The Role of GSK3β in SERCA Dysfunction and the Development of Arrhythmogenic Cardiomyopathy

Abstract

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump catalyzes the active transport of Ca2+ into the sarcoplasmic reticulum and is responsible for over 70% of Ca2+ turnover after muscle contraction. Phospholamban (PLN) is a well-known SERCA regulator, and impaired SERCA function due to reductions in SERCA and/or enhanced inhibitory PLN action has been linked to the development of various cardiomyopathies. Arrhythmogenic cardiomyopathy (ACM) is an inherited, non-ischemic heart disease presenting with ventricular arrhythmias, hypo/dyskinesia, and fibrotic remodeling. Over 60% of ACM cases are caused by pathogenic variants in genes encoding the cardiac desmosome, where Desmoglein-2 (DSG2) is the second most prevalent desmosomal gene variant in the ACM patient population. Using Dsg2 mutant (Dsg2mut/mut) mice, previous studies showed that the enzyme glycogen synthase kinase-3β (GSK3β), plays a major role in ACM pathogenesis. Inhibiting GSK3β in these mice improved cardiac contractility and ACM pathological phenotypes. We have shown that GSK3β inhibition in cardiomyocytes can also improve SERCA function in wild-type (WT) mice; however, the role of GSK3β activity on SERCA function in Dsg2mut/mut mice is currently unknown. Here, we sought to examine the effects of GSK3β inhibition on SERCA function using a GSK3β-specific inhibitor, SB216763 (SB2), in Dsg2mut/mut mice. At 3 weeks of age, both WT and Dsg2mut/mut mice were treated with SB2 (2.5 mg/kg/day) or equivalent volume/kg/day of vehicle (DMSO) for 13 weeks via intraperitoneal injection. After treatment, mice were sacrificed and hearts were collected and homogenized. Our results show that cardiomyocytes obtained from vehicle treated Dsg2mut/mut mice had significantly lower SERCA2a expression (-52%, p<0.01) compared to vehicle treated WT mice, leading to reduced SERCA2a:PLN (-65%, p=0.04). Maximal SERCA activity was also significantly lower in vehicle treated Dsg2mut/mut mice (-65%, p=0.001). However, in myocardium from SB2 treated Dsg2mut/mut mice, maximal SERCA activity, SERCA2a expression, and SERCA2a:PLN ratio were all restored to near WT levels. We also examined the effects of heterozygous and homozygous GSK3β S9A mutation in the Dsg2mut/mut mice, which prevents phosphorylation and maintains GSK3β in its constitutively active form. When comparing WT with Dsg2mut/mutheterozygous and homozygous GSK3β S9A mutants, we saw a significant but non-progressive decrease in maximal SERCA activity (-60%, p=0.02 and -58%, p=0.03 respectively). In conclusion, these results show that SERCA function is negatively impacted in Dsg2mut/mut cardiomyocytes. Conversely, GSK3β inhibition with SB2 treatment improved SERCA content and activity to healthy WT levels. These results further illuminate the role of GSK3β in ACM pathogenesis and support the use of GSK3β inhibitors in its treatment, as our new promising results indicate this therapeutic strategy can also improve Ca2+ handling.