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

Received July 2, 2009; accepted October 5, 2009. Heart failure (HF) is a major health problem in Western countries. Despite significant progress in pharmacological and device-based treatment, the disease burden imposed continues to increase, particularly as the population ages. HF incidence approaches 10 per 1000 after age 65 years.1 Congestive HF is the final consequence of diverse cardiovascular disorders, including atherosclerosis, cardiomyopathy, and hypertension. Described as a complex pathophysiological syndrome that involves interactions of the circulatory, neurohormonal, and renal systems, HF is first a disease of the myocardium, although it soon induces defects in other systems. Current treatments for HF, focused on blocking neurohormonal pathways, improve survival, but they do not halt the progression of HF. Late-stage HF has a poor prognosis, and therapeutic options are limited. Faced with these challenges, researchers are exploring novel therapeutic options. Chronic HF is associated with increased sympathetic outflow, which may be compensatory early on, but long-term neurohormonal activation induces significant damage to the heart; in addition, it results in multiple alterations in the β-adrenergic receptor (β-AR) signaling cascade, including receptor downregulation, upregulation of receptor kinases, and increased inhibitory G-protein function.2 The amplitude and velocity of Ca2+ cycling are regulated by a dynamic balance of phosphorylation and dephosphorylation through kinases and phosphatases. Activation of β-ARs stimulates cAMP production and results in protein kinase A (PKA) phosphorylation of key regulators of excitation-contraction coupling, such as L-type Ca2+ channels, phospholamban, troponin I, ryanodine receptors (RyR), myosin-binding protein C, and protein phosphatase inhibitor-1 (I-1; Figure), which leads to increased amplitude and velocity of Ca2+ cycling and increased contractility on a beat-to-beat basis.3 Protein phosphatases PP1 and PP2A counterbalance phosphorylation of these proteins. There is clear evidence that alterations in sarcoplasmic reticulum (SR) Ca2+ cycling are a component of the impaired …

See related article, pages 617–626 The heart, beating constantly over the course of the human lifetime, operates continuously through the process of excitation-contraction coupling. Initiated by a depolarizing influx of Na, a small Ca2+ flux across the sarcolemma causes a large release of Ca2+ from the sarcoplasmic reticulum (SR). Release of this Ca2+ takes place via the ryanodine receptor (RyR), a Ca2+ channel within the SR membrane which is gated by cytoplasmic Ca2+ ([Ca2+]i).1 This gating takes place to a small but significant extent even at the relatively low [Ca2+]i found during diastole within the myocyte (SR Ca2+ leak). Among the striking features of this SR Ca2+ release is its steep nonlinear dependence on the total concentration of Ca2+ found in the lumen of the SR ([Ca2+]SRT). The degree of release at diastolic [Ca2+]i is very low when the [Ca2+]SRT is approximately 50% of the level usually found in an isolated cardiac myocyte.2 However, it increases dramatically as [Ca2+]SRT increases toward its normal level. Of special interest is the effect of SR [Ca2+] on the SR Ca2+ leak through the RyR. In the cardiac ventricular myocyte, this leak takes place just under the sarcolemmal membrane, the location of many proteins whose effects are regulated by Ca2+. The magnitude of SR Ca2+ leak may alter the activity of these proteins. Among the candidate proteins which may be affected by the subsarocolemmal Ca2+ is the sodium-calcium exchanger (NCX). This may be particularly relevant in cells from hearts undergoing chronic failure because the myocytes from these heart have a higher leak rate than normal.2 These …

The cardiac ryanodine receptor (RyR2)/Ca2+ release channel on the sarcoplasmic reticulum (SR) is regulated by evolutionarily highly conserved signaling pathways that control excitation-contraction (EC) coupling in the heart. Phosphorylation of RyR2 by cAMP-dependent protein kinase (PKA) plays a key role in regulating the channel in response to stress via activation of the sympathetic nervous system (the “fight-or-flight response”).1 Maladaptive PKA hyperphosphorylation of RyR2 in failing hearts alters channel function, which may cause depletion of SR Ca2+ and diastolic release of SR Ca2+. This can initiate delayed afterdepolarizations that trigger ventricular arrhythmias.1 Mutations in RyR2 recently have been identified in patients with catecholaminergic induced sudden cardiac death (SCD).2–4⇓⇓ There may be a direct link between the PKA hyperphosphorylation of RyR2 that occurs during the progression of heart failure and fatal cardiac arrhythmias. Regulation of cardiac EC coupling by the release of Ca2+ from the SR via RyR2 in cardiomyocytes, known as Ca2+-induced Ca2+ release (CICR), has been appreciated for more than a decade.5,6⇓ Furthermore, it is well known that the amplitude of the Ca2+ transient generated by SR Ca2+ release determines contractile force in cardiomyocytes. The systems that regulate SR Ca2+ release include: (1) the triggers (predominantly Ca2+ influx through the voltage-gated Ca2+ channel on the plasma membrane); (2) the SR Ca2+ release channel or type 2 RyR2; and (3) the SR Ca2+ reuptake pump (SERCA2a) and its regulator phospholamban. These systems (trigger, release, and reuptake) are modulated by signaling pathways, including the β-adrenergic receptor (β-AR) signaling pathway (ie, phosphorylation by PKA). Activation of the sympathetic nervous system in response to stress results in elevation of cAMP levels and activation of PKA. Phosphorylation of RyR2 may not correlate directly …

Store overload‐induced Ca²⁺ release as a triggering mechanism for CPVT and MH episodes caused by mutations in <i>RYR</i> and <i>CASQ</i> genes

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.

In heart failure, the amplitude and rate of decay of both contraction and the underlying systolic Ca2+ transient are reduced. A current debate concerns the mechanism of these alterations of calcium handling. One theory invokes decreased activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)1 while another focuses on alterations in the SR Ca2+ release channel (ryanodine receptor, RyR).2 A significant contribution to this debate is made by Jiang et al3 in this issue of Circulation Research . Calcium that activates contraction comes from two sources: (1) the extracellular fluid, largely via the L-type Ca2+ current ( I Ca); and (2) the SR, by release through the RyR. Because the latter is generally larger and the amplitude of I Ca is not consistently altered in failure (see review4), work has focused on release from the SR. Release occurs via calcium-induced calcium release (CICR) whereby Ca2+ entry increases the probability of opening of a closely apposed RyR (see general reviews5,6⇓). Relaxation requires that [Ca2+]i be lowered by the combined effects of SERCA and Na+-Ca2+ exchange (NCX). The activity of SERCA is depressed by the accessory protein phospholamban and this inhibition is removed by phosphorylation, providing a mechanism whereby sympathetic stimulation can increase SR Ca2+ content and hence Ca2+ release from the SR. Importantly, depression of SERCA activity will not only decrease the amplitude of the Ca2+ transient (by decreasing SR content) but will also directly slow the rate of decay. The RyR can be phosphorylated,7 and there is an important interaction between an auxiliary protein (FKBP), phosphorylation, and RyR opening. Briefly, FKBP stabilizes interactions between RyRs such that …

How to shut down Ca²⁺‐induced Ca²⁺ release?

Heart failure is a complex disorder involving maladaptive responses that result in defective regulation and function of multiple biological systems. Central to our understanding of heart failure and to the ability to design and test novel therapeutic approaches that will prolong survival and improve quality of life for the millions of individuals worldwide is the need to gain a better understanding of the molecular pathogenesis of the disorder. In the search for molecular physiological defects in failing hearts, it is logical to examine the mechanism of excitation-contraction (EC) coupling in which cardiomyocyte membrane depolarization, because of the cardiac action potential, is translated into mechanical contraction in the heart. This system requires the normal function of 3 key elements: (1) calcium (Ca2+) entry via the voltage-gated Ca2+ channel (VGCC) on the plasma membrane (transverse tubule); (2) Ca2+ release via the ryanodine receptor/Ca2+ release channel (RyR2); and (3) Ca2+ uptake via the Ca2+-ATPase on the sarcoplasmic reticulum (SR) (Figure 1). Figure 1. Regulation of key molecules in cardiac EC coupling by stress activated pathways. The normal fight or flight stress response activates 3 key molecules involved in cardiac EC coupling via PKA phosphorylation: (1) the trigger for cardiac EC coupling, the voltage-gated Ca2+ channel (VGCC); (2) the SR Ca2+ release channel RyR2; and (3) the Ca2+ uptake pathway (via PKA phosphorylation of phospholamban which reduces inhibition of the Ca2+-ATPase SERCA2a). These regulatory events conspire to increase systolic SR Ca2+ release and thereby increase contractility, providing increased cardiac output to meet metabolic demands of stressful conditions. In failing hearts, this system becomes defective because of the maladaptive chronic hyperadrenergic state of heart failure, resulting in PKA-hyperphosphorylated RyR2 channels that cause a diastolic SR Ca2+ leak1 that conspires with …

Recent advances in understanding skeletal and cardiac muscle function have evolved with recognition of the active role played by the intracellular sarcoplasmic reticulum (SR) Ca2+ store in contraction. The key proteins in this store are the Ca2+ binding protein calsequestrin (CSQ), the ryanodine receptor (RyR) Ca2+ release channel and triadin and junctin (Beard et al. 2004). The CSQ–triadin–junctin–RyR complex (Fig. 1) in the SR lumen forms a ‘Ca2+ transduction machine’ that is central to EC coupling and to normal muscle development. Other proteins in the lumen of the SR, including the histidine rich calcium binding protein (HRC) (Suk et al. 1999), JP-45 (Anderson et al. 2003) and SRP-27 (Bleunven et al. 2008), must also contribute to control of SR intraluminal Ca2+ load, but the precise nature of their role remains undetermined. JP-45 in particular is ideally placed to communicate store load to the excitation–contraction (EC) coupling process as it binds to both CSQ and the dihydropyridine receptor (DHPR) in the surface/transverse tubule membrane. The importance of the luminal proteins has been underlined by the recent discovery that changes in Ca2+ signalling due to mutations in CSQ or to lack of its expression can result in sudden cardiac death (Viatchenko-Karpinski et al. 2004). Furthermore, studies in animal models show that changes in CSQ, junctin, triadin and HRC expression can lead to defective Ca2+ signalling. The review by Pritchard & Kranius (2009) examines the roles of junctin and HRC in Ca2+ cycling and their potential significance in heart failure. This commentary focuses on protein-protein interactions between CSQ, triadin, junctin and RyR proteins that may underlie their roles in Ca2+ cycling. Figure 1 Illustration of the possible interactions between triadin, junctin, CSQ1 and RyR1 in the lumen of the SR

Enhanced Ryanodine Receptor-Mediated Calcium Leak Determines Reduced Sarcoplasmic Reticulum Calcium Content in Chronic Canine Heart Failure

Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation–contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1–10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 +/- 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 +/- 1.91-, 1.42 +/- 0.08-, 2.99 +/- 0.32-, and 1.91 +/- 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load.

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

On the Loose: Uncaging Ca2+-induced Ca2+ Release in Smooth Muscle