Abstract Body: Cardiac disease is one of the leading causes of death around the world due to its complex etiology involving multiple molecular and cellular dysfunctions which the existing therapies fail to simultaneously resolve. Impaired calcium signaling and reduced cardiomyocyte survival underlie most cases with Heat Shock Protein 90β (HSP90β) being relevant in both the pathways. Past studies have shown that Heat Shock Protein 90β (HSP90β) at the sarcoplasmic reticulum (SR) interacts with HCLS1-associated protein X-1 (HAX-1) and Phospholamban (PLN) to inhibit sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) pump, the key regulator of cardiac calcium cycling and contractility. HSP90β also maintains crucial cardioprotective ER stress response during disease conditions like ischemia/reperfusion (I/R) injury by regulating inositol requiring enzyme 1 (IRE-1). Previous studies indicate that upregulated HAX-1 levels reduce HSP90β-IRE-1 interactions. Therefore, our goal is to examine if modulating the interaction of HSP90β with HAX1 using peptide-interference can improve cardiac contractility and cardiomyocyte survival under stress.
Employing recombinant protein binding assays, we determined the minimum binding region of HSP90β on HAX-1, which was then used as the interfering peptide for the HSP90β/HAX-1 interaction. To facilitate peptide entry into the cardiomyocytes, we tagged the Antennapedia sequence to the C-terminal end. Our in vitro studies show that the interfering peptide application significantly enhanced contractility and calcium kinetics in cardiomyocytes derived from human induced pluripotent stem cells, suggesting its potential benefits as an inotropic agent. Next, we examined the effect of the peptide upon Brefeldin A and hydrogen peroxide stresses and observed a significant improvement in cell viability upon peptide application. As HAX-1 shows both ER/SR and mitochondrial localizations, we generated AAV6 vectors coding for the interfering peptide targeted to the SR/ER, mitochondrial intermembrane space or matrix and tested their effects under stress. Our data indicates that the ER/SR targeted interfering peptide exerted the most protection under stress conditions. Thus, our findings suggest that HSP90/HAX-1 interaction at the ER/SR compartment may serve as a target to enhance cardioprotection and contractility under stress.