Structural and functional interactions between the EF hand domain and S2-S3 loop in the type-1 ryanodine receptor ion channel.

Previous cryo-electron micrographs suggested that the skeletal muscle Ca release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface.

Point mutations in RyR2 Ca2+-binding residues of human cardiomyocytes cause cellular remodelling of cardiac excitation contraction-coupling.

Aims: CRISPR/Cas9 gene edits of cardiac ryanodine receptor (RyR2) in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide a novel platform for introducing mutations in RyR2 Ca2+-binding residues and examining the resulting excitation contraction (EC)-coupling remodelling consequences.

Methods And Results: Ca2+-signalling phenotypes of mutations in RyR2 Ca2+-binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and total internal reflection fluorescence-imaged wild type and mutant cardiomyocytes infected with sarcoplasmic reticulum (SR)-targeted ER-GCaMP6 probe. (i) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocytes; (ii) spontaneous beating (Fura-2 Ca2+ transients) persisted in mutant cells without the SR-release signals; (iii) while 5-20 mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; (iv) and 20 mM caffeine transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red.

Do CPVT-linked mutations alter RYR2 regulation by cytosolic Ca2+ in cardiomyocytes?

It is controversial whether the cardiac type-2 ryanodine receptor harboring a catecholaminergic polymorphic ventricular tachycardia-associated point mutation is regulated by luminal or cytosolic Ca. This commentary discusses new findings supporting the cytosolic Ca-dependent regulation.

Molecular Insights into Calcium Dependent Regulation of Ryanodine Receptor Calcium Release Channels.

Ryanodine receptor calcium release channels (RyRs) play central roles in controlling intracellular calcium concentrations in excitable and non-excitable cells. RyRs are located in the sarcoplasmic or endoplasmic reticulum, intracellular Ca storage compartment, and release Ca during cellular action potentials or in response to other cellular stimuli. Mammalian cells express three structurally related isoforms of RyR.

A central core disease mutation in the Ca-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation.

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca-mediated activation of skeletal muscle Ca release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca release measurements, single-channel recordings, and computational methods.

The effect of reactive oxygen species on cardiomyocyte differentiation of pluripotent stem cells.

The coordination of metabolic shift with genetic circuits is critical to cell specification, but the metabolic mechanisms that drive cardiac development are largely unknown. Reactive oxygen species (ROS) are not only the by-product of mitochondrial metabolism, but play a critical role in signalling cascade of cardiac development as a second messenger. Various levels of ROS appear differential and even oppose effect on selfrenewal and cardiac differentiation of pluripotent stem cells (PSCs) at each stage of differentiation.

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin.

The mammalian ryanodine receptor Ca release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca. This suggests isoform-specific domains are involved in RyR regulation by CaM.

Mechanisms of spontaneous pacing: sinoatrial nodal cells, neonatal cardiomyocytes, and human stem cell derived cardiomyocytes.

The sinoatrial (SA) node is the primary site from which the mammalian heart is paced, but the mechanisms underlying the pacemaking still remain clouded. It is generally believed that the hyperpolarization-activated current I, encoded by hyperpolarization-activated cyclic nucleotide-gated (HCN) genes, contributes significantly to pacing, which in tandem with inward current generated by efflux of Ca via the Na-Ca exchanger (NCX), resulting from the released Ca, mediates the diastolic depolarization. Here, we review the data that implicate I as the "pacemaker current" and conclude that there is not only a significant discrepancy between the range of diastolic depolarization potential (-60 to -40 mV) and the activation potential of I (negative to -70 mV), but that also the kinetics of I and its pharmacology are incompatible with the frequency of a heartbeat in rodents and humans.

Cardiac progenitor cells engineered with Pim-1 (CPCeP) develop cardiac phenotypic electrophysiological properties as they are co-cultured with neonatal myocytes.

Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation-contraction coupling. This study aims to determine electrophysiology and Ca(2+)-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment.

Hypoxic regulation of cardiac Ca2+ channel: possible role of haem oxygenase.

Acute and chronic hypoxias are common cardiac diseases that lead often to arrhythmia and impaired contractility. At the cellular level it is unclear whether the suppression of cardiac Ca(2+) channels (Ca(V)1.2) results directly from oxygen deprivation on the channel protein or is mediated by intermediary proteins affecting the channel.