Arrhythmogenic Ca2 Research Articles

I CaL inhibition prevents arrhythmogenic Ca2+ waves caused by abnormal Ca2+ sensitivity of RyR or SR Ca2+ accumulation

Arrhythmogenic Ca(2+) waves result from uncontrolled Ca(2+) release from the sarcoplasmic reticulum (SR) that occurs with increased Ca(2+) sensitivity of the ryanodine receptor (RyR) or excessive Ca(2+) accumulation during β-adrenergic stimulation. We hypothesized that inhibition of the L-type Ca(2+) current (I(CaL)) could prevent such Ca(2+) waves in both situations. Ca(2+) waves were induced in mouse left ventricular cardiomyocytes by isoproterenol combined with caffeine to increase RyR Ca(2+) sensitivity. I(CaL) inhibition by verapamil (0.5 µM) reduced Ca(2+) wave probability in cardiomyocytes during electrostimulation, and during a 10 s rest period after ceasing stimulation. A separate type of Ca(2+) release events occurred during the decay phase of the Ca(2+) transient and was not prevented by verapamil. Verapamil decreased Ca(2+) spark frequency, but not in permeabilized cells, indicating that this was not due to direct effects on RyR. The antiarrhythmic effect of verapamil was due to reduced SR Ca(2+) content following I(CaL) inhibition. Computational modelling supported that the level of I(CaL) inhibition obtained experimentally was sufficient to reduce the SR Ca(2+) content. Ca(2+) wave prevention through reduced SR Ca(2+) content was also effective in heterozygous ankyrin B knockout mice with excessive SR Ca(2+) accumulation during β-adrenergic stimulation. I(CaL) inhibition prevents diastolic Ca(2+) waves caused by increased Ca(2+) sensitivity of RyR or excessive SR Ca(2+) accumulation during β-adrenergic stimulation. In contrast, unstimulated early Ca(2+) release during the decay of the Ca(2+) transient is not prevented, and merits further study to understand the full antiarrhythmic potential of I(CaL) inhibition.

Reacting to too much excitement: ROS overproduction elicits arrhythmogenic Ca2+ waves in the heart

The cardiac cycle is tightly regulated by a number of proteins that function to transfer the electrical stimulus to the contracting chambers of the heart. Movement of calcium ions (Ca2+) within the cell is central to the excitation–contraction coupling, and a key component in the modulation of the strength of each cycle. Increasing demand for greater cardiac output – through changes in myocardial loading or from exogenous stressors (e.g. exercise) – activates the sympathetic nervous system to initiate compensatory mechanisms to augment inotropic function. This occurs through cardiomyocyte β-adrenoreceptor activation. While this response ensures adequate cardiac output, there is evidence that chronic activation of β-adrenoreceptors can initiate maladaptive responses within contractile cells.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Global and Local Effects of Intracellular pH on Ca2+ Waves in Rat Ventricular Myocytes

Arrhythmogenic Ca2+ signalling may be influenced by alterations in intracellular pH (pHi), as found in myocardial ischaemia. Here we characterise the effects of altering pHi on Ca2+ waves in rat isolated ventricular myocytes.Ca2+ waves were induced by raising [Ca2+]o, and imaged confocally using fluo-3. Parallel measurements of pHi were made using cSNARF-1. pHi changes were induced by superfusion of acetate (20-80mM) or trimethylamine (20mM).Reducing pHi acutely decreased (to 16±6% of control) then increased (to 360±62% by 60s) wave frequency. Inhibiting the sarcolemmal Na+/H+ exchanger (NHE) with 30μM 5-(N,N-dimethyl)amiloride suppressed this secondary increase. A linear relationship was found between pHi and wave velocity: intracellular acidosis increased velocity up to 140±3%. Acidosis also slowed wave decay and increased diastolic Ca2+, while alkalosis decreased diastolic Ca2+.A stable longitudinal pHi gradient (pHi 6.6-7.3) was generated in single myocytes by microperfusing half of the cell with normal Tyrode and half with 80mM acetate, using a double-barrelled micropipette. With NHE inhibited, a decrease in wave frequency was seen in the acidic microdomain and an increase in the more alkaline microdomain. Conversely, wave velocity was increased in acidic and decreased in alkaline microdomains. Thus, wave properties map onto a spatial gradient of pHi.Wave velocity may be influenced by multiple pH-dependent components. Computational modelling of Ca2+ waves predicts that, at steady-state, sarco/endoplasmic reticulum Ca2+ ATPase inhibition decreases wave velocity, due to a fall in sarcoplasmic reticulum Ca2+ content (Swietach et al, 2010). However, decreasing the kon of Ca2+ buffers predicted an increase in wave velocity, suggesting the effect of pHi changes on velocity is multifactorial.We conclude that pHi heterogeneity locally regulates Ca2+ waves, suggesting that it may contribute to the heterogeneity of Ca2+ signalling observed in myocardial ischaemia.

Rate-Dependent X-Ros Signaling in Cardiac Ventricular Myocytes

X-ROS is the novel signaling pathway seen in ventricular myocytes1 and skeletal muscle2 that arises when cell stretch triggers local ROS (reactive oxygen species) production by NADPH oxidase 2. A sustained (10-20s) stretch produces a transient rise in ROS with a half-time of decay of ∼5 seconds. In cardiomyocytes, this ROS sensitizes local Ca2+ release channels (ryanodine receptors) in the sarcoplasmic reticulum. In normal heart cells, this produces a transient burst of Ca2+ sparks and ensures the fidelity of excitation-contraction coupling, but can trigger arrhythmogenic Ca2+ waves in diverse pathologies.In reports to date, X-ROS has been characterized in cells that were stretched and held at a constant length. However, this is not how a cardiomyocyte is stretched in situ - instead it is stretched (during diastole) and shortens (during systole) in rhythmic fashion. Additionally, this stretch is regulated by two factors that vary with changing physiologic demand: 1) preload (the amount of blood that fills the ventricles during diastole) grades the magnitude of cell stretch, and 2) heart rate varies the frequency of cell stretch. Thus we investigated how the magnitude and frequency of stretch affect X-ROS signaling. Briefly, we find a critical effect: rhythmic stretch elevates the steady state level of ROS production in the cell, and this level is graded by both the magnitude and frequency of stretch. In turn, the elevated ROS proportionately modulates Ca2+ spark rate. Thus our findings hold the critical implication that the redox state and Ca2+ signaling sensitivity of cardiomyocytes is coupled to mechanical changes and graded by both preload and heart rate.1. Prosser et al. “X-ROS signaling: Rapid mechano-chemo transduction in heart.” Science. 333, 1440 (2011 9 Sept).2. Khairallah et al. “Microtubules underlie dysfunction in Duchenne muscular dystrophy.” Sci. Signal. 5, ra56 (2012).

Inhibition of CaMKII phosphorylation of RyR2 prevents inducible ventricular arrhythmias in mice with Duchenne muscular dystrophy

Ventricular tachycardia (VT) is the second most common cause of death in patients with Duchenne muscular dystrophy (DMD). Recent studies have implicated enhanced sarcoplasmic reticulum (SR) Ca(2+) leak via type 2 ryanodine receptor (RyR2) as a cause of VT in the mdx mouse model of DMD. However, the signaling mechanisms underlying induction of SR Ca(2+) leak and VT are poorly understood. To test whether enhanced Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2 underlies SR Ca(2+) leak and induction of VT in mdx mice. Programmed electrical stimulation was performed on anesthetized mice and confocal imaging of Ca(2+) release events in isolated ventricular myocytes. Programmed electrical stimulation revealed inducible VT in mdx mice, which was inhibited by CaMKII inhibition or mutation S2814A in RyR2. Myocytes from mdx mice exhibited more Ca(2+) sparks and Ca(2+) waves compared with wild-type mice, in particular at faster pacing rates. Arrhythmogenic Ca(2+) waves were inhibited by CaMKII but not by protein kinase A inhibition. Moreover, mutation S2814A but not S2808A in RyR2 suppressed spontaneous Ca(2+) waves in myocytes from mdx mice. CaMKII blockade and genetic inhibition of RyR2-S2814 phosphorylation prevent VT induction in a mouse model of DMD. In ventricular myocytes from mdx mice, spontaneous Ca(2+) sparks and Ca(2+) waves can be suppressed by CaMKII inhibition or mutation S2814A in RyR2. Thus, the inhibition of CaMKII-induced SR Ca(2+) leak might be a new strategy to prevent arrhythmias in patients with DMD without heart failure.

Abstract 189: Activation of Voltage-Dependent Anion Channel 2 Suppresses Ca 2+ -Induced Cardiac Arrhythmia

Abnormal Ca2+ handling in cardiac muscle cells is associated with a wide range of human cardiac diseases, including heart failure and cardiac arrhythmias. Zebrafish tremblor (tre) mutant embryos manifest unsynchronized cardiac contractions due to a Ca2+ extrusion defect in cardiomyocytes and thus are used as an animal model for aberrant Ca2+ homeostasis-induced cardiac arrhythmia. To further dissect molecular mechanisms regulating cardiac Ca2+ homeostasis, we conducted a chemical suppressor screen on tre and found that efsevin, a synthetic compound, potently suppresses cardiac fibrillation and restores rhythmic cardiac contractions in tre embryos. In addition, the treatment with efsevin blocks the propagation of arrhythmogenic Ca2+ waves and accelerates the decay phase of Ca2+ sparks in adult murine cardiomyocytes under Ca2+ overload conditions, demonstrating that efsevin modulates Ca2+ handling in both embryonic and adult cardiac tissues. Through a biochemical pulldown assay, we identified a direct interaction between efsevin and VDAC2, a mitochondrial outer membrane voltage dependent anion channel. Overexpression of VDAC2 restores synchronized cardiac contraction in tre and knocking down VDAC2 activity abolishes the rescue effect of efsevin on tre, suggesting that efsevin modulates cardiac Ca2+ homeostasis by potentiating VDAC2 activity. We further showed that enhancing mitochondria Ca2+ uptake by overexpressing MICU or MCU suppresses cardiac fibrillation in tre just like VDAC2 does. Interestingly, this suppressive effect is absent in tre/vdac2 double deficient embryos and co-expression of VDAC2 and MICU or MCU results in synergistic rescue effect on tre, indicating a critical role for mitochondria in regulating cardiac Ca2+ handling and rhythmicity and suggesting that VDAC2 functions as a gate for transporting Ca2+ across the outer membrane. Taken together, our findings identify efsevin as a potent pharmacological tool to modulate cardiac Ca2+ handling, suggest a critical role of mitochondria in the control of cardiac rhythmicity and establish VDAC2 as a modulator of cardiac Ca2+ handling and a potential therapeutic target for the treatment of arrhythmias.

Reactive oxygen species contribute to the development of arrhythmogenic Ca2+ waves during β‐adrenergic receptor stimulation in rabbit cardiomyocytes

While β-adrenergic receptor (β-AR) stimulation leads to positive inotropic effects, it can also induce arrhythmogenic Ca2+ waves. β-AR stimulation increases mitochondrial oxygen consumption and, thereby, the production of reactive oxygen species (ROS). We therefore investigated the role of ROS in the generation of Ca2+ waves during β-AR stimulation in rabbit ventricular myocytes. Isoproterenol (ISO) increased Ca2+ transient amplitude during systole, sarcoplasmic reticulum (SR) Ca2+ load and the occurrence of Ca2+ waves during diastole. These effects, however, developed at different time points during ISO application.While SR Ca2+ release and load reached a maximum level after 3 min, Ca2+ waves occurred at the highest frequency only after 6 min of ISO application.Measurement of intra-SR-free Ca2+ concentration ([Ca2+]SR) showed an initial increase of SR Ca2+ load followed by a gradual decline over time during ISO application. This decline of [Ca2+]SR was not due to decreased SR Ca2+ uptake, but instead was the result of increased SRCa2+ leak mainly in the form of Ca2+ waves. ISO application led to significant RyR phosphorylation at the protein kinase A (PKA)-specific site, which remained relatively stable throughout β-AR activation.Moreover, β-AR stimulation significantly increased ROS production after 4–6 min of ISO application. The ROS scavenger Tiron and the superoxide dismutase mimetic MnTBPA abolished the ISO-mediated ROS production. The mitochondria-specific antioxidant Mito-Tempo and an inhibitor of the electron transport chain, rotenone, also effectively prevented the ISO-mediated ROS production. Scavenging ROS during ISO application decreased the occurrence of Ca2+ waves and partially prevented augmentation of SRCa2+ leak, but did not affect the increase of Ca2+ transient amplitude. Treatment of myocytes with ISO for 15 min significantly reduced the free thiol content in RyRs. These data suggest that increased mitochondrial ROS production during β-AR stimulation causes RyR oxidation. Together with RyR phosphorylation, oxidation of RyRs increases diastolic SR Ca2+ leak to a critical level leading to the generation of arrhythmogenic Ca2+ waves.

Ca 2+ signaling amplification by oligomerization of L-type Ca v 1.2 channels

Ca(2+) influx via L-type Ca(v)1.2 channels is essential for multiple physiological processes, including gene expression, excitability, and contraction. Amplification of the Ca(2+) signals produced by the opening of these channels is a hallmark of many intracellular signaling cascades, including excitation-contraction coupling in heart. Using optogenetic approaches, we discovered that Ca(v)1.2 channels form clusters of varied sizes in ventricular myocytes. Physical interaction between these channels via their C-tails renders them capable of coordinating their gating, thereby amplifying Ca(2+) influx and excitation-contraction coupling. Light-induced fusion of WT Ca(v)1.2 channels with Ca(v)1.2 channels carrying a gain-of-function mutation that causes arrhythmias and autism in humans with Timothy syndrome (Ca(v)1.2-TS) increased Ca(2+) currents, diastolic and systolic Ca(2+) levels, contractility and the frequency of arrhythmogenic Ca(2+) fluctuations in ventricular myocytes. Our data indicate that these changes in Ca(2+) signaling resulted from Ca(v)1.2-TS increasing the activity of adjoining WT Ca(v)1.2 channels. Collectively, these data support the concept that oligomerization of Ca(v)1.2 channels via their C termini can result in the amplification of Ca(2+) influx into excitable cells.

Stretch-Dependent Sub-Cellular Ca2+ Signaling in Atrial Myocytes

Atrial fibrillation (AF) is a multifactorial disease whose underlying mechanisms are still incompletely understood. Atrial myocytes undergo similar stretch during diastolic filling as ventricular myocytes and acute atrial stretch can induce AF. Our group recently identified a novel mechanism linking cellular stretch to the tuning of Ca2+ release from the sarcoplasmic reticulum (SR) in ventricular myocytes (Prosser et al. Science 201;333:1440-5). This mechanism, “X-ROS signaling”, depends on NOX2 (NADPH oxidase) to generate reactive oxygen species (ROS), which oxidizes the local RyR2 Ca2+ release channels of the SR and increases their Ca2+ sensitivity. Stretch mediates X-ROS signaling through microtubules, which interact with NOX2 located in transverse tubules and the surface sarcolemmal membranes.Interestingly, in atrial myocytes from AF patients both increased ROS levels and arrhythmogenic Ca2+ signaling instability are present. We evaluated the effect of acute and repeated stretch on sub-cellular Ca2+ sparks in murine atrial myocytes (C57/B6) with instrumentation (WPI, Sarasota, FL) and methods of Prosser et al. (2011). Ca2+ sparks were recorded before and after acute and repeated stretch (8% of cell length). Here, we report an increase in stretch-dependent Ca2+ spark frequency in single murine atrial myocytes. Thus, stretch-dependent Ca2+ release mechanisms are also operative in atrial myocytes and likely to play a role in Ca2+ dependent arrhythmias like AF. Our ongoing work will interrogate the components of the X-ROS pathway to determine the relative contribution of this pathway in atrial myocytes.

Inhibition of Akt Attenuates Isoproterenol-Induced RyR-Dependent Diastolic Ca2+ Release in Rabbit Ventricular Myocytes

Cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca2+ release is increased by beta-adrenergic receptor (ß-AR) stimulation. Increased diastolic RyR activity can lead to arrhythmogenic spontaneous SR Ca2+ release. Our studies have shown that calmodulin-dependent protein kinase II (CaMKII) activation by nitric oxide (NO) implicates nitric oxide synthase (NOS) in the pathway. Here we investigate the role Akt may play in activating NOS, thus increasing diastolic SR Ca2+ release during ß-AR stimulation in isolated rabbit ventricular myocytes. Western blot analysis showed a dose-dependent increase in phosphorylated Akt in response to the β-AR agonist isoproterenol (ISO). Cytosolic Ca2+ was measured using fluo-4 loaded intact myocytes. SR Ca2+ was varied by field-stimulated to steady state at different frequencies. Tetracaine (1mM) was used to rapidly and reversibly block the RyR. The tetracaine-dependent shift of Ca2+ from the cytosol to the SR is proportional to diastolic Ca2+ release. Primary myocytes were treated with 250nM ISO with and without Akt inhibitor-X (AIX; 5μM; 30min pre-incubation and continuous perfusion). ISO-induced increase in the diastolic SR Ca2+ release was abolished by the treatment of myocytes with AIX. When data were selected such that SR[Ca2+] was matched in each group (ISO: 155.22±5.1μM; ISO+AIX: 153.92±3.8μM), myocytes treated with ISO had significantly higher tetracaine-dependent increase in diastolic SR[Ca2+] (12.43±3.8μM) vs. those treated with ISO and AIX (1±2.2μM) (P=0.01, t-test). The results suggest that the ISO-dependent increase in diastolic SR Ca2+ release is dependent upon Akt. To further test this hypothesis myocytes were cultured for 24 hours with adenovirus to express a dominant-negative Akt construct (Akt-dn). Preliminary evidence shows reduced diastolic SR[Ca2+] release for a given SR[Ca2+] in Akt-dn myocytes. This evidence indicates that Akt activation may be an important upstream effector involved in the β-AR-induced diastolic SR Ca2+ release.

Ryanodine Receptor Use-Dependent Block Suppresses Ca2+ Waves in Permeabilized Casq2-/- and RyR2-R4496C Myocytes

Background-Casq2 and RyR2 mutations can both cause catecholaminergic-polymorphic ventricular tachycardia (CPVT). Leaky RyR2s (increased open probability) generate Ca2+ waves, delayed afterdepolarizations and ventricular arrhythmias. We hypothesize that the RyR2 open-state inhibitors flecainide (FLEC) and R-propafenone (RPROP) abolish this sequence of events in both CPVT genotypes.Methods and Results-Using confocal microscopy, we recorded Ca2+ waves in permeabilized myocytes from casq2-/-, RyR2-R4496C and wild-type (WT) mice. Casq2-/- myocytes showed the highest wave frequency and speed and the lowest amplitude, RyR2-R4496C exhibited intermediate values and WT had the lowest values (Figure). We next obtained FLEC and RPROP concentration-response curves and calculated IC50 and efficacy of wave inhibition. Both FLEC and RPROP reduced all wave parameters with higher potency (lower IC50) and efficacy in casq2-/- and RyR4496C compared to WT (FLEC: IC50 12μM vs 22μM vs 49μM; efficacy 43% vs 29% vs 21% for casq2-/-, RyR2-R4496C and WT respectively. RPROP: IC50 18μM vs 28μM; efficacy 36% vs 17% for RyR2-R4496C and WT respectively; n=10-60 cells per group).Conclusion-RyR2 activity determines the potency and efficacy of open-state blockers for suppressing arrhythmogenic Ca2+ waves in permeabilized myocytes from casq2-/- and RyR2-R4496C CPVT models.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Self-Organized Criticality Underlies Arrhythmogenic Calcium Waves in Cardiac Myocytes

In cardiac myocytes, calcium (Ca)-induced Ca release can give rise to propagating Ca waves that promote cardiac arrhythmias. To understand the mechanism underlying the transition from localized Ca sparks to global Ca waves, we developed a mathematical model of a spatially-distributed network of diffusively coupled Ca release units (CRUs) comprised of junctional/longitudinal sarcoplasmic reticulum and dyadic/cytoplasmic spaces with physiologically realistic dimensions. As total Ca was increased in the model, Ca spark cluster sizes initially exhibited an exponential distribution, which transitioned to a scale-free power-law (f-β) distribution near the threshold at which random Ca sparks transitioned to Ca waves. The power-law relationship indicates that Ca release patterns in the CRU network are governed by a dynamical mechanism called self-organized criticality, the same process underlying many natural world phenomena such as avalanches and earthquakes. We tested this prediction experimentally in saponin-permeabilized cardiac myocytes. As free Ca, buffered with 0.5 mM EGTA, was increased, Ca spark cluster size transitioned from an exponential distribution to a power-law distribution at a free Ca concentration of 400 nM. Consistent with self-organized criticality, this Ca concentration was near the threshold for spark-to-wave transition since fully propagating waves were observed above 400 nM Ca. Below this concentration, Ca was released mostly as individual sparks, and spark cluster sizes followed an exponential rather than power-law distribution. In conclusion, our findings provide both theoretical and experimental evidence that the transition from Ca sparks to arrhythmogenic Ca waves in cardiac myocytes is mediated by the dynamical process of self-organized criticality, common to many natural phenomena. This provides a theoretical framework for developing interventions which modulate the Ca spark-to-wave transition threshold as a potential therapeutic strategy for preventing arrhythmias.

Dantrolene prevents arrhythmogenic Ca2+release in heart failure

In heart failure (HF), arrhythmogenic Ca(2+) release and chronic Ca(2+) depletion of the sarcoplasmic reticulum (SR) arise due to altered function of the ryanodine receptor (RyR) SR Ca(2+)-release channel. Dantrolene, a therapeutic agent used to treat malignant hyperthermia associated with mutations of the skeletal muscle type 1 RyR (RyR1), has recently been suggested to have effects on the cardiac type 2 RyR (RyR2). In this investigation, we tested the hypothesis that dantrolene exerts antiarrhythmic and inotropic effects on HF ventricular myocytes by examining multiple aspects of intracellular Ca(2+) handling. In normal rabbit myocytes, dantrolene (1 μM) had no effect on SR Ca(2+) load, postrest decay of SR Ca(2+) content, the threshold for spontaneous Ca(2+) wave initiation (i.e., the SR Ca(2+) content at which spontaneous waves initiate) and Ca(2+) spark frequency. In cardiomyocytes from failing rabbit hearts, SR Ca(2+) load and the wave initiation threshold were decreased compared with normal myocytes, Ca(2+) spark frequency was increased, and the postrest decay was potentiated. Using a novel approach of measuring cytosolic and intra-SR Ca(2+) concentration (using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR), we showed that treatment of HF cardiomyocytes with dantrolene rescued postrest decay and increased the wave initiation threshold. Additionally, dantrolene decreased Ca(2+) spark frequency while increasing the SR Ca(2+) content in HF myocytes. These data suggest that dantrolene exerts antiarrhythmic effects and preserves inotropy in HF cardiomyocytes by decreasing the incidence of diastolic Ca(2+) sparks, increasing the intra-SR Ca(2+) threshold at which spontaneous Ca(2+) waves occur, and decreasing the loss of Ca(2+) from the SR. Furthermore, the observation that dantrolene reduces arrhythmogenicity while at the same time preserves inotropy suggests that dantrolene is a potentially useful drug in the treatment of arrhythmia associated with HF.

Abstract 18314: Dysfunction of Junctional NCX Following JPH2 Knockdown Alters Spatial Properties of Ca 2+ Sparks and Contributes to Arrhythmogenesis in Mice

Background. Na + /Ca 2+ exchanger (NCX) is essential in tuning intracellular Ca 2+ and the development of heart failure (HF). We reported that loss of Junctophilin 2 (JPH2) in adult mouse hearts leads to reduced NCX activity in addition to hyperactive RyR2s and more arrhythmogenic Ca 2+ waves. NCX has been found to co-localize with RyR2 recently. However, what is the physiological significance of junctional NCX and whether the reduced junctional NCX activity following JPH2 knockdown contributes to the arrhythmogenesis is still unknown. Hypothesis. Dysfunction of Junctional NCX in response to JPH2 knockdown alters spatial properties of Ca 2+ sparks and contributes to arrhythmogenesis. Methods. Tamoxifen-treated MCM and MCM-shJPH2 (KD) mice were studied. Contractile function was measured by echocardiography. Open probability of single RyR2 was evaluated by lipid bilayer. Ca 2+ spark were assessed with confocal microscopy. NCX activity was measured by exponential fitting of the declining phase of caffeine-induced Ca 2+ transient. Results. JPH2 knockdown increased spark size (MCM: 2.55 ± 0.14; KD: 4.25 ± 0.16, P < 0.001), suggesting an increased occurrence of proarrhythmic macrosparks. Cardiac Contractile function (EF% ) is negatively correlated with Ca 2+ spark size (n = 8, P < 0.05). Increased Open probability of RyR2 (MCM: 0.038 ± 0.018 KD: 0.274 ± 0.142, P < 0.01) is unlikely the reason for the ~70% increase in spark size, since it is not observed in 3 other mouse models with hyperactive RyR2s but normal JPH2 levels (RyR2:S2814D, mdx and FKBP12.6 -/- ). Loss of JPH2 also slows Ca 2+ efflux via NCX by ~50% (MCM: 2.70 ± 0.23 vs. KD: 5.11 ± 0.61 s, P < 0.01) without altering NCX expression level. Spark size and NCX activity are negatively correlated (n = 8 mice, P < 0.05), and partial inhibition (~30%) of NCX with 1 mM Cd 2+ significantly increased spark size by 14.0 ± 2.8% (n = 8, P <0.05) in wt mice. Conclusion. For the first time we showed that reduced NCX activity increases spatial properties of Ca 2+ sparks and thus arrhythmogenesis. Since reduced JPH2 is associated with HF and NCX plays more important role in human than in murine, reduced junctional NCX activity following loss of JPH2 could be a novel mechanism underlying the development of HF and associated ventricular arrhythmias.

The interaction of Ca2+ with sarcomeric proteins: role in function and dysfunction of the heart.

The hallmarks of the normal heartbeat are both rapid onset of contraction and rapid relaxation as well as an inotropic response to both increased end-diastolic volume and increased heart rate. At the microscopic level, Ca(2+) plays a crucial role in normal cardiac contraction. This paper reviews the cycle of Ca(2+) fluxes during the normal heartbeat, which underlie the coupling between excitation and contraction and permit a highly synchronized action of cardiac sarcomeres. Length dependence of the response of the regulatory sarcomeric proteins mediates the Frank-Starling Law of the heart. However, Ca(2+) transport may go astray in heart disease such as in congestive heart failure, and both jeopardize systole and diastole and triggering arrhythmias. The interaction between weak and strong segments in nonuniform cardiac muscle allows partial preservation of force of contraction but may further lead to mechanoelectric feedback or reverse excitation-contraction coupling mediating an early diastolic Ca(2+) transient caused by the rapid force decrease during the relaxation phase. These rapid force changes in nonuniform muscle may cause arrhythmogenic Ca(2+) waves to propagate by the activation of neighboring sarcoplasmic reticulum by diffusing Ca(2+) ions.

S100A1 Genetically Targeted Therapy Reverses Dysfunction of Human Failing Cardiomyocytes

This study investigated the hypothesis whether S100A1 gene therapy can improve pathological key features in human failing ventricular cardiomyocytes (HFCMs). Depletion of the Ca²⁺-sensor protein S100A1 drives deterioration of cardiac performance toward heart failure (HF) in experimental animal models. Targeted repair of this molecular defect by cardiac-specific S100A1 gene therapy rescued cardiac performance, raising the immanent question of its effects in human failing myocardium. Enzymatically isolated HFCMs from hearts with severe systolic HF were subjected to S100A1 and control adenoviral gene transfer and contractile performance, calcium handling, signaling, and energy homeostasis were analyzed by video-edge-detection, FURA2-based epifluorescent microscopy, phosphorylation site-specific antibodies, and mitochondrial assays, respectively. Genetically targeted therapy employing the human S100A1 cDNA normalized decreased S100A1 protein levels in HFCMs, reversed both contractile dysfunction and negative force-frequency relationship, and improved contractile reserve under beta-adrenergic receptor (β-AR) stimulation independent of cAMP-dependent (PKA) and calmodulin-dependent (CaMKII) kinase activity. S100A1 reversed underlying Ca²⁺ handling abnormalities basally and under β-AR stimulation shown by improved SR Ca²⁺ handling, intracellular Ca²⁺ transients, diastolic Ca²⁺ overload, and diminished susceptibility to arrhythmogenic SR Ca²⁺ leak, respectively. Moreover, S100A1 ameliorated compromised mitochondrial function and restored the phosphocreatine/adenosine-triphosphate ratio. Our results demonstrate for the first time the therapeutic efficacy of genetically reconstituted S100A1 protein levels in HFCMs by reversing pathophysiological features that characterize human failing myocardium. Our findings close a gap in our understanding of S100A1's effects in human cardiomyocytes and strengthen the rationale for future molecular-guided therapy of human HF.

Catecholaminergic-induced arrhythmias in failing cardiomyocytes associated with human HRCS96A variant overexpression

The histidine-rich calcium binding protein (HRC) Ser96Ala polymorphism was shown to correlate with ventricular arrhythmias and sudden death only in dilated cardiomyopathy patients but not in healthy human carriers. In the present study, we assessed the molecular and cellular mechanisms underlying human arrhythmias by adenoviral expression of the human wild-type (HRC(WT)) or mutant HRC (HRC(S96A)) in adult rat ventricular cardiomyocytes. Total HRC protein was increased by ∼50% in both HRC(WT)- and HRC(S96A)-infected cells. The HRC(S96A) mutant exacerbated the inhibitory effects of HRC(WT) on the amplitude of Ca(2+) transients, prolongation of Ca(2+) decay time, and caffeine-induced sarcoplasmic reticulum Ca(2+) release. Consistent with these findings, HRC(S96A) reduced maximal sarcoplasmic reticulum calcium uptake rate to a higher extent than HRC(WT). Furthermore, the frequency of spontaneous Ca(2+) sparks, which was reduced by HRC(WT), was increased by mutant HRC(S96A) under resting conditions although there were no spontaneous Ca(2+) waves under stress conditions. However, expression of the HRC(S96A) genetic variant in cardiomyocytes from a rat model of postmyocardial infarction heart failure induced dramatic disturbances of rhythmic Ca(2+) transients. These findings indicate that the HRC Ser96Ala variant increases the propensity of arrhythmogenic Ca(2+) waves in the stressed failing heart, suggesting a link between this genetic variant and life-threatening ventricular arrhythmias in human carriers.

172 Increasing anti-oxidant capacity reverses iron overload mediated dysfunction in cardiomyocytes

IntroductionIron overload-cardiomyopathy (IOCM) is an increasing clinical problem worldwide. 70% of patients who receive compulsory blood transfusion die of IOCM, with increased susceptibility to arrhythmias and sudden death. We have...

Electromechanical coupling in the cardiac myocyte; stretch-arrhythmia feedback

The macroscopic hallmarks of the normal heartbeat are rapid onset of contraction and rapid relaxation and an inotropic response to both increased end diastolic volume and increased heart rate. At the microscopic level, the calcium ion (Ca(2+)) plays a crucial role in normal cardiac contraction. This paper reviews the cycle of Ca(2+) fluxes during the normal heartbeat, which underlie the coupling between excitation and contraction (ECC) and permit a highly synchronized action of cardiac sarcomeres. Length dependence of the response of the regulatory sarcomeric proteins mediates the Frank-Starling Law of the heart. However, Ca(2+) transport may go astray in heart disease and both jeopardize the exquisite mechanism of systole and diastole and triggering arrhythmias. The interplay between weakened and strong segments in nonuniform cardiac muscle may further lead to mechanoelectric feedback-or reverse excitation contraction coupling (RECC) mediating an early diastolic Ca(2+) transient caused by the rapid force decrease during the relaxation phase. These rapid force changes in nonuniform muscle may cause arrhythmogenic Ca(2+) waves to propagate by activation of neighbouring SR by diffusing Ca(2+) ions.