47 research outputs found
Ryanodine Receptor Use-Dependent Block Suppresses Ca2+ Waves in Permeabilized Casq2-/- and RyR2-R4496C Myocytes
Slow Diffusion Across Cell Membrane Delays Onset of RyR2-Channel Block and Ca Wave Suppression by Flecainide in Intact Myocytes
Genetically De-Sensitizing Myofilaments to Ca in a Mouse Model for Hypertrophic Cardiomyopathy Decreases Arrhythmia Incidence
Ca2+ Sensitizing Troponin T Mutations Linked To Hypertrophic Cardiomyopathy Increase Apparent Cytosolic Ca2+ Binding
Inhibition of Cardiac Ca2+ Release Channels by Class I Anti Arrhythmic Drugs as Therapy for Arrhythmia
Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca 2+ -binding protein changes in CSQ2 knockout mice
Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n = 8) contained 24 ± 2 ÎŒmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430 ± 20 ÎŒmol/kg (with SR Ca2+ likely <250 ÎŒmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of âŒ10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (âŒ1 mmol Ca2+/kg ventricular tissue, assuming a maximum of âŒ40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidinerich Ca2+-binding protein was upregulated âŒ35% in ventricular tissue, possibly in compensation
Genetic ablation of ryanodine receptor 2 phosphorylation at Serâ2808 aggravates Ca 2+ âdependent cardiomyopathy by exacerbating diastolic Ca 2+ release
Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Serâ2808 is suggested to mediate the physiological âfight or flightâ 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 Serâ2808 in the progression of Ca 2+ âdependent 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 Serâ2808 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 Serâ2808 than in mice with complete ablation. Wildâtype (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Serâ2808. 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 Serâ2808 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+ âdependent 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
Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin-Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral-mediated delivery to alleviate arrhythmias in non-CaM-related CPVT