Functional Role of Calstabin2 in Age-related Cardiac Alterations

Abstract

Calstabin2 is a component of the cardiac ryanodine receptor (RyR2) macromolecular complex, which modulates Ca2+ release from the sarcoplasmic reticulum in cardiomyocytes. Previous reports implied that genetic deletion of Calstabin2 leads to phenotypes related to cardiac aging. However, the mechanistic role of Calstabin2 in the process of cardiac aging remains unclear. To assess whether Calstabin2 is involved in age-related heart dysfunction, we studied Calstabin2 knockout (KO) and control wild-type (WT) mice. We found a significant association between deletion of Calstabin2 and cardiac aging. Indeed, aged Calstabin2 KO mice exhibited a markedly impaired cardiac function compared with WT littermates. Calstabin2 deletion resulted also in increased levels of cell cycle inhibitors p16 and p19, augmented cardiac fibrosis, cell death, and shorter telomeres. Eventually, we demonstrated that Calstabin2 deletion resulted in AKT phosphorylation, augmented mTOR activity, and impaired autophagy in the heart. Taken together, our results identify Calstabin2 as a key modulator of cardiac aging and indicate that the activation of the AKT/mTOR pathway plays a mechanistic role in such a process.

Calcium release channel RyR2 regulates insulin release and glucose homeostasis

“…Islet homogenates were lysed in 150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% NP-40, 0.4% deoxycholic acid, 1 mM Na 3 VO 4 , and complete protease inhibitors. RyR2 was immunoprecipitated from precleared lysates with 4 μg anti-RyR2 antibody (22,26,74). After 3 hours incubation, 30 μl of 50% slurry of protein A-Sepharose were added to each sample and incubated for an additional hour at 4°C.…”

“…Oxidation and Cys-nitrosylation of RyR were determined as described (21). Immunoblotting was performed as previously described (26,75,76) using the following antibodies: calstabin2 (catalog sc-98742, Santa Cruz Biotechnology), Kir6.2 (catalog ab95317, Abcam), and GAPDH (catalog 2118, Cell Signaling Technology).…”

“…Ca 2+ measurements. Ca 2+ imaging assays were performed as previously described (21,22,26,27,73). Briefly, islets were dispersed using 5 mg/ml dispase (Roche) attached on poly-l-ornithine-coated (PORNcoated) (Sigma-Aldrich) glass-bottom culture dishes (MatTek Corp.) loaded with Fura-2 AM (5 μM, 15 minutes, 37°C Ryanodine (20 μM) was used to prove that the RyR channel was the specific source of the leak.…”

“…We studied knockin mice harboring RyR2 containing mutations that were discovered in humans with a genetic form of exercise-induced sudden cardiac death syndrome known as catecholaminergic polymorphic ventricular tachycardia (CPVT), which has a 50% mortality rate by 35 years of age (23)(24)(25). Each RyR2 protomer binds one calstabin2, a subunit that stabilizes the closed state of the channel and prevents the pathological leak of Ca 2+ (18,19,26). Dissociation of calstabin2 from RyR2 results in intracellular Ca 2+ leak via RyR2 channels (20,23,27).…”

Nitric oxide mediates stretch-induced Ca2+ oscillation in smooth muscle

“…Previous studies have demonstrated that RYR gating underlies Ca 2+ release in smooth muscle (Herrera and Nelson, 2002;Nelson et al, 1995;Yuan et al, 2014;Zheng et al, 2010) and stretch-induced Ca 2+ sparks (Ibrahim et al, 2012;Iribe et al, 2009;Ji et al, 2002;Wei et al, 2008), as these events are eliminated by pre-exposure of myocytes to ryanodine. In the present study, we used a similar approach to determine whether stretch-induced Ca 2+ oscillations were inhibited by the application of ryanodine and/or the selective IP3R inhibitor xestospongin C in mouse bladder smooth muscle.…”