Duane D. Hall, Ph.D. - US grants

PROJECT SUMMARY Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is an inherited disease characterized by fibro-fatty infiltration of the heart, life-threatening ventricular arrhythmias and sudden cardiac death, particularly in response to sympathetic stress. ARVC is a leading cause of sudden cardiac death among young athletes with no prior symptoms or diagnosis of cardiovascular disease. Desmosome cell-adhesion gene mutations constitute the majority of familial ARVC cases, but it remains largely unknown how catecholamine-sensitive ventricular tachycardia and cardiac remodeling processes are facilitated by desmosome dysfunction. Patient management is therefore limited to improving quality of life by reducing arrhythmic symptoms or cardiac transplantation upon advanced heart failure. To begin to uncover mechanisms inherent to ARVC, we performed unbiased mass spectrometry analysis of ventricular samples from patients with different desmosomal gene mutations. Our preliminary data demonstrate that integrin ?1D is significantly downregulated in ARVC resulting in de- stabilization of RyR2 ryanodine receptor Ca2+ channels that localize to cardiac dyad junctions. Mechanistically, we find that ERK1/2 activation in response to desmosome loss results in ubiquitin-dependent degradation of integrin ?1D, RyR2 Ser-2030 phosphorylation, sarcoplasmic reticulum Ca2+ leak and arrhythmogenesis. Importantly, hearts of our integrin ?1D knockout mice exhibit ARVC-like disease with increased RyR2 phosphorylation, catecholamine-induced ventricular arrhythmias and cardiac fibrosis. We hypothesize that communication between desmosome junctions and cardiac dyads is essential for maintaining Ca2+ homeostasis and that ERK1/2 activation-induced loss of integrin ?1D impairs this ?desmosome-dyad crosstalk? thereby promoting RyR2-dependent and catecholamine-sensitive arrhythmogenesis and fibrotic infiltration in ARVC. We further hypothesize that interventions targeting this pathway may offer a promising approach for treating ARVC. To test our hypothesis, we have generated three congenic knock-in mouse models using CRISPR-Cas9 that contain mutations equivalent to those we identified from human ARVC patients. In Aim 1, we will determine the pathogenicity of knock-in ARVC mutations in recapitulating cardiac remodeling, catecholamine-induced arrhythmogenesis and desmosome-dyad crosstalk in mutant ARVC mice. In Aims 2 and 3, we will test whether mutation-induced ARVC phenotypes can be effectively prevented through ERK1/2 (Aim 2) and RyR2 (Aim 3) inhibition. We expect our studies to show that life-threatening ventricular arrhythmias and heart failure from ARVC can be therapeutically managed by modulating desmosome-dyad crosstalk and attenuating Ca2+ handling dysfunction.

PROJECT SUMMARY Junctophilin 2 (JP2) is an essential structural protein required for the formation of junctional couplings (i.e., cardiac dyads) between the transverse (T)-tubule membrane and the sarcoplasmic reticulum (SR). JP2 function is therefore fundamental for the local control of Ca2+-induced Ca2+ release and efficient contraction in ventricular myocytes during cardiac excitation-contraction (E-C) coupling. JP2 protein levels progressively decline in failing human hearts and in animal models of heart failure leading to T-tubule remodeling and loss of E-C coupling function. The downregulation of JP2 at E-C coupling sites is in part due to specific cleavage by the Ca2+-activated protease calpain that is implicated in a variety of heart diseases. During the previous funding period, we demonstrated that stress- and calpain-dependent cleavage of JP2 liberates a novel, nuclear translocating, N- terminal fragment (JP2NT) that represses maladaptive transcriptional reprogramming in diseased hearts, thus transducing E-C uncoupling information into a unique cardio-protective excitation-transcription (E-T) coupling signal to the nucleus. However, how JP2-mediated E-C and E-T coupling phenomena are mechanistically regulated remains to be determined. Our new preliminary results show that JP2 is reproducibly phosphorylated in stressed hearts near regions responsible for JP2 cleavage and the subcellular localization of JP2NT. In this competitive renewal application, we aim to define how stress-induced post-translational modifications regulate the structure, localization, and function of JP2/JP2NT. We hypothesize that JP2NT-mediated E-T coupling is tightly regulated by cardiac stress-dependent phosphorylation of JP2 that determines JP2 sensitivity to calpain and JP2NT nuclear translocation and transcriptional activity. To test our hypothesis, in Aim 1, we will use mutation analysis and cell models to determine how JP2 phosphorylation regulates E-C coupling and cleavage-induced JP2NT generation, nuclear translocation and transcriptional regulation. In Aim 2, we will utilize our novel JP2 calpain resistant mice in combination with JP2NT overexpression to determine how these targeted approaches modulate cardiac responses to stress in vivo. We will determine how E-C coupling structure/function and cardiac gene transcription are altered in these mice in response to pressure overload and myocardial infarction. We expect our studies will provide significant insights into the regulatory mechanisms governing JP2/JP2NT function and their salutary contribution toward heart disease pathogenesis.