No abstract

The major underpinning of massive cell death associated with myocardial infarction involves opening of the mitochondrial permeability transition pore (mPTP), resulting in disruption of mitochondria membrane integrity and programmed necrosis. Studies in human lymphocytes suggested that the hematopoietic-substrate-1 associated protein X-1 (HAX-1) is linked to regulation of mitochondrial membrane function, but its role in controlling mPTP activity remains obscure. Herein we used models with altered HAX-1 expression levels in the heart and uncovered an unexpected role of HAX-1 in regulation of mPTP and cardiomyocyte survival. Cardiac-specific HAX-1 overexpression was associated with resistance against loss of mitochondrial membrane potential, induced by oxidative stress, whereas HAX-1 heterozygous deficiency exacerbated vulnerability. The protective effects of HAX-1 were attributed to specific down-regulation of cyclophilin-D levels leading to reduction in mPTP activation. Accordingly, cyclophilin-D and mPTP were increased in heterozygous hearts, but genetic ablation of cyclophilin-D in these hearts significantly alleviated their susceptibility to ischemia/reperfusion injury. Mechanistically, alterations in cyclophilin-D levels by HAX-1 were contributed by the ubiquitin-proteosomal degradation pathway. HAX-1 overexpression enhanced cyclophilin-D ubiquitination, whereas proteosomal inhibition restored cyclophilin-D levels. The regulatory effects of HAX-1 were mediated through interference of cyclophilin-D binding to heat shock protein-90 (Hsp90) in mitochondria, rendering it susceptible to degradation. Accordingly, enhanced Hsp90 expression in HAX-1 overexpressing cardiomyocytes increased cyclophilin-D levels, as well as mPTP activation upon oxidative stress. Taken together, our findings reveal the role of HAX-1 in regulating cyclophilin-D levels via an Hsp90-dependent mechanism, resulting in protection against activation of mPTP and subsequent cell death responses.HAX-1 | necrosis | mitochondrial permeability transition | cyclophilin-D | heat shock protein-90

The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.

HSPB6/Hsp20 (heat shock protein family B [small] member 6) has emerged as a novel cardioprotector against stress-induced injury. We identified a human mutant of HSPB6 (HSPB6) exclusively present in dilated cardiomyopathy (DCM) patients. Cardiac expression of this mutant in mouse hearts resulted in remodeling and dysfunction, which progressed to heart failure and early death. These detrimental effects were associated with reduced interaction of mutant HSPB6 with BECN1/Beclin 1, leading to BECN1 ubiquitination and its proteosomal degradation. As a result, autophagy flux was substantially inhibited and apoptosis was increased in HSPB6-mutant hearts. In contrast, overexpression of wild-type HSPB6 (HSPB6 WT) not only increased BECN1 levels, but also competitively suppressed binding of BECN1 to BCL2, resulting in stimulated autophagy. Indeed, preinhibition of autophagy attenuated the cardioprotective effects of HSPB6 WT. Taken together, these findings reveal a new regulatory mechanism of HSPB6 in cell survival through its interaction with BECN1. Furthermore, Ser10 appears to be crucial for the protective effects of HSPB6 and transversion of this amino acid to Phe contributes to cardiomyopathy.