Project Summary The purpose of this project is to provide research and career development training for Valeria Gomez, a current PhD student at the University of Florida. This project is a logical extension of the parent project, ‘Role of SR-mitochondria interplay in calcium-dependent arrhythmias’, thus furthering its overarching mission. Additionally, it will facilitate career growth for the candidate, aligning with NIH’s interest in advancing diversity. The release of calcium (Ca) from the sarcoplasmic reticulum (SR) via the ryanodine receptors (RyR2) regulates the heartbeat. This Ca release process is tightly controlled in healthy hearts but goes awry in diseased hearts due to genetic or acquired defects of the RyR2 channel complex. These defects typically make the channel complex hyperactive or leaky, thus giving rise to aberrant Ca release (ACR). RyR2 dysfunction and resultant ACR is implicated in a range of cardiac dysfunctions including heart failure, metabolic heart, and cardiac arrhythmias. For instance, catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic arrhythmia syndrome directly caused by mutations in RyR2 or its accessory proteins. Interestingly, recent studies have shown that mitochondria in CPVT appear to play a protective role by absorbing RyR2-derived ACR. Indeed, strategies aimed at directing more Ca to mitochondria by targeting mitochondrial Ca uptake mechanisms appear to be effective in alleviating arrhythmias in CPVT. SR and mitochondria form structural tethering known as mitochondria-associated membranes (MAMs), which are the primary sites facilitating SR-mitochondria Ca transfer. The parent project focuses on targeting one of the endogenous tether proteins, mitofusin 2, to manipulate MAMs and assess its impact on arrhythmogenesis. In this project, Ms. Gomez aims to introduce an engineered cardiac muscle-specific MAMs linker to CPVT hearts. She will test the hypothesis that the expression of the engineered MAMs linker inhibits arrhythmias in CPVT by promoting MAMs. Aim 1 will test the hypothesis that the expression of the engineered MAMs linker promotes MAMs in CPVT hearts. Adeno-associated virus 9 (AAV9)-mediated gene transfer will be employed to introduce the MAMs linker. The formation of MAMs will be assessed through transmission electron microscopy and cellular assays, including immunofluorescence and proximity ligation assay. Aim 2 will test the hypothesis that the expression of the engineered MAMs linker inhibits cellular arrhythmias. Confocal imaging experiments will be conducted to evaluate the frequency of arrhythmogenic Ca waves in myocytes. Aim 3 aims to test the hypothesis that the expression of the engineered MAMs linker inhibits arrhythmias in vivo, assessed through surface electrocardiogram measurements. The proposed project will explore innovative strategies for targeting mitochondria to inhibit Ca-dependent arrhythmias. Moreover, it will offer exceptional training opportunities for the...