Friday, April 26, 2024
4/26/2024
The maintenance of redox homeostasis is a crucial prerequisite for cardiovascular function as oxidative and reductive extremes produce pathophysiology. Although aberrant microRNA (miRNA) responses underlie heart failure (HF) progression, miRNA-mediated regulation of redox disequilibrium during HF remains elusive. This proposal aims to determine the role of miR-671 in myocardial reductive stress (RS). In contrast to oxidative stress, RS is characterized by an abnormal surplus of reducing equivalents which abrogate physiological reactive oxygen species (ROS) signaling and cause pathological cardiac remodeling. We have previously shown that constitutive activation of nuclear factor, erythroid 2 like 2 (Nfe2l2/Nrf2), a master transcriptional regulator of the antioxidant response element (ARE), catalyzes RS in the heart. Our preliminary data now indicate that both the 5' (miR-671-5p) and 3' (miR-671-3p) mature products of the precursor miR-671 transcript (pre-miR-671) are increased in the heart in an Nrf2 dose-dependent manner. Moreover, pilot transfections of miRNA mimics indicate that each of these ARE-harboring miRNAs have the capacity to augment glutathione redox ratio (GSH:GSSG) in cardiomyocytes. Therefore, the proposed research will test the hypothesis that synergistic augmentation of miRs 671-5p and 671-3p causes RS and pathological cardiac hypertrophy. First, mechanistic in vitro studies will be performed to tease apart the individual and synergistic effects of miR- 671 gain-of-function in the acute (48hr) and chronic (10-day) setting using HL-1 cardiomyocytes. Using an inducible pre-miR-671 lentiviral overexpression system, single-stranded inhibitors will be delivered to study the effect of: 1) miR-671-5p, 2) miR-671-3p, and 3) miR-671-5p and miR-671-3p co-expression. Through this strategy, GSH:GSSG, ROS levels, and hypertrophy will be evaluated in stably transduced cells. Next, a more physiologically relevant model will be used to test the hypothesis that chronic induction of miRs 671-5p and 671-3p cause pathological cardiac remodeling and dysfunction in vivo. C57BL6/J mice will be administered adeno-associated virus serotype 9 containing miR-671 transgene driven by the troponin T promoter (AAV9:cTNT:miR-671) to facilitate cardiac-specific miR-671 overexpression. Relative to control mice expressing luciferase, myocardial GSH:GSSG, hypertrophy as well as systolic and diastolic function will be measured by transthoracic echocardiography and pulse wave Doppler imaging, respectively. The presence of RS will be further confirmed by FACs analysis of ROS in freshly isolated AAV9:cTNT:miR-671 cardiomyocytes. While miR-671 is endogenously expressed in cardiomyocytes, its specific role in the heart has not been studied. Accordingly, this proposal is expected to provide novel insight into a relevant aspect of cardiac pathogenesis. The current training plan has been constructed to afford a promising pre-doctoral candidate several new technical proficiencies in molecular biology, redox biochemistry and physiology. Accordingly, Mr. Quiles will be remarkably prepared as he transitions into his postdoctoral studies in cardiovascular medicine.
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