Kafa Walweel

Divergent Regulation of Ryanodine Receptor 2 Calcium Release Channels by Arrhythmogenic Human Calmodulin Missense Mutants

Rationale: Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. Objective: To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. Methods and Results: We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. Conclusions: A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca2+ release. Although dantrolene inhibits Ca2+ release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca2+ release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca2+] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca2+ release in the absence of CaM, but reduced Ca2+ wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

Divergent Regulation of Ryr2 Calcium Release Channels by Arrhythmogenic Human Calmodulin Missense Mutants

Rationale: Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. Objective: To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. Methods and Results: We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. Conclusions: A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.

The emerging role of calmodulin regulation of RyR2 in controlling heart rhythm, the progression of heart failure and the antiarrhythmic action of dantrolene.

Cardiac output and rhythm depend on the release and the take‐up of calcium from the sarcoplasmic reticulum (SR). Excessive diastolic calcium leak from the SR due to dysfunctional calcium release channels (RyR2) contributes to the formation of delayed after‐depolarizations, which underlie the fatal arrhythmias that occur in heart failure and inherited syndromes. Calmodulin (CaM) is a calcium‐binding protein that regulates target proteins and acts as a calcium sensor. CaM is comprised of two calcium‐binding EF‐hand domains and a flexible linker. CaM is an accessory protein that partially inhibits RyR2 channel activity. CaM is critical for normal cardiac function, and altered CaM binding and efficacy may contribute to defects in SR calcium release. The present paper reviews CaM binding to RyR2 and how it regulates RyR2 channel activity. It then goes on to review how mutations in the CaM amino acid sequence give rise to inherited syndromes such as Catecholaminergic Polymorphic Ventricular Tachychardia (CPVT) and long QT syndrome (LQTS). In addition, the role of reduced CaM binding to RyR2 that results from RyR2 phosphorylation or from oxidation of either RyR2 or CaM contributes to the progression of heart failure is reviewed. Finally, this manuscript reviews recent evidence that CaM binding to RyR2 is required for the inhibitory action of a pharmaceutical agent (dantrolene) on RyR2. Dantrolene is a clinically used muscle relaxant that has recently been found to exert antiarrhythmic effects against SR Ca2+ overload arrhythmias.

Calmodulin Mutants Linked to Catecholaminergic Polymorphic Ventricular Tachycardia Fail to Inhibit Human RyR2 Channels

Calmodulin (CaM) is a calcium-binding protein that can directly inhibit cardiac ryanodine receptor calcium release channels (ryanodine receptor 2 [RyR2]) [(1)][1]. CaM mutations can cause an autosomal-dominant form of catecholaminergic polymorphic ventricular tachycardia (CPVT), a syndrome

Mechanisms of SR calcium release in healthy and failing human hearts

Normal heart contraction and rhythm relies on the proper flow of calcium ions (Ca2+) into cardiac cells and between their intracellular organelles, and any disruption can lead to arrhythmia and sudden cardiac death. Electrical excitation of the surface membrane activates voltage-dependent L-type Ca2+ channels to open and allow Ca2+ to enter the cytoplasm. The subsequent increase in cytoplasmic Ca2+ concentration activates calcium release channels (RyR2) located at specialised Ca2+ release sites in the sarcoplasmic reticulum (SR), which serves as an intracellular Ca2+ store. Animal models have provided valuable insights into how intracellular Ca2+ transport mechanisms are altered in human heart failure. The aim of this review is to examine how Ca2+ release sites are remodelled in heart failure and how this affects intracellular Ca2+ transport with an emphasis on Ca2+ release mechanisms in the SR. Current knowledge on how heart failure alters the regulation of RyR2 by Ca2+ and Mg2+ and how these mechanisms control the activity of RyR2 in the confines of the Ca2+ release sites is reviewed.

Divergent Regulation of Ryanodine Receptor 2 Calcium Release Channels by Arrhythmogenic Human Calmodulin Missense MutantsNovelty and Significance

Rationale: Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. Objective: To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. Methods and Results: We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. Conclusions: A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.