Investigating the pathological heart and its regenerative potential

Abstract: Cardiovascular disease is a leading contributor to mortality the world over, affecting millions of people each year. This, combined with its associated monetary and societal costs, has made the investigation of the etiology and pathophysiology of CVD a scientific priority. Researchers have developed numerous tools to aid in these studies, many of which have been successful in developing effective clinical therapies. However, while technical and medical advances have helped slow the spread of this burgeoning epidemic, CVD-related deaths are still on the rise globally. It is for this reason that the doctoral thesis presented here aims to develop a better understanding of the pathogenesis of CVD and to determine the magnitude of cardiac regenerative potential. In PAPER I we present clear methods for the isolation, culture, and functional characterization of adult murine cardiomyocytes for in vitro studies. We show that our method results in a single-cell suspension of electrochemically active, rod-shaped adult cardiomyocytes that maintain calcium sensitivity and contractile function. We further present proper cell culture conditions, optimized methods for whole-cell patch clamp and adenovirus-mediated gene delivery, as well as a clear protocol for the collection and analysis of contractility traces and calcium transients. In PAPER II we identify estrogen-related receptor β (ESRRβ) as a causative factor in the pathogenesis of dilated cardiomyopathy (DCM). We show that mice lacking ESRRβ develop DCM in mid-life and die prematurely. We further show that human myocardial tissue samples from DCM patients lack the expected nuclear ESRRβ localization, suggesting a role for ESRRβ also in human disease onset. In PAPER III we describe the role of SGK1 in the development of the fatal ventricular arrhythmias commonly associated with heart failure. Using transgenic mice expressing either constitutively active or dominant negative forms of SGK1, we show that chronic SGK1 activation in disease results in fatal ventricular arrhythmias due to an increased persistent sodium current, INaL, leading to prolonged action potential duration. We further show a direct interaction between SGK1 and the primary cardiac voltage-gated sodium channel, Nav1.5, and have identified putative phosphorylation sites contained in the SCN5a gene. Together, these findings suggest direct SGK1 regulation of sodium channel kinetics and gating. In PAPER IV we compare transcriptional profiles from three zebrafish models of cardiac regeneration to determine common genetic drivers of the regenerative response. By comparing the transcriptional profile of our genetic cardiomyocyte ablation model to those of two apical resection models and one cryoablation model, we have identified 16 genes to be up-regulated in all screens and 1 gene downregulated. Gene ontology analysis of this gene set reveals significant enrichment in biological processes associated with the cell cycle and mitosis, and pathway analysis has provided a list of putative upstream regulators of these genes. Further analysis of all transcription profiles also indicated a large number of model-specific genes, indicating that these disease models, and thus the regenerative responses activated viii therein, should not be considered interchangeably, but should be regarded as genetically distinct models of cardiac regeneration. Finally, in PAPER V we use 14C dating to determine the magnitude and dynamics of human cardiac cell turnover in dilated cardiomyopathy. The amount of 14C contained in postmortem cardiac tissue from patients diagnosed with DCM was compared to atmospheric levels at the time of the patient’s birth to determine the rate of cardiac cell turnover. Our mathematical model predicts that cardiomyocyte turnover is at least maintained in DCM. Importantly, though we report a significant increase in cardiomyocyte ploidy with disease onset, this was not sufficient to entirely account for the increased 14C concentration. Together, this work provides valuable information regarding the pathogenesis of both DCM and arrhythmogenic heart failure, and contributes to the growing literature surrounding potential regenerative therapies for cardiovascular disease.