Modeling the Effects of Genetic Manipulations of Calsequestrin on Local Calcium Release and Depletion in Cardiac Myocytes

Cardiac calsequestrin (CASQ2), a Ca buffer localized to the junctional SR (jSR) of cardiac myocytes, is known to bind to the RyR-triadin-junctin complex, participate in the luminal regulation of RyRs, and modulate Ca spark activity..

Genetic ablation of ryanodine receptor 2 phosphorylation at Ser‐2808 aggravates Ca 2+ ‐dependent cardiomyopathy by exacerbating diastolic Ca 2+ release

Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Ser‐2808 is suggested to mediate the physiological ‘fight or flight’ response and contribute to heart failure by rendering the sarcoplasmic reticulum (SR) leaky for Ca 2+ . In the present study, we examined the potential role of RyR2 phosphorylation at Ser‐2808 in the progression of Ca 2+ ‐dependent cardiomyopathy (CCM) by using mice genetically modified to feature elevated SR Ca 2+ leak while expressing RyR2s that cannot be phosphorylated at this site (S2808A). Surprisingly, rather than alleviating the disease phenotype, constitutive dephosphorylation of Ser‐2808 aggravated CCM as manifested by shortened survival, deteriorated in vivo cardiac function, exacerbated SR Ca 2+ leak and mitochondrial injury. Notably, the deteriorations of cardiac function, myocyte Ca 2+ handling, and mitochondria integrity were consistently worse in mice with heterozygous ablation of Ser‐2808 than in mice with complete ablation. Wild‐type (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Ser‐2808. Exposure of these CCM cells to protein phosphatase 1 caused a transitory increase in Ca 2+ leak attributable to partial dephosphorylation of RyR2 tetramers at Ser‐2808 from more fully phosphorylated state. Thus, exacerbated Ca 2+ leak through partially dephosphorylated RyR2s accounts for the prevalence of the disease phenotype in the heterozygous S2808A CCM mice. These results do not support the importance of RyR2 hyperphosphorylation in Ca 2+ ‐dependent heart disease, and rather suggest roles for the opposite process, the RyR2 dephosphorylation at this residue in physiological and pathophysiological Ca 2+ signalling.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106986/1/tjp6067.pd

Advanced glycation end product cross-link breaker attenuates diabetes-induced cardiac dysfunction by improving sarcoplasmic reticulum calcium handling

Diabetic heart disease is a distinct clinical entity that can progress to heart failure and sudden death. However, the mechanisms responsible for the alterations in excitation-contraction coupling leading to cardiac dysfunction during diabetes are not well known. Hyperglycemia, the landmark of diabetes, leads to the formation of advanced glycation end products (AGEs) on long-lived proteins, including sarcoplasmic reticulum (SR) Ca2+ regulatory proteins. However, their pathogenic role on SR Ca2+ handling in cardiac myocytes is unknown. Therefore, we investigated whether an AGE cross-link breaker could prevent the alterations in SR Ca2+ cycling that lead to in vivo cardiac dysfunction during diabetes. Streptozotocin-induced diabetic rats were treated with alagebrium chloride (ALT-711) for 8 weeks and compared to age-matched placebo-treated diabetic rats and healthy rats. Cardiac function was assessed by echocardiographic examination. Ventricular myocytes were isolated to assess SR Ca2+ cycling by confocal imaging and quantitative Western blots. Diabetes resulted in in vivo cardiac dysfunction and ALT-711 therapy partially alleviated diastolic dysfunction by decreasing isovolumetric relaxation time and myocardial performance index (MPI) (by 27 and 41% vs. untreated diabetic rats, respectively, P < 0.05). In cardiac myocytes, diabetes-induced prolongation of cytosolic Ca2+ transient clearance by 43% and decreased SR Ca2+ load by 25% (P < 0.05); these parameters were partially improved after ALT-711 therapy. SERCA2a and RyR2 protein expression was significantly decreased in the myocardium of untreated diabetic rats (by 64 and 36% vs. controls, respectively, P < 0.05), but preserved in the treated diabetic group compared to controls. Collectively, our results suggest that, in a model of type 1 diabetes, AGE accumulation primarily impairs SR Ca2+ reuptake in cardiac myocytes and that long-term treatment with an AGE cross-link breaker partially normalized SR Ca2+ handling and improved diabetic cardiomyopathy.Peer reviewedPhysiological Science

Sexual Dimorphism in Bidirectional Sr-Mitochondria Crosstalk in Ventricular Cardiomyocytes

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females

Protein Phosphatases Decrease Sarcoplasmic Reticulum Calcium Content by Stimulating Calcium Release in Cardiac Myocytes

Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation−contraction coupling; furthermore abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca2+ sparks and SR Ca2+ load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca2+ sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca2+ stores, as determined by application of caffeine. These changes in Ca2+ release and SR Ca2+ load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR-mediated SR Ca2+ release leading to depleted SR Ca2+ stores in cardiac myocytes