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• Introducing W4645R-RyR2 mutation in hiPSC CMs via CRISPR/Cas9 unveiled insights into RyR2 Ca2+-binding regulation and its interplay with the caffeine-binding site. • W4645R-RyR2 cardiomyocytes show similar I Ca magnitude but exhibit a slight reduction in CICR compared to WT myocytes. • W4645R-RyR2 cardiomyocytes uniquely lack caffeine-triggered SR Ca2+ release, pointing to a disruption in intracellular calcium regulation. • W4645R-RyR2 mutation results in a significantly larger SR Ca2+ leak, causing a 40 % reduction in SR Ca2+ content, as observed with 4-CmC. • W4645R-RyR2 cardiomyocytes have Diminished Calcium Sparks and Spontaneous SR Ca2+ Releases. Previous studies have identified RyR2 W4645R mutation, located in the caffeine-binding site, to associate with CPVT1 pathology. Caffeine binding to its site is thought to displace the carboxyl-terminal domain to Ca2+-binding, allowing the tryptophan residue (W4645) to regulate Ca2+ sensitivity of RyR2. To gain insights into regulation of RyR2 Ca2+-binding and its interaction with caffeine-binding site, we introduced W4645R-RyR2 point mutation via CRISPR/Cas9 gene-editing in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC CMs) and characterized their Ca2+-signaling phenotype compared to WT hiPSC CMs. W4645R-RyR2 cardiomyocytes had: (1) no significant change in I Ca magnitude or voltage-dependence; (2) slightly reduced CICR; (3) altered relaxation kinetics of Ca2+-transients with no change in isoproterenol sensitivity; (4) complete loss of caffeine-triggered Ca2+ release; (5) larger SR Ca2+ leak resulting in 40 % lower SR Ca2+ content, as determined by myocytes' response to 4-CmC; (6) lower incidence of calcium sparks and asynchronous spontaneous SR Ca2+ releases. W4645R-RyR2 mutation induces loss of caffeine-triggered SR Ca2+ release and enhances SR Ca2+ leak that underlie asynchronous spontaneous Ca2+ releases, triggering arrhythmia and impairing cardiac function. [Display omitted] [ABSTRACT FROM AUTHOR]

Highlights • Human Induced Pluripotent Stem Cell–derived CMs express two cell populations based on inactivation kinetics of L-type I Ca (τ i 20ms & 50ms). • The diverse expression of slowly and rapidly inactivating I Ca in hiPSC-CMs is responsible for variability in action potential morphology. • Suppressive effects of hypoxia were larger in slowly vs rapidly inactivating I Ca , suggesting CDI protects the channels against hypoxia. • The varied inhibition of I Ca in both cell types disappeared when Ba2+ was the charge carrier, suggesting critical role for CDI in hypoxic response. • Inhibition of focal Ca2+ transients by hypoxia or acidosis was more consistent with suppressive effects of these interventions on SR Ca2+ content. Abstract Aims The effects of acute (100 s) hypoxia and/or acidosis on Ca2+ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time. Methods and results 1) hiPSC-CMs express two cell populations: rapidly-inactivating I Ca myocytes (τ i <40 ms, in 4–5 day cultures) and slowly-inactivating I Ca (τ i ≥ 40 ms, in 6–8 day cultures). 2) Hypoxia suppressed I Ca by 10–20% in rapidly- and 40–55% in slowly-inactivating I Ca cells. 3) Isoproterenol enhanced I Ca in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba2+ was the charge carrier through the calcium channels, implicating Ca2+-dependent inactivation in O 2 sensing. 5) Acidosis suppressed I Ca by ∼35% and ∼25% in rapidly and slowly inactivating I Ca cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ∼25% in global and ∼37% in RyR2 Ca2+-microdomains in either cell type, whereas with hypoxia suppression was ∼20% and ∼25% respectively in global and RyR2-microdomaine in rapidly and ∼35% and ∼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells. Conclusions Variability in I Ca inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca2+-signaling in the two-cell types arises from differential Ca2+-dependent inactivation of the Ca2+-channel caused by proximity of Ca2+-release stores to the Ca2+ channels. [ABSTRACT FROM AUTHOR]

• CRISPR/Cas9 gene-editing at the RyR2 FKBP binding site (N771D-RyR2) in hiPSC CMs. • The parallel skeletal mutation (N760D-RyR1) is related to CCD and arrhythmogenesis. • The N771D-RyR2 mutation induced cellular arrhythmogenesis. • Calcium signaling from N771D-RyR2 show suppressed I Ca , SR Ca2+ content and leak. Aims: To gain insights into FKBP regulation of cardiac ryanodine receptor (RyR2) and Ca2+ signaling, we introduced the point mutation (N771D-RyR2) corresponding to skeletal muscle mutation (N760D-RyR1) associated with central core disease (CCD) via CRISPR/Cas9 gene-editing in the RyR2 FKBP binding site expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC CMs). Patients inflicted with CCD and other hereditary skeletal muscle diseases often show higher incidence of atrial or ventricular arrhythmias. Methods and Results: Ca2+ imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSC CMs showed: (1) ∼30% suppressed I Ca with no significant changes in the gating kinetics of I Ca ; (2) 29% lower SR Ca2+ content and 33% lower RyR2 Ca2+ leak; (3) higher CICR gain and 30–35% increased efficiency of I Ca -triggered Ca 2± release ; (4) higher incidence of aberrant SR Ca2+ releases, DADs, and Ca2+ sparks; (5) no change in fractional Ca2+-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern. Conclusions: The more frequent spontaneous Ca2+ releases and longer Ca2+ sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Ca2± leak and SR content result from suppressed I Ca that is compensated by higher CICR gain. [Display omitted] [ABSTRACT FROM AUTHOR]

It is known that chronic ethanol (EtOH) consumption leads to hypertension development and has been associated with deleterious effects on the cardiovascular system. Whether this condition alters calcium (Ca 2+ ) signaling and exocytosis in adrenal chromaffin cells (CCs) as the case is for genetic hypertension, is unknown. We explored this question in four randomized experimental groups, male Wistar Kyoto (WKY/EtOH) and Spontaneously Hypertensive (SHR/EtOH) rats were subjected to the intake of increasing EtOH concentrations (5–20%, for 30 days) and their respective controls (WKY/Control and SHR/Control) received water. WKY/EtOH developed hypertension and cardiac hypertrophy; blood aldehyde dehydrogenase (ALDH) and H 2 O 2 were also augmented. In comparison with WKY/Control, CCs from WKY/EtOH had the following features: (i) depolarization and higher frequency of spontaneous action potentials; (ii) decreased Ca 2+ currents with slower inactivation; (iii) decreased K + currents; (iv) augmented K + -elicited cytosolic Ca 2+ transients ([Ca 2+ ] c ); (v) enhanced K + -elicited catecholamine release. These cardiovascular, blood and CCs changes were qualitatively similar to those undergone by SHR/Control and SHR/EtOH. The results suggest that the hypertension elicited by chronic EtOH has pathogenic features common to genetic hypertension namely, augmented [Ca 2+ ] c transients and catecholamine release from their CCs. [ABSTRACT FROM AUTHOR]

Abstract: The cardiovascular protecting effect of the grape fruit trans-resveratrol has been explained among other factors, through augmentation of nitric oxide (NO) production in cardiovascular tissues. Another effect of low resveratrol concentration is the inhibition of single-vesicle quantal release of catecholamine from bovine adrenal chromaffin cells, that was recently suggested to be an additional factor contributing to its beneficial cardiovascular effects. We have investigated here the effects of a low concentration of trans-resveratrol (1μM) on Ca2+ and NO signaling pathways in bovine chromaffin cells, in an attempt to understand the mechanism underlying its previously reported inhibitory effects on quantal secretion. In cells loaded with fura-2 acetoxymethyl ester (fura-2), we have found that 1μM resveratrol produces a transient elevation of the cytosolic Ca2+ concentration ([Ca2+]c). This Ca2+ transient was drastically reduced when the Ca2+ store was depleted by ryanodine and dantrolene; it was also inhibited by Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Furthermore, the Ca2+ transient was mimicked by NO donor S-nitroso-N-acetyl-penicillamine (SNAP). Resveratrol also enhanced the production of nitrites and NO, and L-NAME blocked both responses; in contrast, augmentation by SNAP of nitrites and NO was unaffected by ODQ and was only partially inhibited by L-NAME. On the basis of these results, we are proposing that resveratrol is mitigating the catecholamine surge occurring during stress, through its ability to elicit mild local [Ca2+]c transients and enhanced NO production, that blocks the last steps of exocytosis. [Copyright &y& Elsevier]