- Email: [email protected]
Circulating Factors Contribute to PDE5-Mediated Pathological Myocardial Remodeling in Single Ventricle Congenital Heart Disease
146
Abstracts
cardioprotection. Methods: Male Sprague-Dawley rats were subjected to 30 minutes of coronary occlusion followed by 120 minutes reperfusion. Before ischaemia, a laparotomy with or without application of capsaicin cream (0.1%, a TRPV1 activator), was performed. Additional sets of rats were given morphine (3 mg/kg) with or without capsaicin. Further, capsazepine (3 mg/kg, a classical TRPV1 inhibitor), and P5 (3 mg/kg, a peptide analgesic and TRPV1 inhibitor), were given prior to the laparotomy or morphine. Myocardial infarct size was determined. Results: A laparotomy or capsaicin reduced infarct size versus controls (to 44±2%* or 49±1%* vs. 66±1%, infarct size/area at risk %, n=6, *Pb0.0001). When combining laparotomy and capsaicin, cardioprotection was not additive. Morphine also reduced infarct size (37±3%* vs. 61±2%, n=6, Pb0.0001), with no additive effect when combined with capsaicin. The TRPV1 inhibitors capsazepine and P5 abolished cardioprotection induced by either a laparotomy (58±1%+ and 65±2%+ vs. 44±2%+, respectively) or morphine (62±3%+ and 58±2%+ vs. 37±3%, respectively, n=6, + Pb0.0001). Conclusions: Infarct size reduction by a laparotomy or morphine is blocked by inhibiting TRPV1. Drugs given which inhibit TRPV1 may impede organ protection and this requires further study. doi:10.1016/j.yjmcc.2017.07.048
036 Circulating Factors Contribute to PDE5-Mediated Pathological Myocardial Remodeling in Single Ventricle Congenital Heart Disease Anastacia Garciaa,b, Stephanie Nakanoa,b, Anis Karimpour-Farda, Brian Stauffera,c, Carmen Sucharova, Shelley Miyamotoa,b a
University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, USA Children’s Hospital Colorado, Aurora, CO, USA c Denver Health and Hospital Authority, Denver, CO, USA b
Introduction: Single ventricle congenital heart disease (SV) is fatal without intervention, and maladaptive myocardial remodeling and eventual heart failure are a major cause of morbidity and mortality in this population. Our previous and current studies demonstrate gene expression changes indicative of pathological myocardial remodeling and increased phosphodiesterase-5 (PDE5) expression and activity in SV myocardium relative to non-failing (NF) myocardium. Interestingly, Sildenafil, a PDE5 inhibitor (PDE5i), is increasingly utilized in SV patients, to target the pulmonary vasculature. However, our data suggest potential myocardial effects of PDE5i. We are utilizing a cell-based model (neonatal rat ventricular myocytes, NRVMs) in combination with an ongoing human heart and blood bank to determine if PDE5 has a role in the induction of pathological gene expression changes seen in SV myocardium. Methods: PDE5 levels in NF and SV sera were measured using an ELISA. NRVMs were treated for 72 hours with sera or serum-derived exosomes from NF and SV patients +/sildenafil. qRTPCR was performed for the targets of interest and RNAseq was performed on a subset of serum-treated NRVMs +/sildenafil. Results: Circulating levels of PDE5 are significantly higher in SV patients compared to NF controls. Additionally, treatment of NRVMs with serum or exosomes from SV patients recapitulates the pathologic gene expression pattern seen in the human SV myocardium, and PDE5i blunts this response. Further, transcriptome analysis of serum-treated NRVMs +/- sildenafil revealed that SV circulating factors significantly impact the expression of 1,449 genes involved in multiple cellular processes including fibrosis, hypertrophy, lipid metabolism, and cell death, and PDE5i reverses the expression of 65% (1088) of these genes. Conclusion: Together, these data suggest that SV circulating factors contribute to PDE5mediated myocyte remodeling, and in addition to effects on the
pulmonary vasculature, PDE5i may be a direct myocardial target of therapy for the treatment of SV. doi:10.1016/j.yjmcc.2017.07.049
038 Function Beyond RNA Splicing for RBFox Family Members in Heart Chen Gaoa,b, Jing Huc, Chaoliang Weid, Yunhua Esther Hsiaoe, Shuxun Rena,b, Yuanchao Xuec, Yu Zhouc, Jianlin Zhangf, Ju Chenf, Xinshu Xiaoe, Xiang-Dong Fuc,e, Yi Xingg, Yibin Wanga,b a Division of Molecular Medicine, Cardiovascular Research Laboratories, University of California, Los Angeles, Los Angeles, California, USA b Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA c Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, California, USA d Department of Cell Biology and Medical Genetics, School of Medicine, Shenzhen University, Shenzhen, China e Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, California, USA f Department of Medicine, University of California, San Diego, San Diego, California, USA g Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA
Background: RNA metabolism, from synthesis, processing, translation to degradation is an integrated part of gene regulation that ultimately determines the overall cardiac transcriptome complexity. The RBFox family members of RNA splicing regulator, including RBFox1 and 2, are dramatically downregulated in the diseased hearts and are found to have critical roles in cardiac development and pathological remodeling. In addition to expected alternative RNA splicing and transcription regulation targeted by the nuclear RBFox1 and RBFox2, both genes also produce cytosolic localized splicing variants, raising interesting questions about additional roles in the cytosol for RNA processing. Goal and Methods: This study investigated the functional impact of two RBFox family members- RBFox1 and RBFox2 in cytosol in cardiomyocytes. Results: Cardiac specific KO of RBFox1 exacerbated pressure-overload induced heart failure and fibrotic remodeling while cardiac specific expression of the RBFox1 cytosolic isoform (RBFox1c) in mouse heart was sufficient to protect against pressure-overload induced cardiac dysfunction and fibrotic remodeling. RNA-seq analysis in cultured myocytes revealed a significant suppression of proinflammatory genes upon RBFox1c expression, and motif analysis identified significant enrichment of RBFox binding sites in the 3’UTR of the affected genes mRNA. By both mass-spec and co-IP analysis, we also detected specific interaction between RBFox1c and RNA degradation complex protein, implicating a molecular link between RBFox1c and targeted RNA degradation. For RBFox2, cardiac specific and inducible KO of RBFox2 in adult mouse hearts was sufficient to trigger dilated cardiomyopathy with disorganized T-tubules structure associated with targeted inhibition of Jph2 expression at protein level but not at mRNA level. Loss of RBFox2 repressed Jph2 protein expression due to competitive binding on the Jph2 3’-UTR with miR34a. Inactivating miR34a partially rescued heart failure induced by RBFox2 KO in heart by restoring Jph2 expression, normalizing cardiac function and T-tubule organization. Conclusion: Both RBFox1 and RBFox2 have potentially important function beyond splicing regulation in cardiac muscle cells. In cytosol, RBFox1 suppresses inflammatory gene expression and cardiac fibrosis by binding to 3’UTR, and potentially modulating target mRNA stability. In contrast, RBFox2 alters Jph2 protein expression by interacting with Jph2 3’UTR and competing with
Abstracts
cardioprotection. Methods: Male Sprague-Dawley rats were subjected to 30 minutes of coronary occlusion followed by 120 minutes reperfusion. Before ischaemia, a laparotomy with or without application of capsaicin cream (0.1%, a TRPV1 activator), was performed. Additional sets of rats were given morphine (3 mg/kg) with or without capsaicin. Further, capsazepine (3 mg/kg, a classical TRPV1 inhibitor), and P5 (3 mg/kg, a peptide analgesic and TRPV1 inhibitor), were given prior to the laparotomy or morphine. Myocardial infarct size was determined. Results: A laparotomy or capsaicin reduced infarct size versus controls (to 44±2%* or 49±1%* vs. 66±1%, infarct size/area at risk %, n=6, *Pb0.0001). When combining laparotomy and capsaicin, cardioprotection was not additive. Morphine also reduced infarct size (37±3%* vs. 61±2%, n=6, Pb0.0001), with no additive effect when combined with capsaicin. The TRPV1 inhibitors capsazepine and P5 abolished cardioprotection induced by either a laparotomy (58±1%+ and 65±2%+ vs. 44±2%+, respectively) or morphine (62±3%+ and 58±2%+ vs. 37±3%, respectively, n=6, + Pb0.0001). Conclusions: Infarct size reduction by a laparotomy or morphine is blocked by inhibiting TRPV1. Drugs given which inhibit TRPV1 may impede organ protection and this requires further study. doi:10.1016/j.yjmcc.2017.07.048
036 Circulating Factors Contribute to PDE5-Mediated Pathological Myocardial Remodeling in Single Ventricle Congenital Heart Disease Anastacia Garciaa,b, Stephanie Nakanoa,b, Anis Karimpour-Farda, Brian Stauffera,c, Carmen Sucharova, Shelley Miyamotoa,b a
University of Colorado, Denver Anschutz Medical Campus, Aurora, CO, USA Children’s Hospital Colorado, Aurora, CO, USA c Denver Health and Hospital Authority, Denver, CO, USA b
Introduction: Single ventricle congenital heart disease (SV) is fatal without intervention, and maladaptive myocardial remodeling and eventual heart failure are a major cause of morbidity and mortality in this population. Our previous and current studies demonstrate gene expression changes indicative of pathological myocardial remodeling and increased phosphodiesterase-5 (PDE5) expression and activity in SV myocardium relative to non-failing (NF) myocardium. Interestingly, Sildenafil, a PDE5 inhibitor (PDE5i), is increasingly utilized in SV patients, to target the pulmonary vasculature. However, our data suggest potential myocardial effects of PDE5i. We are utilizing a cell-based model (neonatal rat ventricular myocytes, NRVMs) in combination with an ongoing human heart and blood bank to determine if PDE5 has a role in the induction of pathological gene expression changes seen in SV myocardium. Methods: PDE5 levels in NF and SV sera were measured using an ELISA. NRVMs were treated for 72 hours with sera or serum-derived exosomes from NF and SV patients +/sildenafil. qRTPCR was performed for the targets of interest and RNAseq was performed on a subset of serum-treated NRVMs +/sildenafil. Results: Circulating levels of PDE5 are significantly higher in SV patients compared to NF controls. Additionally, treatment of NRVMs with serum or exosomes from SV patients recapitulates the pathologic gene expression pattern seen in the human SV myocardium, and PDE5i blunts this response. Further, transcriptome analysis of serum-treated NRVMs +/- sildenafil revealed that SV circulating factors significantly impact the expression of 1,449 genes involved in multiple cellular processes including fibrosis, hypertrophy, lipid metabolism, and cell death, and PDE5i reverses the expression of 65% (1088) of these genes. Conclusion: Together, these data suggest that SV circulating factors contribute to PDE5mediated myocyte remodeling, and in addition to effects on the
pulmonary vasculature, PDE5i may be a direct myocardial target of therapy for the treatment of SV. doi:10.1016/j.yjmcc.2017.07.049
038 Function Beyond RNA Splicing for RBFox Family Members in Heart Chen Gaoa,b, Jing Huc, Chaoliang Weid, Yunhua Esther Hsiaoe, Shuxun Rena,b, Yuanchao Xuec, Yu Zhouc, Jianlin Zhangf, Ju Chenf, Xinshu Xiaoe, Xiang-Dong Fuc,e, Yi Xingg, Yibin Wanga,b a Division of Molecular Medicine, Cardiovascular Research Laboratories, University of California, Los Angeles, Los Angeles, California, USA b Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA c Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, California, USA d Department of Cell Biology and Medical Genetics, School of Medicine, Shenzhen University, Shenzhen, China e Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, California, USA f Department of Medicine, University of California, San Diego, San Diego, California, USA g Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA
Background: RNA metabolism, from synthesis, processing, translation to degradation is an integrated part of gene regulation that ultimately determines the overall cardiac transcriptome complexity. The RBFox family members of RNA splicing regulator, including RBFox1 and 2, are dramatically downregulated in the diseased hearts and are found to have critical roles in cardiac development and pathological remodeling. In addition to expected alternative RNA splicing and transcription regulation targeted by the nuclear RBFox1 and RBFox2, both genes also produce cytosolic localized splicing variants, raising interesting questions about additional roles in the cytosol for RNA processing. Goal and Methods: This study investigated the functional impact of two RBFox family members- RBFox1 and RBFox2 in cytosol in cardiomyocytes. Results: Cardiac specific KO of RBFox1 exacerbated pressure-overload induced heart failure and fibrotic remodeling while cardiac specific expression of the RBFox1 cytosolic isoform (RBFox1c) in mouse heart was sufficient to protect against pressure-overload induced cardiac dysfunction and fibrotic remodeling. RNA-seq analysis in cultured myocytes revealed a significant suppression of proinflammatory genes upon RBFox1c expression, and motif analysis identified significant enrichment of RBFox binding sites in the 3’UTR of the affected genes mRNA. By both mass-spec and co-IP analysis, we also detected specific interaction between RBFox1c and RNA degradation complex protein, implicating a molecular link between RBFox1c and targeted RNA degradation. For RBFox2, cardiac specific and inducible KO of RBFox2 in adult mouse hearts was sufficient to trigger dilated cardiomyopathy with disorganized T-tubules structure associated with targeted inhibition of Jph2 expression at protein level but not at mRNA level. Loss of RBFox2 repressed Jph2 protein expression due to competitive binding on the Jph2 3’-UTR with miR34a. Inactivating miR34a partially rescued heart failure induced by RBFox2 KO in heart by restoring Jph2 expression, normalizing cardiac function and T-tubule organization. Conclusion: Both RBFox1 and RBFox2 have potentially important function beyond splicing regulation in cardiac muscle cells. In cytosol, RBFox1 suppresses inflammatory gene expression and cardiac fibrosis by binding to 3’UTR, and potentially modulating target mRNA stability. In contrast, RBFox2 alters Jph2 protein expression by interacting with Jph2 3’UTR and competing with