Epigenetic Regulation of Cardiac Mitochondrial Function by Long Noncoding RNA: Investigating the Impact of Ketosis and Intermittent Fasting in Heart Failure

Project Summary: Heart failure, a leading cause of death worldwide, is increasingly recognized as a metabolic disorder linked to mitochondrial dysfunction and energy deficits. The long-term goal of this project is to prevent heart failure and develop effective therapies to improve and extend the lives of cardiac patients. Metabolic interventions, such as ketosis and intermittent fasting, have shown promise in improving mitochondrial function and heart failure outcomes. Long noncoding RNAs (lncRNAs) regulate gene expression by interacting with chromatin, transcription factors, or other RNA molecules. While lncRNAs are known to regulate mitochondrial function and are altered in heart failure, their specific roles, particularly under ketotic conditions, remain poorly understood. The preliminary results of this project indicate that a six-month ketogenic diet in C57BL/6 mice significantly increased the expression of lncRNAs Anril and Malat1, along with proteins involved in mitochondrial protection and biogenesis (Nrf2 and Pgc1α) in mice heart. In vitro ketosis in AC16 human cardiomyocytes, induced by β-hydroxybutyrate also upregulated MALAT1 and key genes involved in ketone oxidation, mitochondrial biogenesis, and antioxidant defense. siRNA-mediated knockdown of MALAT1 significantly reduced cellular ATP levels, as well as the expression of the rate-limiting ketone oxidation enzyme SCOT and the antioxidant gene NRF2 in human cardiomyocytes. Computational analysis using LncRRIsearch identified interactions between MALAT1 and the mRNAs of NRF2 and PGC1α. From these results, it is hypothesized that ketosis-induced upregulation of MALAT1 improves mitochondrial function and reduces oxidative stress in heart failure by regulating mitochondrial genes. Aim 1 will investigate how lncRNA MALAT1 regulates mitochondrial function in human cardiomyocytes, focusing on genes involved in biogenesis, dynamics, and metabolism. MALAT1 will be modulated via siRNA knockdown and plasmid overexpression. RNA Chromatin Immunoprecipitation-sequencing, qPCR, and RNA Immunoprecipitation will be used to examine its interactions with mitochondrial gene promoters. Mitochondrial function will be assessed using the Seahorse XF Analyzer and assays for reactive oxygen species, membrane potential, and ATP levels. Aim 2 will assess the therapeutic potential of modulating MALAT1 in a mouse model of heart failure with preserved ejection fraction. MALAT1 will be overexpressed or knocked down using adeno-associated viral vectors, and metabolic interventions like ketone ester supplementation and intermittent fasting will be tested for combined effects on mitochondrial function. Cardiac function will be evaluated via echocardiography, myocardial fibrosis with Masson's trichrome staining, and mitochondrial bioenergetics and biogenesis through Seahorse XF Analyzer, qPCR, Western blotting, and immunofluorescence. This innovative approach, using lifestyle or ketone body-mediated metabolic interventions, will identify how the lncRNA MALAT1 regulates mitochondrial function and aims to develop novel strategies for treating heart failure.