Search for cardiac calcium cycling gene mutations in familial ventricular arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia

<p>Abstract</p> <p>Background</p> <p>Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe inherited cardiac disorder caused by mutations predominantly in the ryanodine receptor (<it>RyR2</it>) gene. We sought to identify mutations in genes affecting cardiac calcium cycling in patients with CPVT and in less typical familial exercise-related ventricular arrhythmias.</p> <p>Methods and Results</p> <p>We recruited 33 consecutive patients with frequent ventricular premature complexes (VPCs) without structural heart disease and often history of syncope or sudden death in family. Sixteen of the patients featured a phenotype typical of CPVT. In 17 patients, VPCs emerged also at rest. Exercise stress test and echocardiography were performed to each patient and 232 family members. Familial background was evident in 42% of cases (n = 14). We sequenced all the coding exons of the <it>RyR2</it>, <it>FKBP1B</it>, <it>ATP2A2 </it>and <it>SLC8A1 </it>genes from the index patients. Single channel recordings of a mutant RyR2 were performed in planar lipid bilayers. Two novel <it>RyR2 </it>missense mutations (R1051P and S616L) and two <it>RyR2 </it>exon 3 deletions were identified, explaining 25% of the CPVT phenotypes. A rare variant (N3308S) with open probabilities similar to the wild type channels <it>in vitro</it>, was evident in a patient with resting VPCs. No disease-causing variants were detectable in the <it>FKBP1B</it>, <it>ATP2A2 </it>or <it>SLC8A1 </it>genes.</p> <p>Conclusion</p> <p>We report two novel CPVT-causing <it>RyR2 </it>mutations and a novel <it>RyR2 </it>variant of uncertain clinical significance in a patient with abundant resting VPCs. Our data also strengthen the previous assumption that exon 3 deletions of <it>RyR2 </it>should screened for in CPVT and related phenotypes.</p

Ca2+-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca2+ Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca2+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca2+. We show that hyperphosphorylation by exogenous PKA increases open probability (Po) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca2+; lowering [Ca2+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca2+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg2+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca2+. We show that the endogenous kinase is both Ca2+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca2+-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca2+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca2+ regulation

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: A coupled electromechanical modeling study

Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa;L); and the enhancement in SERCA pump activity via phosphorylation of PLB.Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated -adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa;L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa;L as the key mechanisms underlying the aforementioned positive FFR

Angiotensin II type 2 receptor agonists: where should they be applied?

Introduction: Angiotensin II, the active endproduct of the renin-angiotensin system (RAS), exerts its effects via angiotensin II type 1 and type 2 (AT(1), AT(2)) receptors. AT(1) receptors mediate all well-known effects of angiotensin II, ranging from vasoconstriction to tissue remodeling. Thus, to treat cardiovascular disease, RAS blockade aims at preventing angiotensin II-AT(1) receptor interaction. Yet RAS blockade is often accompanied by rises in angiotensin II, which may exert beneficial effects via AT(2) receptors. Areas covered: This review summarizes our current knowledge on AT(2) receptors, describing their location, function(s), endogenous agonist(s) and intracellular signaling cascades. It discusses the beneficial effects obtained with C21, a recently developed AT(2) receptor agonist. Important questions that are addressed are do these receptors truly antagonize AT(1) receptor-mediated effects? What about their role in the diseased state and their heterodimerization with other receptors? Expert opinion: The general view that AT(2) receptors exclusively exert beneficial effects has been challenged, and in pathological models, their function sometimes mimics that of AT(1) receptors, for example, inducing vasoconstriction and cardiac hypertrophy. Yet given its upregulation in various pathological conditions, the AT(2) receptor remains a promising target for treatment, allowing effects beyond blood pressure-lowering, for example, in stroke, aneurysm formation, inflammation and myoc

Calcium signalling: dynamics, homeostasis and remodelling

Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease