calpain and Arrhythmias--Cardiac

Calcium (Ca

Topics: Animals; Arrhythmias, Cardiac; Calcineurin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calpain; Computer Simulation; Electrophysiological Phenomena; Electrophysiology; Excitation Contraction Coupling; Humans; In Vitro Techniques; Membrane Proteins; Mice; Myocardial Contraction; Myocytes, Cardiac; Phosphoproteins; Phosphorylation; Protein Kinase C; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder with variable genetic etiologies. Here we focused on understanding the precise molecular pathology of a single clinical variant in DSP, the gene encoding desmoplakin. We initially identified a novel missense desmoplakin variant (p.R451G) in a patient diagnosed with biventricular ACM. An extensive single-family ACM cohort was assembled, revealing a pattern of coinheritance for R451G desmoplakin and the ACM phenotype. An in vitro model system using patient-derived induced pluripotent stem cell lines showed depressed levels of desmoplakin in the absence of abnormal electrical propagation. Molecular dynamics simulations of desmoplakin R451G revealed no overt structural changes, but a significant loss of intramolecular interactions surrounding a putative calpain target site was observed. Protein degradation assays of recombinant desmoplakin R451G confirmed increased calpain vulnerability. In silico screening identified a subset of 3 additional ACM-linked desmoplakin missense mutations with apparent enhanced calpain susceptibility, predictions that were confirmed experimentally. Like R451G, these mutations are found in families with biventricular ACM. We conclude that augmented calpain-mediated degradation of desmoplakin represents a shared pathological mechanism for select ACM-linked missense variants. This approach for identifying variants with shared molecular pathologies may represent a powerful new strategy for understanding and treating inherited cardiomyopathies.

Topics: Adult; Arrhythmias, Cardiac; Calpain; Cardiomyopathies; Desmoplakins; Female; Genetic Predisposition to Disease; Glycine; Heart; Heart Failure; Humans; Male; Middle Aged; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Mutation, Missense; Pedigree; Phenotype; Recombinant Proteins; Stem Cells

During the cardiac cycle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR2) channel complex is controlled by the levels of cytosolic and luminal Ca(2+) and alterations in these regulatory processes have been implicated in cardiac disease including arrhythmia. To better understand the mechanisms of regulation of SR Ca(2+) release by Ca(2+) on both sides of the SR membrane, we investigated SR Ca(2+) release in a wide range of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt); 1-100 microm) in permeabilized canine ventricular myocytes by monitoring [Ca(2+)] inside the SR ([Ca(2+)](SR)). Exposing myocytes to activating [Ca(2+)](cyt) resulted in spontaneous oscillations of [Ca(2+)](SR) due to periodic opening and closing of the RyR2s. Elevating [Ca(2+)](cyt) (up to 10 microm) increased the frequency of [Ca(2+)](SR) oscillations; however at higher [Ca(2+)](cyt) (>50 microm) the oscillations diminished due to RyR2s staying perpetually open, resulting in depleted SR. Ablation of cardiac calsequestrin (CASQ2) altered the [Ca(2+)](cyt) dependence of Ca(2+) release oscillations such that oscillations were highly frequent at low [Ca(2+)](cyt) (100 nm) but became diminished at moderate [Ca(2+)](cyt) (10 microm), as determined in myocytes from calsequestrin-null versus wild-type mice. Our results suggest that under conditions of continuous activation by cytosolic Ca(2+), RyR2s can periodically cycle between open and deactivated states due to effects of luminal Ca(2+). Deactivation at reduced [Ca(2+)]SR appears to involve reduction of sensitivity to cytosolic Ca(2+) and might be mediated by CASQ2. Inactivation by cytosolic Ca(2+) plays no detectable role in controlling SR Ca(2+) release.

Topics: Animals; Arrhythmias, Cardiac; Caffeine; Calcium; Calcium Signaling; Calpain; Calsequestrin; Central Nervous System Stimulants; Dogs; Humans; Male; Mice; Myocytes, Cardiac; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

Topics: Animals; Arrhythmias, Cardiac; Caffeine; Calcium; Calcium Signaling; Calpain; Calsequestrin; Central Nervous System Stimulants; Dogs; Humans; Male; Mice; Myocytes, Cardiac; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum