Sensitivity Of Ryanodine Receptors Research Articles

Cardiac Protein Kinase D1 ablation alters the myocytes β-adrenergic response

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

Ryanodine receptor-active non-dioxin-like polychlorinated biphenyls cause neurobehavioral deficits in larval zebrafish.

Although their production was banned in the United States in 1977, polychlorinated biphenyls (PCBs) continue to pose significant risks to the developing nervous system. Perinatal exposure to PCBs is associated with increased risk of neuropsychiatric disorders, perhaps due to altered patterns of dendritic arborization of central neurons. Non-dioxin-like (NDL) PCB congeners enhance dendritic arborization of developing mammalian neurons via sensitization of ryanodine receptors (RYR). Structure-activity relationships (SAR) of RYR sensitization by PCBs have been demonstrated using mammalian and rainbow trout (Oncorhynchus mykiss) tissue homogenates. The purpose of this study is to determine whether this SAR translates to developmental neurotoxicity (DNT) of PCBs in vivo, a question that has yet to be tested. To address this gap, we leveraged a zebrafish model to evaluate the developmental neurotoxicity potential of PCBs 28, 66, 84, 95, 138, and 153, congeners previously shown to have broadly different potencies towards sensitizing RYR. We first confirmed that these PCB congeners exhibited differing potency in sensitizing RYR in zebrafish muscle ranging from negligible (PCB 66) to moderate (PCB 153) to high (PCB 95) RYR activity. Next, enzymatically dechorionated embryos were statically exposed to varying concentrations (0.1–10 μM) of each PCB congener from 6 h post-fertilization to 5 days post-fertilization (dpf). Embryos were observed daily using stereomicroscopy to assess mortality and gross malformations and photomotor behavior was assessed in larval zebrafish at 3, 4, and 5 dpf. The body burden of each PCB was measured by gas chromatography. The key findings are: 1) None of these PCBs caused death or overt teratology at the concentrations tested; 2) A subset of these PCB congeners altered photomotor behavior in larval zebrafish and the SAR for PCB behavioral effects mirrored the SAR for RYR sensitization; and 3) Quantification of PCB levels in larval zebrafish ruled out the possibility that congener-specific effects on behavior were due to differential uptake of PCB congeners. Collectively, the findings from this study provide in vivo evidence in support of the hypothesis that RYR sensitization contributes to the DNT of PCBs.

Enhanced Klotho availability protects against cardiac dysfunction induced by uraemic cardiomyopathy by regulating Ca2+ handling.

Klotho is a membrane-bound or soluble protein, originally identified as an age-suppressing factor and regulator of mineral metabolism. Klotho deficiency is associated with the development of renal disease, but its role in cardiac function in the context of uraemic cardiomyopathy is unknown. We explored the effects of Klotho on cardiac Ca2+ cycling. We analysed Ca2+ handling in adult cardiomyocytes from Klotho-deficient (kl/kl) mice and from a murine model of 5/6 nephrectomy (Nfx). We also studied the effect of exogenous Klotho supplementation, by chronic recombinant Klotho treatment, or endogenous Klotho overexpression, using transgenic mice overexpressing Klotho (Tg-Kl), on uraemic cardiomyopathy. Hearts from Nfx mice were used to study Ca2+ sensitivity of ryanodine receptors and their phosphorylation state. Cardiomyocytes from kl/kl mice showed decreased amplitude of intracellular Ca2+ transients and cellular shortening together with an increase in pro-arrhythmic Ca2+ events compared with cells from wild-type mice. Cardiomyocytes from Nfx mice exhibited the same impairment in Ca2+ cycling as kl/kl mice. Changes in Nfx cardiomyocytes were explained by higher sensitivity of ryanodine receptors to Ca2+ and their increased phosphorylation at the calmodulin kinase type II and protein kinase A sites. Ca2+ mishandling in Nfx-treated mice was fully prevented by chronic recombinant Klotho administration or transgenic Klotho overexpression. Klotho emerges as an attractive therapeutic tool to improve cardiac Ca2+ mishandling observed in uraemic cardiomyopathy. Strategies that improve Klotho availability are good candidates to protect the heart from functional cardiac alterations in renal disease.

Ivabradine is as effective as metoprolol in the prevention of ventricular arrhythmias in acute non-reperfused myocardial infarction in the rat

Ventricular arrhythmias are a major source of early mortality in acute myocardial infarction (MI) and remain a major therapeutic challenge. Thus we investigated effects of ivabradine, a presumably specific bradycardic agent versus metoprolol, a β-blocker, at doses offering the same heart rate (HR) reduction, on ventricular arrhythmias in the acute non-reperfused MI in the rat. Immediately after MI induction a single dose of ivabradine/ metoprolol was given. ECG was continuously recorded and ventricular arrhythmias were analyzed. After 6 h epicardial monophasic action potentials (MAPs) were recorded and cardiomyocyte Ca2+ handling was assessed. Both ivabradine and metoprolol reduced HR by 17% and arrhythmic mortality (14% and 19%, respectively, versus 33% in MI, p < 0.05) and ventricular arrhythmias in post-MI rats. Both drugs reduced QTc prolongation and decreased sensitivity of ryanodine receptors in isolated cardiomyocytes, but otherwise had no effect on Ca2+ handling, velocity of conduction or repolarization. We did not find any effects of potential IKr inhibition by ivabradine in this setting. Thus Ivabradine is an equally effective antiarrhythmic agent as metoprolol in early MI in the rat. It could be potentially tested as an alternative antiarrhythmic agent in acute MI when β-blockers are contraindicated.

Ontogenetic peculiarities of the effect of dantrolene and caffeine on the cardiac, respiratory and motor performance of intact and poisoned by physostigmine of rats

Activities of the somatomotor (MA), cardiovascular and respiratory systems were investigated in conditions of activation and blockade of ryanodine receptors (RyR) impaired cholinergic regulation in rat pups aged 3, 16 and 30 days. It was found that blockage of RyR by dantrolene has a negative chronotropic effect, the severity of which decreases with age, reduces the respiratory rate in rats on P3 and practically does not change in older animals. The amplitude of EMG and the power of motor bursts decreases in rats on P3-P16 and increases on P30. The duration of MA in the activity- rest cycle remains unchanged at P3 and decreases at P16-P30, which indicates a different change in the pattern of MA in rats of different ages after the blockade of RyR. Caffeine-induced activation of RyR is accompanied by an increase in heart rate and respiratory rate in rats on P30 and does not cause significant changes in these parameters on P3-P16. There has been an increase in all MA indices, especially in P3. Certain age-related regularities were revealed in the conditions of acetylcholinesterase inhibition by physostigmine, carried out against the background of RyR inhibition or activation. Maximum sensitivity to such effects was found in rats on P3. It was shown that in early postnatal ontogenesis of rats the sensitivity of RyR, including RyR2, to dantrolene is higher than in adult animals. Changes in RyR activity level do not prevent the development of pathological forms of heart rhythm that developing in rats after the physostigmine injection.

Prenatal caffeine exposure induces down-regulation of the protein kinase A/ryanodine receptor/large-conductance Ca2+-activated K+ pathway in the cerebral arteries of old offspring rats

The current study investigated the long-term effects of prenatal caffeine (Caf) exposure on cerebral vessels of old offspring rats. Pregnant rats were treated with Caf (20 mg/kg, twice daily) or 0.9% normal saline during gestational days 3.5-19.5, and offspring were tested at 24 months old. Vascular functions of middle cerebral arteries and ion channel activities in smooth muscle cells were examined using myograph system and patch-clamp. Prenatal Caf exposure decreased isoprenaline (β-adrenergic agonist)-induced dilatation of the middle cerebral artery in the offspring. Treatment with protein kinase A (PKA) inhibitor reduced isoprenaline-mediated vasodilatation to a greater extent in the control. Forskolin-mediated vasodilatation and membrane hyperpolarization were reduced in the Caf group. Large-conductance Ca-activated K (BKCa) channel inhibitor iberiotoxin significantly attenuated forskolin-induced vasodilatation and reduced depolarization in the control, not in the Caf group. The PKA agonist-activated cell-attached single BKCa currents to a greater extent in the control. The mRNA and protein expression levels of PKA-Cα were decreased. The sensitivity of ryanodine receptors to the PKA agonist was blunted in the Caf group, whereas the mRNA expression of ryanodine receptor 2 subunit was reduced. Voltage/Ca sensitivity of BKCa was decreased accompanied by reduced mRNA and protein expression of BKCa-β1 subunits in the Caf group. PKA agonist-stimulated inside-out BKCa currents were weaker in the Caf group. Prenatal exposure to Caf-affected isoprenaline/forskolin-mediated vascular functions in aged cerebral arteries, related to dysfunction of the PKA/ryanodine receptors/BKCa signaling pathway.

Role of FK506-binding protein in Ca2+ spark regulation

The elementary Ca2+ release events, Ca2+ sparks, has been found for a quarter of century. However, the molecular regulation of the spark generator, the ryanodine receptor (RyR) on the sarcoplasmic reticulum, remains obscure. Although each subunit of the RyR homotetramer has a site for FK506-binding protein (FKBP), the role of FKBPs in modifying RyR Ca2+ sparks has been debated for long. One of the reasons behind the controversy is that most previous studies detect spontaneous sparks, where the mixture with out-of-focus events and local wavelets prevents an accurate characterization of Ca2+ sparks. In the present study, we detected Ca2+ sparks triggered by single L-type Ca2+ channels (LCCs) under loose-seal patch clamp conditions in FK506-treated or FKBP12.6 knockout cardiomyocytes. We found that FKBP dissociation both by FK506 and by rapamycin decreased the Ca2+ spark amplitude in ventricular cardiomyocytes. This change was neither due to decreased releasable Ca2+ in the sarcoplasmic reticulum, nor explained by changed RyR sensitivity. Actually FK506 increased the LCC-RyR coupling probability and curtailed the latency for an LCC to trigger a RyR Ca2+ spark. FKBP12.6 knockout had similar effects as FK506/rapamycin treatment, indicating that the decreased spark amplitude was attributable to the dissociation of FKBP12.6 rather than FKBP12. We also explained how decreased amplitude of spontaneous sparks after FKBP dissociation sometimes appears to be increased or unchanged due to inappropriate data processing. Our results provided firm evidence that without the inter-RyR coordination by functional FKBP12.6, the RyR recruitment during a Ca2+ spark would be compromised despite the sensitization of individual RyRs.

A novel mechanism of tandem activation of ryanodine receptors by cytosolic and SR luminal Ca2+ during excitation-contraction coupling in atrial myocytes.

In atrial myocytes excitation-contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca2+ release starts in the cell periphery and propagates towards the cell centre by Ca2+ -induced Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ store. The cytosolic Ca2+ sensitivity of the ryanodine receptor (RyRs) Ca2+ release channel is low and it is unclear how Ca2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra-SR calcium revealed a transient elevation of store Ca2+ that we termed 'Ca2+ sensitization signal'. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca2+ through a 'fire-diffuse-uptake-fire' (or FDUF) mechanism: Ca2+ uptake by SR Ca2+ pumps at the propagation front elevates Ca2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca2+ activation threshold. In atrial myocytes Ca2+ release during excitation-contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca2+ store into the peripheral subsarcolemmnal junctional (j-SR) and the much more abundant central non-junctional (nj-SR) SR. Action potential (AP)-induced Ca2+ entry activates Ca2+ -induced Ca2+ release (CICR) from j-SR ryanodine receptor (RyR) Ca2+ release channels. Peripheral elevation of [Ca2+ ]i initiates CICR from nj-SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca2+ ]i ; with the fluorescent Ca2+ indicator rhod-2) and intra-SR ([Ca2+ ]SR ; fluo-5N) Ca2+ in rabbit atrial myocytes revealed that Ca2+ release from j-SR resulted in a cytosolic Ca2+ transient of higher amplitude compared to release from nj-SR; however, the degree of depletion of j-SR [Ca2+ ]SR was smaller than nj-SR [Ca2+ ]SR . Similarly, Ca2+ signals from individual release sites of the j-SR showed a larger cytosolic amplitude (Ca2+ sparks) but smaller depletion (Ca2+ blinks) than release from nj-SR. During AP-induced Ca2+ release the rise of [Ca2+ ]i detected at individual release sites of the nj-SR preceded the depletion of [Ca2+ ]SR , and during this latency period a transient elevation of [Ca2+ ]SR occurred. We propose that Ca2+ release from nj-SR is activated by cytosolic and luminal Ca2+ (tandem RyR activation) via a novel 'fire-diffuse-uptake-fire' (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca2+ uptake by sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) at the propagation front elevates local [Ca2+ ]SR , leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR.

Dampened Activity of Single Ryanodine Receptor Channels in Mice Devoid of TRIC-A

TRIC-A and TRIC-B are proteins localized to endo/sarcoplasmic reticulum (ER/SR). Their physiological roles are not established although there is evidence that they can function as monovalent cation channels (Yazawa et al., 2007). Mice lacking both TRIC-A and TRIC-B die in embryonic heart failure displaying serious defects in SR Ca2+-release (Yazawa et al., 2007). TRIC-A knockout (KO) mice survive but exhibit hypertension and abnormalities in cardiac and skeletal excitation-contraction coupling. We incorporated SR vesicles derived from the skeletal muscle of wild type (WT) and TRIC-A KO mice into planar lipid bilayers under voltage-clamp conditions to investigate if the single-channel properties of ryanodine receptors (RyR) and SR K+ channels are altered.We found no significant difference in single channel conductance, open probability (Po) and voltage-dependence of SR K+ channels from wild type (WT) or TRIC-A KO mice. [3H]ryanodine binding studies indicate that the Ca2+ sensitivity of RyR derived from TRIC-A KO or WT mice is similar (WT EC50 =4.14 µM; TRIC-A KO EC50 =3.65 µM; n=6). This was confirmed by the similar Po of RyR from WT (0.0023±0.0011, SEM, n=14) and TRIC-A KO (0.0017±0.0006, SEM, n=21) mice with 10 µM cytosolic Ca2+ as the sole activator. However, in the presence of 1 mM free Mg2+, 3 mM ATP was significantly less effective at activating RyR from TRIC-A KO mice (Po=0.0464±0.0133; SEM, n=21) than from WT mice (Po =0.1149±0.0290; n=14, SEM; p<0.05). Our results indicate that TRIC-A indirectly modulates RyR channel function either by reducing the inhibitory effect of Mg2+ or by increasing sensitivity to the effects of ATP and may, at least in part, explain why SR Ca2+-release is impaired in TRIC-A KO mice.Funded by the BHF.

Ryanodine Receptor Sensitivity Governs the Stability and Synchrony of Local Calcium Release during Cardiac Excitation-Contraction Coupling

Synchronous coordination of calcium-induced calcium release (CICR) across thousands of calcium release units (CRUs) within the cardiomyocyte is critical for normal excitation-contraction coupling (ECC). Here we utilize a novel “local control” mathematical model of ECC, designed to simulate a mouse cardiomyocyte, to investigate physiological and pathophysiological calcium (Ca2+) signaling. The model contains 20,000 independent CRUs each composed of 6 stochastically gated L-type Ca2+ channels (LCCs) in the transverse tubule membrane that are positioned across a local “dyadic subspace” from a junctional sarcoplasmic reticulum (JSR) compartment containing a cluster of 50 stochastically gated ryanodine receptors (RyR2s). Action potential depolarization of the sarcolemmal membrane activates LCC openings which elevate subspace [Ca2+] ([Ca2+]ds) across each of the cell's 20,000 CRUs. This rise in [Ca2+]ds promotes RyR2 opening to initiate the fundamental element of triggered Ca2+ release, the Ca2+ spark. The mechanistic design and true SR Ca2+ pump/leak balance displayed by our model allows us to quantify ECC fidelity and Ca2+ spark fidelity (the probability that a LCC opening or RyR2 opening induces a Ca2+ spark, respectively). We study the effect of excess SR Ca2+ leak in the context of “catecholaminergic polymorphic ventricular tachycardia (CPVT)” caused by the R33Q mutation in calsequestrin (CASQ2). CPVT is simulated by increasing RyR2 sensitivity to [Ca2+]i and reducing JSR Ca2+ buffering capacity to levels identified by prior studies of the CASQ2-R33Q “knock-in” mice. Our model effectively reproduces both normal and arrhythmogenic CPVT RyR2 [Ca2+]i sensitivity, local Ca2+ spark activity, and global Ca2+ signals. Under CPVT conditions enhanced RyR2 open probability is critical for the development of “unstable”, quiescent Ca2+ sparks that fail to terminate robustly as well as asynchronous systolic Ca2+ spark activity and increased diastolic SR Ca2+ leak.

Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling

Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca2+]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca2+) channels (LCCs) and sarcoplasmic reticulum (SR) Ca2+ release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca2+] within each of the cell's 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca2+ sparks, the fundamental unit of triggered Ca2+ release. Synchronization of Ca2+ sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca2+ sparks (ECC fidelity). The detailed design and true SR Ca2+ pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca2+ spark fidelity, the balance between visible (Ca2+ spark) and invisible (Ca2+ quark/sub-spark) SR Ca2+ release events. Excess SR Ca2+ leak is examined as a disease mechanism in the context of “catecholaminergic polymorphic ventricular tachycardia (CPVT)”, a Ca2+-dependent arrhythmia. We find that that RyR2s (and therefore Ca2+ sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca2+ sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca2+]i sensitivity during CPVT leads to increased Ca2+ spark fidelity resulting in asynchronous systolic Ca2+ spark activity. It also produces increased diastolic SR Ca2+ leak with some prolonged Ca2+ sparks that at times become “metastable” and fail to efficiently terminate. There is a huge margin of safety for stable Ca2+ handling within the cell and this novel mechanistic model provides insight into the molecular signaling characteristics that help maintain overall Ca2+ stability even under the conditions of high SR Ca2+ leak during CPVT. Finally, this model should provide tools for investigators to examine normal and pathological Ca2+ signaling characteristics in the heart.

The developmental neurotoxicity of non-dioxin-like PCBs: Sensitization of ryanodine receptors interferes with neurodevelopmental processes that determine neuronal connectivity

The developmental neurotoxicity of non-dioxin-like PCBs: Sensitization of ryanodine receptors interferes with neurodevelopmental processes that determine neuronal connectivity

Ryanodine receptor sensitization results in abnormal calcium signaling in airway smooth muscle cells.

Intracellular Ca(2+) dynamics of airway smooth muscle cells (ASMCs) are believed to play a major role in airway hyperresponsiveness and remodeling in asthma. Prior studies have underscored a prominent role for inositol 1,4,5-triphosphate (IP3) receptors in normal agonist-induced Ca(2+) oscillations, whereas ryanodine receptors (RyRs) appear to remain closed during such Ca(2+) oscillations, which mediate ASMC contraction. Nevertheless, RyRs have been hypothesized to play a role in hyperresponsive Ca(2+) signaling. This could be explained by RyRs being "sensitized" to open more frequently by certain compounds. We investigate the implications of RyR sensitization on Ca(2+) dynamics in ASMC using a combination of mathematical modeling and experiments with mouse precision-cut lung slices. Caffeine is used to increase the sensitivity of RyRs to cytosolic Ca(2+) concentration ([Ca(2+)]i) and sarcoplasmic reticulum Ca(2+) ([Ca(2+)]SR). In ASMCs, high caffeine concentrations (>10 mM) induce a sustained elevation of [Ca(2+)]i. Our mathematical model accounts for this by the activation of store-operated Ca(2+) entry that results from a large increase in the RyR sensitivity to [Ca(2+)]SR and the associated Ca(2+) release, which leads to a reduction of [Ca(2+)]SR. Importantly, our model also predicts that: (1) moderate RyR sensitization induces slow Ca(2+) oscillations, a result experimentally confirmed with low concentrations of caffeine; and (2) high RyR sensitization suppresses fast, agonist-induced Ca(2+) oscillations by inducing substantial store-operated Ca(2+) entry and elevated [Ca(2+)]i. These results suggest that RyR sensitization could play a role in ASMC proliferation (by inducing slow Ca(2+) oscillations) and in airway hyperresponsiveness (by inducing greater mean [Ca(2+)]i for similar levels of contractile agonist).

Decreased inward rectifying K+ current and increased ryanodine receptor sensitivity synergistically contribute to sustained focal arrhythmia in the intact rabbit heart.

Heart failure leads to dramatic electrophysiological remodelling as a result of numerous cellular and tissue-level changes. Important cellular changes include increased sensitivity of ryanodine receptors (RyRs) to Ca(2+) release and down-regulation of the inward rectifying K(+) current (IK1), both of which contribute to triggered action potentials in isolated cells. We studied the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non-failing rabbit heart using optical mapping and pharmacological manipulation of RyRs and IK1. Neither increased RyR sensitivity or decreased IK1 alone led to significant increases in arrhythmia following local sympathetic stimulation; however, in combination, these two factors led to a significant increase in premature ventricular complexes and focal ventricular tachycardia. These results suggest synergism between increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact heart and may provide important insights into novel anti-arrhythmic treatments in heart failure. Heart failure (HF) results in dramatic electrophysiological remodelling, including increased sensitivity of ryanodine receptors (RyRs) and decreased inward rectifying K(+) current (IK1), which predisposes HF myocytes to delayed afterdepolarizations and triggered activity. Therefore, we sought to determine the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non-failing heart. Optical mapping of transmembrane potential and intracellular Ca(2+) was performed in Langendorff-perfused rabbit hearts (n = 15). Local β-adrenergic receptor stimulation with noradrenaline (norepinephrine; NA, 50 μl, 250 μM) was applied to elicit focal activity (premature ventricular complexes (PVCs) or ventricular tachycardia (VT ≥ 3 beats)). NA was administered under control conditions (CTL) and following pretreatment with 50 μM BaCl2 to reduce IK1, or 200 μM caffeine (Caff) to sensitize RyRs, both alone and in combination. Local NA injection resulted in Ca(2+)-driven PVCs arising from the injection site in all hearts studied. No increase in NA-mediated PVCs was observed following pretreatment with either BaCl2 or Caff alone (CTL: 1.1 ± 0.7, BaCl2: 1.0 ± 0.7, Caff: 1.3 ± 0.8 PVCs/injection, P not significant). However, pretreatment with the combination of BaCl2 + Caff resulted in a significant increase in PVCs (2.3 ± 2.8 PVCs/injection, P < 0.05 vs. CTL, BaCl2, Caff). Additionally, pretreatment with BaCl2 + Caff led to sustained monomorphic VT arising from the NA application site in all hearts studied, which lasted up to 6 min following a single NA injection. VT was never observed under any other condition suggesting synergism between increased RyR sensitivity and decreased IK1 in contributing to focal activity. These findings may have important implications for the understanding and prevention of focal arrhythmia in HF.

Maximal acceleration of Ca2+ release refractoriness by β-adrenergic stimulation requires dual activation of kinases PKA and CaMKII in mouse ventricular myocytes.

Refractoriness of calcium release in heart cells is altered in several disease states, but the physiological mechanisms that regulate this process are incompletely understood. We examined refractoriness of calcium release in mouse ventricular myocytes and investigated how activation of different intracellular signalling pathways influenced this process. We found that refractoriness of calcium release is abbreviated by stimulation of the 'fight-or-flight' response, and that simultaneous activation of multiple intracellular signalling pathways contributes to this response. Data obtained under several conditions at the subcellular, microscopic level were consistent with results obtained at the cellular level. The results provide insight into regulation of cardiac calcium release and how alterations to this process may increase arrhythmia risk under different conditions. Time-dependent refractoriness of calcium (Ca(2+)) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca(2+) spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca(2+) spark refractoriness in mouse ventricular myocytes by examining how β-adrenergic stimulation influenced sequences of Ca(2+) sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) β-adrenergic stimulation with isoproterenol (isoprenaline) accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark-to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with β-adrenergic stimulation; (5) inhibiting either PKA or Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) during β-adrenergic stimulation prevented the decrease in spark-to-spark delays seen without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into β-adrenergic regulation of Ca(2+) release refractoriness in mouse myocytes.

Ivabradine Protects Against Ventricular Arrhythmias in Acute Myocardial Infarction in the Rat

Ventricular arrhythmias are an important cause of mortality in the acute myocardial infarction (MI). To elucidate effect of ivabradine, pure heart rate (HR) reducing drug, on ventricular arrhythmias within 24 h after non-reperfused MI in the rat. ECG was recorded for 24 h after MI in untreated and ivabradine treated rats and episodes of ventricular tachycardia/fibrillation (VT/VF) were identified. Forty-five minutes and twenty-four hours after MI epicardial monophasic action potentials (MAPs) were recorded, cardiomyocyte Ca(2+) handling was assessed and expression and function of ion channels were studied. Ivabradine reduced average HR by 17%. Combined VT/VF incidence and arrhythmic mortality were higher in MI versus MI + Ivabradine rats. MI resulted in (1) increase of Ca(2+) sensitivity of ryanodine receptors 24 h after MI; (2) increase of HCN4 expression in the left ventricle (LV) and funny current (IF) in LV cardiomyocytes 24 h after MI, and (3) dispersion of MAP duration both 45 min and 24 h after MI. Ivabradine partially prevented all these three potential proarrhythmic effects of MI. Ivabradine is antiarrhythmic in the acute MI in the rat. Potential mechanisms include prevention of: diastolic Ca(2+)-leak from sarcoplasmic reticulum, upregulation of IF current in LV and dispersion of cardiac repolarization. Ivabradine could be an attractive antiarrhythmic agent in the setting of acute MI.

The Environmental Neurotoxicant PCB 95 Promotes Synaptogenesis via Ryanodine Receptor-Dependent miR132 Upregulation

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are widespread environmental contaminants linked to neuropsychological dysfunction in children. NDL PCBs increase spontaneous Ca(2+) oscillations in neurons by stabilizing ryanodine receptor (RyR) calcium release channels in the open configuration, which results in CREB-dependent dendritic outgrowth. In this study, we address the question of whether activation of CREB by NDL PCBs also triggers dendritic spine formation. Nanomolar concentrations of PCB 95, a NDL congener with potent RyR activity, significantly increased spine density and the frequency of miniature EPSCs in primary dissociated rat hippocampal cultures coincident with upregulation of miR132. Inhibition of RyR, CREB, or miR132 as well as expression of a mutant p250GAP cDNA construct that is not suppressed by miR132 blocked PCB 95 effects on spines and miniature EPSCs. PCB 95 also induced spine formation via RyR- and miR132-dependent mechanisms in hippocampal slice cultures. These data demonstrate a novel mechanism of PCB developmental neurotoxicity whereby RyR sensitization modulates spine formation and synaptogenesis via CREB-mediated miR132 upregulation, which in turn suppresses the translation of p250GAP, a negative regulator of synaptogenesis. In light of recent evidence implicating miR132 dysregulation in Rett syndrome and schizophrenia, these findings identify NDL PCBs as potential environmental risk factors for neurodevelopmental disorders.

Activity of Both PKA and Camkii is Required for Maximal RyR Sensitivity Under Beta-Adrenergic Stimulation

In cardiac myocytes, calcium sparks exhibit time-dependent refractoriness such that triggering of a second spark soon after an initial spark has terminated is improbable. Recent studies in rat ventricular myocytes suggested that spark amplitude recovery is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and ryanodine receptor (RyR) sensitivity.Here we examined spark refractoriness in mouse ventricular myocytes by exposing Fluo-3 loaded quiescent cells to 50 nM ryanodine, recording sparks with a confocal microscope, and analyzing the repeated sparks that were produced at singular RyR clusters.Beta-adrenergic stimulation accelerated spark amplitude recovery and decreased median spark-to-spark delay compared to control. Spark amplitude recovery was also accelerated/decelerated, respectively, by either activating or inhibiting PKA with forskolin or H89. Spark-to-spark delays were not affected by forskolin but increased in H89. Experiments performed on S2808A mice with a mutated PKA phosphorylation site corroborate these results.Forskolin results suggest that during beta-adrenergic stimulation pathways other than PKA can be activated. Beta-adrenergic stimulation in the presence of PKA or CaMKII blockers (H89 and KN93, respectively) were studied. In both conditions, time constant of amplitude recovery was increased and spark-to-spark delay was slightly higher than control. Employing a mathematical model allowed us to gain further insight into experimental results. Under control conditions SR refilling is enhanced due to activation of SERCA via endogenous PKA activity. Additional activation of either PKA or CaMKII is sufficient to accelerate spark amplitude restitution through faster refilling. However, inhibition of either pathway prevents RyR sensitivity to be increased. Thus, activity of both kinases is necessary to explain the changes in RyR gating observed during beta-adrenergic stimulation.

Effect of dantrolene on prevention of malignant hyperthermia crisis by decreasing the sensitivity of ryanodine receptor 1

Effect of dantrolene on prevention of malignant hyperthermia crisis by decreasing the sensitivity of ryanodine receptor 1

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.