Dissection and Rescue of Mechanical and Transcriptional Defects in Desmoplakin Cardiomyopathy

文摘:

Abstract: Variants in the gene desmoplakin (DSP) are one of the more common genetic causes of dilated cardiomyopathy. DSP variants cause an arrhythmogenic form of cardiomyopathy that can lead to both lethal ventricular arrhythmias and progressive heart failure, and no treatments are available. DSP encodes a critical structural protein that transduces force from the contractile machinery to intercellular junctions. Prior work has demonstrated the DSP cardiomyopathy is almost always caused by truncating genetic variants that cause a loss of function through reduced DSP mRNA abundance. Distinct to DSP cardiomyopathy, these truncating variants cause cardiac fibrosis to develop early in the disease course, preceding development of left ventricular systolic dysfunction. Based on the rationale that fibrosis occurs due to the cardiac injury-repair response, we hypothesize that reduced DSP abundance due to truncating mutations renders heart muscle tissue susceptible to injury and fibrotic repair due to an incapacity to normally handle the cardiac workload. Our primary objective is to test this mechanism in vitro and in vivo while also building evidence in pre-clinical models for novel treatment strategies that can be used in patients to prevent cardiac injury in DSP patients. Our specific aims will test the following specific hypothesis: (Aim 1) biomechanical stress induced cardiomyocyte damage is a consequence of DSP genetic variants that can be reduced through contractile inhibition as an upstream preventive approach; (Aim 2) loss of function consequences of DSP variants can be completely abrogated through transcriptional rescue of DSP expression. To rigorously examine relationships between biomechanical stress and injury in DSP cardiomyopathy, we will utilize two in vitro bioengineered cardiac muscle tissue platforms that leverage induced pluripotent stem cells (iPSCs) derived from DSP patients. Further, contractile antagonists will be tested as an in vivo preventive approach in a mouse model of DSP cardiomyopathy. Although seemingly paradoxical, these experiments will test whether inhibitory contractile modulation using re-purposed drugs is actually preventive to the development of fibrotic remodeling in DSP cardiomyopathy by reducing biomechanical strain at the cardiomyocyte level. In parallel, we will use these same in vitro and in vivo systems to dissect the relationships between DSP mRNA reduction and impaired biomechanical injury response. CRISPR-Cas9 tools that enable activation and repression of endogenous mRNA expression will be targeted to the DSP promoter. CRISPR-Cas9 activation will be tested in vivo with adeno-associated virus as a novel gene therapy approach with high potential for clinical translation. Taken together, this proposal will yield fundamental insights into the mechanisms by which DSP loss of function genetic variants cause cardiomyocyte injury and fibrosis while directly translating clinical observations towards two novel therapeutic approaches.