Spontaneous Ca2+ release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca2+ release (SOICR) can result in Ca2+ waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here, we show that a point mutation E4872A in the helix bundle crossing (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, RyR2 Ca2+ activation. Introducing metal-binding histidines at this site converts RyR2 into a luminal Ni2+ gated channel. Mouse hearts harboring an RyR2 mutation at this site (E4872Q+/−) are resistant to store overload-induced Ca2+ waves and completely protected against Ca2+-triggered VTs. These data show that the RyR2 gate directly senses store Ca2+, explaining RyR2 store Ca2+ regulation, Ca2+ wave initiation, and Ca2+-triggered arrhythmias. This novel store-sensing gate structure is conserved in all RyRs and inositol 1,4,5-trisphosphate receptors.

Mutants of HERG, the human form of ERG (the ether-a-go-go-related K+ channel gene), are responsible for some forms of the long-QT syndrome, an abnormality of cardiac repolarization. HERG was cloned from brain and has properties similar but not identical to the rapidly activating component of the native cardiac K+ channel current (Ikr). We identified in the mouse an alternatively processed form of ERG (MERG B) that is expressed abundantly in heart but only in trace amounts in brain. MERG B has a unique 36-amino acid NH2-terminal domain that is strongly basic and considerably shorter than the 376-amino acid NH2-terminal domain of HERG. When expressed in Xenopus oocytes, the kinetics of activation and deactivation of the MERG B current were best fit by a biexponential function, with the fast components dominant over the slow components. The fast component of activation had a mean tau value of 163 +/- 16 ms at -20 mV and 8 +/- 4 ms at +20 mV (n = 4). The fast component of deactivation had a mean tau value of 145 +/- 29 ms at -20 mV and 12 +/- 4 ms at -90 mV (n = 4). The MERG B current was blocked by the selective IKr blocker, dofetilide, with an IC50 of 54 nmol/L. In addition, we isolated HERG B, the human homologue of MERG B, which has electrophysiological characteristics qualitatively similar to those of MERG B. We have identified ERG B, an alternatively processed isoform of the ERG gene, expressed selectively in heart and with electrophysiological characteristics similar to those of native cardiac IKr.

Expression of cardiac transient outward current and inwardly rectifying K+ current is age dependent. However, little is known about age-related changes in cardiac delayed rectifier K+ current (IK, with rapidly and slowly activating components, IKr and IKs, respectively). Accordingly, the purpose of the present study was to assess developmental changes in IK channels in fetal, neonatal, and adult mouse ventricles. Three techniques were used: conventional microelectrode to measure the action potential, voltage clamp to record macroscopic currents of IK, and radioligand assay to examine [3H]dofetilide binding sites. The extent of prolongation of action potential duration at 95% repolarization (APD95) by a selective IKr blocker, dofetilide (1 mumol/L), dramatically decreased from fetal (137% +/- 18%) to day-1 (75% +/- 29%) and day-3 (20% +/- 15%) neonatal mouse ventricular tissues (P < .01). Dofetilide did not prolong APD95 in adult myocardium. IKr is the sole component of IK in day-18 fetal mouse ventricular myocytes. However, both IKr and IKs were observed in day-1 neonatal ventricular myocytes. With further development, IKs became the dominant component of IK in day-3 neonates. In adult mouse ventricular myocytes, neither IKr nor IKs was observed. Correspondingly, a high-affinity binding site for [3H]dofetilide was present in fetal mouse ventricles but was absent in adult ventricles. The complementary data from microelectrode, voltage-clamp, and [3H]dofetilide binding studies demonstrate that expression of the IK channel is developmentally regulated in the mouse heart.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) capture patient-specific genotype-phenotype relationships, as well as cell-to-cell variability of cardiac electrical activity r Computational modelling and simulation provide a high throughput approach to reconcile multiple datasets describing physiological variability, and also identify vulnerable parameter regimes r We have developed a whole-cell model of iPSC-CMs, composed of single exponential voltage-dependent gating variable rate constants, parameterized to fit experimental iPSC-CM outputs r We have utilized experimental data across multiple laboratories to model experimental variability and investigate subcellular phenotypic mechanisms in iPSC-CMs r This framework links molecular mechanisms to cellular-level outputs by revealing unique subsets of model parameters linked to known iPSC-CM phenotypes Abstract There is a profound need to develop a strategy for predicting patient-to-patient vulnerability in the emergence of cardiac arrhythmia. A promising in vitro method to address patient-specific proclivity to cardiac disease utilizes induced pluripotent stem cell-derived Divya Kernik is currently a PhD candidate in Biomedical Engineering at the University of California, Davis. She obtained a BS in Biomedical Engineering from Johns Hopkins University. The focus of her PhD work has been the development of computational methods that help to understand human-derived cardiac cells, as reported in the present study. In the future, she aims to continue to use computational modelling to address questions in cardiac physiology and pharmacology, with the underlying goal of incorporating human diversity throughout these efforts.

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An increase in regional QT interval dispersion during class Ia antiarrhythmic drug therapy is associated with torsade de pointes. Chronic amiodarone therapy in patients with a history of class Ia drug-induced torsade de pointes produces comparable maximum QT interval prolongation but does not increase QT interval dispersion. This characteristic may explain its apparent safe use in patients with a history of class Ia drug-induced torsade de pointes.

Developmental changes in the transient outward K+ current (Ito) in mouse ventricular myocytes were assessed by the whole-cell patch-clamp technique. The density of Ito in mouse ventricular myocytes was significantly increased from the day-1 neonate to the adult. At +50 mV, the density of Ito was 3 +/- 1 pA/pF in the day-1 neonate, 15 +/- 3 pA/pF in the day-14 neonate, and 19 +/- 4 pA/pF in the adult (P < .01). Unlike other species, the rate of Ito inactivation significantly slowed in mouse ventricular cells during development. Moreover, the time courses of inactivation and recovery from inactivation of Ito were well described by a monoexponential function in day-1 neonatal cells, whereas they were best fitted by a biexponential function in day-14 neonatal and adult cells. The characteristics of steady state inactivation were also significantly different in day-1 neonatal cells (half-inactivation potential [Vh] = -66 +/- 4 mV, slope factor [k] = 12 +/- 2 mV), in day-14 neonatal cells (Vh = -40 +/- 3 mV, k = 13 +/- 1 mV), and in adult cells (Vh = -34 +/- 4 mV, k = 6 +/- 1 mV). Microelectrode studies revealed that action potential duration progressively decreased in mouse ventricles during normal postnatal development. In addition, 4-aminopyridine (1 mmol/L) prolonged action potential duration more in adult than in neonatal mouse ventricles, suggesting that the developmental increase in the density of Ito contributes to the age-related shortening of action potential duration in mouse ventricles. In conclusion, Ito in adult mouse ventricular myocytes exhibits a higher density, slower inactivation kinetics, and a relatively more positive half-inactivation potential. All these characteristics result in Ito being a physiologically more important repolarizing K+ current in adult than in neonatal mouse hearts.

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1 cleavage in or WT mice that received WT bone marrow than in WT mice that received bone marrow. Intravital microscopy of the kidney in mice 5-6 days after UUO demonstrated the significant recruitment of GFP proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CXCR1 renal phagocytes. , macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1, as well as the formation of AIM2 ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1 levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.