDE regulation, we conducted chromatin immunoprecipitation (CHIP) assays, which showed significant up-regulation of activation histone marker and down-regulation of repressive histone marker in the PDE promoters during maturation process of DCM iPSC-CMs, which closely recapitulated the epigenetic modulation in the ventricle tissues harvested from DCM patients. Conclusions: Patient-specific DCM iPSC-CMs recapitulated impaired beta-adrenergic responsiveness and contractility in diseased heart. Studies on iPSC-CM model revealed a novel epigenetic mechanism that underlies PDE subtype specific regulation and signaling deficiency in DCM pathogenesis, which may serve as a new therapeutic target in the future.
• Churko, J. M., & Laird, D. W. (2013). Gap junction remodeling in skin repair following wounding and disease.. Physiology (Bethesda, Md.), 28(3), 190-8. doi:10.1152/physiol.00058.2012
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  In the present review, we provide an overview of connexin expression during skin development and remodeling in wound healing, and reflect on how loss- or gain-of-function connexin mutations may change cellular phenotypes and lead to diseases of the skin. We also consider the therapeutic value of targeting connexins in wound healing.
• Churko, J. M., Burridge, P. W., & Wu, J. C. (2013). Generation of human iPSCs from human peripheral blood mononuclear cells using non-integrative Sendai virus in chemically defined conditions.. Methods in molecular biology (Clifton, N.J.), 1036, 81-8. doi:10.1007/978-1-62703-511-8_7
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  Human-induced pluripotent stem cells (hiPSCs) have received enormous attention because of their ability to differentiate into multiple cell types that demonstrate the patient's original phenotype. The use of hiPSCs is particularly valuable to the study of cardiac biology, as human cardiomyocytes are difficult to isolate and culture and have a limited proliferative potential. By deriving iPSCs from patients with heart disease and subsequently differentiating these hiPSCs to cardiomyocytes, it is feasible to study cardiac biology in vitro and model cardiac diseases. While there are many different methods for deriving hiPSCs, clinical use of these hiPSCs will require derivation by methods that do not involve modification of the original genome (non-integrative) or incorporate xeno-derived products (such as bovine serum albumin) which may contain xeno-agents. Ideally, this derivation would be carried out under chemically defined conditions to prevent lot-to-lot variability and enhance reproducibility. Additionally, derivation from cell types such as fibroblasts requires extended culture (4-6 weeks), greatly increasing the time required to progress from biopsy to hiPSC. Herein, we outline a method of culturing peripheral blood mononuclear cells (PBMCs) and reprogramming PBMCs into hiPSCs using a non-integrative Sendai virus.
• Churko, J. M., Mantalas, G. L., Snyder, M. P., & Wu, J. C. (2013). Overview of high throughput sequencing technologies to elucidate molecular pathways in cardiovascular diseases.. Circulation research, 112(12), 1613-23. doi:10.1161/circresaha.113.300939
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  High throughput sequencing technologies have become essential in studies on genomics, epigenomics, and transcriptomics. Although sequencing information has traditionally been elucidated using a low throughput technique called Sanger sequencing, high throughput sequencing technologies are capable of sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time. This advantage allows high throughput sequencing to be used to create large data sets, generating more comprehensive insights into the cellular genomic and transcriptomic signatures of various diseases and developmental stages. Within high throughput sequencing technologies, whole exome sequencing can be used to identify novel variants and other mutations that may underlie many genetic cardiac disorders, whereas RNA sequencing can be used to analyze how the transcriptome changes. Chromatin immunoprecipitation sequencing and methylation sequencing can be used to identify epigenetic changes, whereas ribosome sequencing can be used to determine which mRNA transcripts are actively being translated. In this review, we will outline the differences in various sequencing modalities and examine the main sequencing platforms on the market in terms of their relative read depths, speeds, and costs. Finally, we will discuss the development of future sequencing platforms and how these new technologies may improve on current sequencing platforms. Ultimately, these sequencing technologies will be instrumental in further delineating how the cardiovascular system develops and how perturbations in DNA and RNA can lead to cardiovascular disease.
• Mordwinkin, N. M., Gu, M., Kooreman, N. G., Hu, S., Churko, J. M., Diecke, S., Burridge, P. W., He, C., Lee, J., Gold, J. D., & Wu, J. C. (2013). Abstract 16710: Induced Pluripotent Stem Cell-Derived Endothelial Cells From Diet-Induced Obesity Mice Exhibit Decreased Vascular Function in a Murine Model of Hindlimb Ischemia. Circulation, 128.
• Churko, J. M., Kelly, J. J., Macdonald, A., Bai, D., Laird, D. W., Lee, J. K., & Sampson, J. B. (2012). The G60S Cx43 mutant enhances keratinocyte proliferation and differentiation.. Experimental dermatology, 21(8), 612-8. doi:10.1111/j.1600-0625.2012.01532.x
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  Transient knock-down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell-cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild-type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium-induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes.
• Penuela, S., Gyenis, L., Ablack, A., Churko, J. M., Berger, A. C., Lewis, J. E., Litchfield, D. W., & Laird, D. W. (2012). Pannexin 1 knockdown in metastatic melanoma cells induces cell differentiation into a melanocytic phenotype decreasing tumor size and metastasis in vivo. The FASEB Journal, 26.
• Penuela, S., Gyenis, L., Ablack, A., Churko, J. M., Berger, A. C., Litchfield, D. W., Lewis, J. D., & Laird, D. W. (2012). Loss of pannexin 1 attenuates melanoma progression by reversion to a melanocytic phenotype.. The Journal of biological chemistry, 287(34), 29184-93. doi:10.1074/jbc.m112.377176
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  Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and β-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.
• Churko, J. M., Shao, Q., Laird, D. W., & Chan, J. L. (2011). The G60S connexin43 mutant regulates hair growth and hair fiber morphology in a mouse model of human oculodentodigital dysplasia.. The Journal of investigative dermatology, 131(11), 2197-204. doi:10.1038/jid.2011.183
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  Patients expressing mutations in the gene encoding the gap junction protein Cx43 suffer from a disease called oculodentodigital dysplasia (ODDD). Patients with ODDD are often reported to develop hair that is dry, dull, sparse, and slow growing. To evaluate the linkage between Cx43 and hair growth, structure, and follicle density we employed a mouse model of ODDD that harbors a Cx43 G60S point mutant. Regionally sparse and overall dull hair were observed in mutant mice compared with their wild-type (WT) littermates. However, histological analysis of overall hair follicle density in mutant and WT mice did not reveal any significant differences. After epilation, mutant mouse hair grew back slower, and hair growth was asynchronous. In addition, ultrastructural scanning electron microscopic imaging of hair fibers taken from mutant mice and two patients harboring the G143S mutation revealed severe cuticle weathering. Nodule formation was also observed in the proximal region of hair fibers taken from mutant mice. These results suggest that the G60S mutant mouse model mimics the hair phenotype found in at least some ODDD patients and suggests an important role for Cx43 in hair regeneration, growth, and cuticle formation.
• Churko, J. M., Shao, Q., Swoboda, K. J., Bai, D., Laird, D. W., Gong, X. Q., & Sampson, J. B. (2011). Human dermal fibroblasts derived from oculodentodigital dysplasia patients suggest that patients may have wound-healing defects.. Human mutation, 32(4), 456-66. doi:10.1002/humu.21472
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  Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant human disease caused by any one of over 60 mutations in the GJA1 gene encoding the gap junction protein Cx43. In the present study, wound healing was investigated in a G60S ODDD mutant mouse model and by using dermal fibroblasts isolated from two ODDD patients harboring the p.D3N and p.V216L mutants along with dermal fibroblasts isolated from their respective unaffected relatives. Punch biopsies revealed a delay in wound closure in the G60S mutant mice in comparison to wild-type littermates, and this delay appeared to be due to defects in the dermal fibroblasts. Although both the p.D3N and p.V216L mutants reduced gap junctional intercellular communication in human dermal fibroblasts, immunolocalization studies revealed that Cx43 gap junctions were prevalent at the cell surface of p.D3N expressing fibroblasts but greatly reduced in p.V216L expressing fibroblasts. Mutant expressing fibroblasts were further found to have reduced proliferation and migration capabilities. Finally, in response to TGFβ1, mutant expressing fibroblasts expressed significantly less alpha smooth muscle actin suggesting they were inefficient in their ability to differentiate into myofibroblasts. Collectively, our results suggest that ODDD patients may have subclinical defects in wound healing due to impaired function of dermal fibroblasts.
• Bhalla-gehi, R., Penuela, S., Churko, J. M., Shao, Q., & Laird, D. W. (2010). Pannexin1 and pannexin3 delivery, cell surface dynamics, and cytoskeletal interactions.. The Journal of biological chemistry, 285(12), 9147-60. doi:10.1074/jbc.m109.082008
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  Pannexins (Panx) are a class of integral membrane proteins that have been proposed to exhibit characteristics similar to those of connexin family members. In this study, we utilized Cx43-positive BICR-M1R(k) cells to stably express Panx1, Panx3, or Panx1-green fluorescent protein (GFP) to assess their trafficking, cell surface dynamics, and interplay with the cytoskeletal network. Expression of a Sar1 dominant negative mutant revealed that endoplasmic reticulum to Golgi transport of Panx1 and Panx3 was mediated via COPII-dependent vesicles. Distinct from Cx43-GFP, fluorescence recovery after photobleaching studies revealed that both Panx1-GFP and Panx3-GFP remained highly mobile at the cell surface. Unlike Cx43, Panx1-GFP exhibited no detectable interrelationship with microtubules. Conversely, cytochalasin B-induced disruption of microfilaments caused a severe loss of cell surface Panx1-GFP, a reduction in the recoverable fraction of Panx1-GFP that remained at the cell surface, and a decrease in Panx1-GFP vesicular transport. Furthermore, co-immunoprecipitation and co-sedimentation assays revealed actin as a novel binding partner of Panx1. Collectively, we conclude that although Panx1 and Panx3 share a common endoplasmic reticulum to Golgi secretory pathway to Cx43, their ultimate cell surface residency appears to be independent of cell contacts and the need for intact microtubules. Importantly, Panx1 has an interaction with actin microfilaments that regulates its cell surface localization and mobility.
• Celetti, S. J., Cowan, K. N., Penuela, S., Shao, Q., Laird, D. W., & Churko, J. M. (2010). Implications of pannexin 1 and pannexin 3 for keratinocyte differentiation.. Journal of cell science, 123(Pt 8), 1363-72. doi:10.1242/jcs.056093
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  Pannexin (Panx) 1 and Panx3 are integral membrane proteins that share some sequence homology with the innexin family of invertebrate gap junctions. They are expressed in mammalian skin. Pannexins have been reported to form functional mechanosensitive single-membrane channels, but their importance in regulating cellular function is poorly understood. In this study, Panx1 and Panx3 were detected in the epidermis of 13.5 day embryonic mice. Compared with newborn mice, there was less Panx1 expression in both thin and thick murine skin, whereas Panx3 expression was unchanged. To investigate the role of pannexins in keratinocyte differentiation, we employed rat epidermal keratinocytes (REKs) that have the capacity to differentiate into organotypic epidermis, and engineered them to overexpress Panx1, Panx1-GFP or Panx3. The expression of Panx1 or Panx3 resulted in the increased ability of REKs to take up dye, suggesting that cell-surface channels were formed. Compared with monolayer REKs, endogenous Panx1 levels remained unchanged, whereas the 70 kDa immunoreactive species of Panx3 was greatly increased in the organotypic epidermis. In monolayer cultures, ectopic Panx1 and Panx1-GFP localized to the plasma membrane, whereas Panx3 displayed both intracellular and plasma-membrane profiles. Although both pannexins reduced cell proliferation, only Panx1 disrupted the architecture of the organotypic epidermis and markedly dysregulated cytokeratin 14 expression and localization. Furthermore, ectopic expression of only Panx1 reduced the vital layer thickness of the organotypic epidermis. In summary, Panx1 and Panx3 are coexpressed in the mammalian epidermis, and the regulation of Panx1 plays a key role in keratinocyte differentiation.
• Churko, J. M., Langlois, S., Pan, X., Shao, Q., & Laird, D. W. (2010). The potency of the fs260 connexin43 mutant to impair keratinocyte differentiation is distinct from other disease-linked connexin43 mutants.. The Biochemical journal, 429(3), 473-83. doi:10.1042/bj20100155
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  Although there are currently 62 mutants of Cx43 (connexin43) that can cause ODDD (oculodentodigital dysplasia), only two mutants have also been reported to cause palmar plantar hyperkeratosis. To determine how mutants of Cx43 can lead to this skin disease, REKs (rat epidermal keratinocytes) were engineered to express an ODDD-associated Cx43 mutant always linked to skin disease (fs260), an ODDD-linked Cx43 mutant which has been reported to sometimes cause skin disease (fs230), Cx43 mutants which cause ODDD only (G21R, G138R), a mouse Cx43 mutant linked to ODDD (G60S), a non-disease-linked truncated Cx43 mutant that is trapped in the endoplasmic reticulum (Delta244*) or full-length Cx43. When grown in organotypic cultures, of all the mutants investigated, only the fs260-expressing REKs consistently developed a thinner stratum corneum and expressed lower levels of Cx43, Cx26 and loricrin in comparison with REKs overexpressing wild-type Cx43. REKs expressing the fs260 mutant also developed a larger organotypic vital layer after acetone-induced injury and exhibited characteristics of parakeratosis. Collectively, our results suggest that the increased skin disease burden exhibited in ODDD patients harbouring the fs260 mutant is probably due to multiple additive effects cause by the mutant during epidermal differentiation.
• Langlois, S., Churko, J. M., & Laird, D. W. (2010). Optical and biochemical dissection of connexin and disease-linked connexin mutants in 3D organotypic epidermis.. Methods in molecular biology (Clifton, N.J.), 585(Issue), 313-34. doi:10.1007/978-1-60761-380-0_22
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  The epidermis is a complex tissue composed principally of differentiated keratinocytes that form a keratinized stratified squamous epithelium. The gap junction proteins, connexins (Cx), are differentially expressed throughout the stratified layers of the epidermis and their exquisite regulation appears to govern the delicate balance between cell proliferation and differentiation in normal skin homeostasis and in wound healing. In the last 10 years, germ line mutations in the genes encoding five connexin family members have been linked to various types of skin diseases that appear to offset the balance between keratinocyte differentiation and proliferation. Consequently, in order to determine how these connexin gene mutations manifest as skin disease, disease-linked mutants must be expressed in 3D organotypic epidermis reference models that attempt to mimic the human condition. Given the complexity of organotypic epidermis, confocal optical and biochemical dissection of connexin or disease-linked connexin mutants within the regenerated epidermal layer is required. The procedures necessary to assess the architectural characteristics of genetically modified organotypic epidermis and its state of differentiation will be described in this chapter.
• Simek, J., Shao, Q., Laird, D. W., & Churko, J. M. (2009). Cx43 has distinct mobility within plasma-membrane domains, indicative of progressive formation of gap-junction plaques.. Journal of cell science, 122(Pt 4), 554-62. doi:10.1242/jcs.036970
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  Connexin 43 (Cx43) is a dynamic molecule, having a short half-life of only a few hours. In this study, we use fluorescent-protein-tagged Cx43 variants to examine Cx43 delivery to the cell surface, its residency status in various cell-surface membrane domains and its mobility characteristics. Rapid time-lapse imaging led to the identification of Cx43 being delivered to cell-surface domains that lacked a contacting cell, and also to its localization within membrane protrusions. Fluorescence recovery after photobleaching (FRAP) was used to investigate the mobility state of cell-surface-localized Cx43. Cx43 mobility within clustered cell-surface profiles of Cx43 could be categorized into those with generally a high degree of lateral mobility and those with generally a low degree of lateral mobility. Cx43 mobility was independent of cluster size, yet the C-terminal domain of Cx43 regulated the proportion of gap-junction-like clusters that acquired a low Cx43 mobility state. Collectively, these studies show that Cx43 establishes residency at all cell-surface membrane domains, and progressively acquires assembly states that probably reflect differences in either channel packing and/or its interactions with Cx43-binding proteins.
• Manias, J. L., Plante, I., Shao, Q., Bai, D., Laird, D. W., Churko, J. M., & Gong, X. Q. (2008). Fate of connexin43 in cardiac tissue harbouring a disease-linked connexin43 mutant.. Cardiovascular research, 80(3), 385-95. doi:10.1093/cvr/cvn203
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  More than 40 mutations in the GJA1 gene encoding connexin43 (Cx43) have been linked to oculodentodigital dysplasia (ODDD), a pleiotropic, autosomal dominant disorder. We hypothesized that even with a significant reduction in the levels of Cx43 in a mutant mouse model of ODDD (Gja1(Jrt/+)) harbouring a G60S mutation (Cx43(G60S)), cardiomyocyte function may only be moderately compromised given that a majority of mutant mice typically survive..Western blotting and quantitative reverse transcriptase-polymerase chain reaction in conjunction with immunofluorescence were used to assess the expression and localization of Cx43 in hearts and cultured cardiomyocytes from wild-type and Gja1(Jrt/+) mice. Dye-coupling and dual whole cell patch-clamp recordings were also used to assess the gap junction channel status in cultured cardiomyocytes from wild-type and mutant mice. Cardiac tissue from adult Gja1(Jrt/+) mice revealed a 60-80% reduction in Cx43 protein with a preferential loss of the highly phosphorylated forms of Cx43. Compensation via the up-regulation of Cx40 or Cx45 was not observed. Immunofluorescent analysis of cultured cardiomyocytes revealed a trafficking defect, with a decrease in Cx43 plaques and a large population of Cx43 being retained in the Golgi apparatus. However, cultured cardiomyocytes from mutant mice remained beating with a 50% decrease in coupling conductance..These results suggest that the Cx43(G60S) mutant impairs normal trafficking and function of co-expressed Cx43 with no dramatic effect on cardiomyocyte function, suggesting that Cx43 is biosynthesized in excess of an essential need.