Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) inducing Ca²⁺ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca²⁺ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca²⁺ release, leading to a significant increase in the activity of Ca²⁺ sparks and contraction in airway SMCs. The increased Ca²⁺ sparks are independent of VDCCs and the associated extracellular Ca²⁺ influx. This format of local Ca²⁺ release results from a direct activation of G protein-coupled, M₃ muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP₃), inducing initial Ca²⁺ release through IP₃ receptors and then further Ca²⁺ release via RyR2 due to a local Ca²⁺-induced Ca²⁺ release process. These findings demonstrate an important mechanism for Ca²⁺ signaling and attendant physiological function in SMCs.     

Ca2+ channels, Ca2+ sparks, and regulation of arterial smooth muscle function

Summary In cardiac, skeletal, and arterial muscle, transient, spatially localized elevations in [Ca²⁺]_i, termed "Ca²⁺ sparks", have been observed using confocal laser scanning microscopy. Ca²⁺ sparks are thought to represent "elementary" Ca²⁺ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In striated muscle, Ca²⁺ sparks are thought to be key elements of excitation-contraction coupling. In arterial smooth muscle, Ca²⁺ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation. Using a developmental model, we have investigated whether RyRs causing Ca²⁺ sparks and activation of Ca²⁺-activated K⁺ (K_Ca) channels (STOCs) function as "elementary" Ca²⁺ release units that regulate arterial mygenic tone. Whereas increases in the global [Ca²⁺]_i induce sustained constriction of arterial smooth muscle, Ca²⁺ sparks induce vasodilation through the local activation of K_Ca channels. In cerebral arteries, the global bulk [Ca²⁺]_i and a Ca²⁺ spark frequency < 10^-2 Hz/cell do not cause sufficient K_Ca channel activity to regulate membrane potential of smooth muscle cells and myogenic tone. The frequency of Ca²⁺ sparks and STOCs is regulated by agents that modulate protein kinase G and protein kinase A activity. Our findings suggest that "elementary" Ca²⁺ release units may represent novel, important therapeutic targets for regulating function of the intact arterial smooth muscle tissue.     

The ryanodine receptor leak: how a tattered receptor plunges the failing heart into crisis

It has been persuasively shown in the last two decades that the development of heart failure is closely linked to distinct alterations in Ca²⁺ cycling. A crucial point in this respect is an increased spontaneous release of Ca²⁺ out of the sarcoplasmic reticulum during diastole via ryanodine receptors type 2 (RyR2). The consequence is a compromised sarcoplasmic reticulum Ca²⁺ storage capacity, which impairs systolic contractility and possibly diastolic cardiac function due to Ca²⁺ overload. Additionally, leaky RyR2 are more and more regarded to potently induce proarrhythmic triggers. Elimination of spontaneously released Ca²⁺ via RyR2 in diastole can cause a transient sarcolemmal inward current and hence delayed after depolarisations as substrate for cardiac arrhythmias. In this article, the pathological role and consequences of the SR Ca²⁺-leak and its regulation are reviewed with a main focus on protein kinase A and Ca²⁺-calmodulin-dependent kinase II. We summarise clinical consequences of “leaky RyR2” as well as possible therapeutic strategies in order to correct RyR2 dysfunction and discuss the significance of the available data.     

FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel

Calmodulin (CaM) functions as a regulatory subunit of ryanodine receptor (RyR) channels, modulating channel activity in response to changing [Ca²⁺]_i. To investigate the structural basis of CaM regulation of the RyR1 isoform, we used site-directed labeling of channel regulatory subunits and fluorescence resonance energy transfer (FRET). Donor fluorophore was targeted to the RyR1 cytoplasmic assembly by preincubating sarcoplasmic reticulum membranes with a fluorescent FK506-binding protein (FKBP), and FRET was determined following incubations in the presence of fluorescent CaMs in which acceptor fluorophore was attached within the N lobe, central linker, or C lobe. Results demonstrated strong FRET to acceptors attached within CaM's N lobe, whereas substantially weaker FRET was observed when acceptor was attached within CaM's central linker or C lobe. Surprisingly, Ca²⁺ evoked little change in FRET to any of the 3 CaM domains. Donor–acceptor distances derived from our FRET measurements provide insights into CaM's location and orientation within the RyR1 3D architecture and the conformational switching that underlies CaM regulation of the channel. These results establish a powerful new approach to resolving the structure and function of RyR channels.     

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter,RyR structure and calmodulin affinity

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain–domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H₂O₂)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM–RyR2 and FK506-binding protein (FKBP12.6)–RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H₂O₂ was used to mimic oxidative stress. H₂O₂ significantly increased the frequency of Ca^2 + sparks and spontaneous Ca^2 + waves, and dantrolene almost completely blocked these effects. H₂O₂ pretreatment significantly reduced CaM–RyR2 binding, but had no effect on FKBP12.6–RyR2 binding. Dantrolene restored CaM–RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H₂O₂ also accelerated F-DPc10–RyR2 association while dantrolene slowed it. Thus, H₂O₂ causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H₂O₂ treatment markedly reduces the CaM–RyR2 affinity, has no effect on FKBP12.6–RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM–RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.     

Flecainide inhibits arrhythmogenic Ca waves by open state block of ryanodine receptor Ca release channels and reduction of Ca spark mass

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca²⁺ waves and elementary sarcoplasmic reticulum (SR) Ca²⁺ release events, Ca²⁺ sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca²⁺ leak or SR Ca²⁺ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca²⁺ leak, resulting in a significantly increased SR Ca²⁺ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca²⁺ waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca²⁺ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca²⁺ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca²⁺ waves resulting in triggered arrhythmias, such as CPVT.     

Ca(2+) sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle

Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular-sarcoplasmic reticulum junctions, and the unit signal is the Ca(2+) spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RyR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca(2+) sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RyR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca(2+)-induced Ca(2+) release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RyR3 isoform. Because RyR3 does not contact voltage sensors, their opening was probably activated by Ca(2+), secondarily to Ca(2+) release through junctional RyR1. Physiologically voltage-controlled Ca(2+) sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca(2+), and a slave parajunctional RyR3 cohort.     

Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca²⁺ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.     

Pernicious attrition and inter-RyR2 CICR current control in cardiac muscle

In cardiac muscle cells, ryanodine receptor (RyR) mediated Ca^2 + release from the sarcoplasmic reticulum (SR) drives the contractile apparatus. Spontaneous bouts of inter-RyR Ca^2 + induced Ca^2 + release (CICR) generate an elemental unit of SR Ca^2 + release called a spark. Sparks are localized events that terminate soon after they begin. The local control of sparks is not clearly understood. In this article, we review the potential regulatory role that the changing single RyR Ca^2 + current may play. Moreover, we aggregate RyR data into a working scheme of inter-RyR CICR current control of sparks and a potential inter-RyR CICR termination mechanism that we call pernicious attrition. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.     

Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart

Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca²⁺ leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart. Here we show in the intact heart, reduced NOS1 activity increased Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ release, resulting from a combination of decreased RyR2 S-nitrosylation (RyR2-SNO) and increased RyR2 oxidation (RyR-SOx) (i.e., increased reactive oxygen species (ROS) from xanthine oxidoreductase activity) and could be suppressed with xanthine oxidoreductase (XOR) inhibition (i.e., allopurinol) or nitric oxide donors (i.e., S-nitrosoglutathione, GSNO). Surprisingly, we found evidence of NOS1 down-regulation of RyR2 phosphorylation at the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between nitrosylation and phosphorylation of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype by oxidative stress. Our findings suggest that nitroso–redox imbalance is an important mechanism of ventricular arrhythmias in the intact heart under disease conditions (i.e., elevated [Ca²⁺]_i and oxidative stress), and that therapies restoring nitroso–redox balance in the heart could prevent sudden arrhythmic death.Nitric oxide (NO) is an important regulator of cardiac function via both the activation of cyclic guanosine monophosphate-dependent signaling pathways and direct posttranslational modification of protein thiols (S-nitrosylation) (1). NO derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca²⁺ handling proteins (2). In particular, nitrosylation of both skeletal and cardiac muscle ryanodine receptors (RyR1 and RyR2, respectively) alters their release properties, favoring activation (3, 4). Notably, an increase in RyR2 open probability can cause spontaneous SR Ca²⁺ release, which may cause arrhythmias. Recently, it was shown that decreased RyR2 S-nitrosylation (RyR2-SNO) through loss of NOS1, was associated with increased spontaneous SR Ca²⁺ release events in isolated cardiomyocytes, following rapid pacing (5). In a separate study, NOS1 deficiency was shown to decrease spontaneous SR Ca²⁺ sparks and the open probability of RyR2 under resting conditions in cardiomyocytes and lipid bilayers, respectively (6). These studies suggest that NOS1 deficiency has a variable effect on RyR2 function, likely dependent on the underlying physiological state (i.e., rapid heart rate versus quiescence). It remains unknown, however, whether these changes create a substrate for arrhythmogenesis in the intact heart.It is increasingly evident that activities of nitric oxide and reactive oxygen species (ROS) are tightly coupled in cardiomyocytes producing nitroso–redox balance. Elevated ROS production (oxidative stress) is a hallmark of several cardiovascular diseases associated with increased risk of fatal ventricular arrhythmias [e.g., myocardial infarction (MI) and heart failure]. Burger et al. (7) recently demonstrated an increased incidence of ventricular arrhythmias following MI in NOS1-deficient mice. These data suggest that a nitroso–redox imbalance may be arrhythmogenic in the setting of MI. However, the molecular basis of the increased arrhythmogenesis is not known.In the current study, we found that decreased NOS1 activity increased Ca²⁺-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca²⁺]_i. These arrhythmias arose from increased spontaneous SR Ca²⁺ release resulting from a combination of decreased RyR2-SNO and increased RyR2 oxidation (RyR2-SOx) [i.e., increased ROS from xanthine oxidoreductase (XOR) activity] and could be suppressed with xanthine oxidoreductase inhibition (i.e., allopurinol) or nitric oxide donors (i.e., GSNO). Notably, we found evidence of NOS1 regulation of RyR2 phosphorylation at the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between the nitrosylation and phosphorylation states of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype under oxidative stress.     

Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity

During exercise, defects in calcium (Ca²⁺) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca²⁺ release channel required for excitation–contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S-nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in “leaky” channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca²⁺-dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca²⁺ leak via calstabin1-depleted RyR1 channels leads to defective Ca²⁺ signaling, muscle damage, and impaired exercise capacity.     

Ablation of triadin causes loss of cardiac Ca2+ release units,impaired excitation–contraction coupling,and cardiac arrhythmias

Heart muscle excitation–contraction (E-C) coupling is governed by Ca²⁺ release units (CRUs) whereby Ca²⁺ influx via L-type Ca²⁺ channels (Cav1.2) triggers Ca²⁺ release from juxtaposed Ca²⁺ release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn^−/−). The structure and protein composition of the cardiac CRU is significantly altered in Trdn^−/− hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn^−/− hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn^−/− myocytes. CRU function is impaired in Trdn^−/− myocytes, with reduced SR Ca²⁺ release and impaired negative feedback of SR Ca²⁺ release on Cav1.2 Ca²⁺ currents (I_Ca). Uninhibited Ca²⁺ influx via I_Ca likely contributes to Ca²⁺ overload and results in spontaneous SR Ca²⁺ releases upon β-adrenergic receptor stimulation with isoproterenol in Trdn^−/− myocytes, and ventricular arrhythmias in Trdn^−/− mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.     

High-mobility group box 1 (HMGB1) impaired cardiac excitation–contraction coupling by enhancing the sarcoplasmic reticulum (SR) Ca2 + leak through TLR4–ROS signaling in cardiomyocytes

High-mobility group box 1 (HMGB1) is a proinflammatory mediator playing an important role in the pathogenesis of cardiac dysfunction in many diseases. In this study, we explored the effects of HMGB1 on Ca^2 + handling and cellular contractility in cardiomyocytes to seek for the mechanisms underlying HMGB1-induced cardiac dysfunction. Our results show that HMGB1 increased the frequency of Ca^2 + sparks, reduced the sarcoplasmic reticulum (SR) Ca^2 + content, and decreased the amplitude of systolic Ca^2 + transient and myocyte contractility in dose-dependent manners in adult rat ventricular myocytes. Inhibiting high-frequent Ca^2 + sparks with tetracaine largely inhibited the alterations of SR load and Ca^2 + transient. Blocking Toll-like receptor 4 (TLR4) with TAK-242 or knockdown of TLR4 by RNA interference remarkably inhibited HMGB1 induced high-frequent Ca^2 + sparks and restored the SR Ca^2 + content. Concomitantly, the amplitude of systolic Ca^2 + transient and myocyte contractility had significantly increased. Furthermore, HMGB1 increased the level of intracellular reactive oxygen species (ROS) and consequently enhanced oxidative stress and CaMKII-activated phosphorylation (pSer2814) in ryanodine receptor 2 (RyR2). TAK-242 pretreatment significantly decreased intracellular ROS levels and oxidative stress and hyperphosphorylation in RyR2, similar to the effects of antioxidant MnTBAP. Consistently, MnTBAP normalized HMGB1-impaired Ca^2 + handling and myocyte contractility. Taken together, our findings suggest that HMGB1 enhances Ca^2 + spark-mediated SR Ca^2 + leak through TLR4–ROS signaling pathway, which causes partial depletion of SR Ca^2 + content and hence decreases systolic Ca^2 + transient and myocyte contractility. Prevention of SR Ca^2 + leak may be an effective therapeutic strategy for the treatment of cardiac dysfunction related to HMGB1 overproduction.     

基因突变致心肌组织兰尼碱受体功能缺陷的研究进展

兰尼碱受体是心肌细胞上的一种钙离子释放通道,当基因发生突变时可导致延迟后除极致心脏猝死性室性心动过速的发生,但到目前为止,具体的分子生物学机制却不是很清楚,可能与其调节蛋白-FK506结合蛋白的解离、Ca2 激活域值降低及通道内部各功能域之间作用受损等引起的舒张期钙离子渗漏有关,现就关于基因突变所致兰尼碱受体功能障碍发生机制的各种学说做一综述.     

Crystal structure of type I ryanodine receptor amino-terminal β-trefoil domain reveals a disease-associated mutation “hot spot” loop

Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca²⁺. Ca²⁺ is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca²⁺ transient by activation of a class of Ca²⁺ release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca²⁺-ATPases (SERCAs) to terminate the Ca²⁺ transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR_NTD) at 2.5 Å. The RyR_NTD structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP₃Rsup), but lacks most of the long helix-turn-helix segment of the “arm” domain in IP₃Rsup. The N-terminal β-trefoil fold, found in both RyR and IP₃R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation “hot spot” loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyR_NTD, supporting previously described mechanisms whereby mutations perturb protein–protein interactions.     

Ryanodin-Rezeptor-Stabilisatoren

Different disease syndromes including arrhythmias, heart failure, skeletal myopathy, and epilepsy have been associated with abnormally increased Ca²⁺ leak from the intracellular organelles. In the heart, intracellular Ca²⁺ release is controlled by cardiac ryanodine receptors (RyR2s) which are Ca²⁺ release channels situated in the membranes of the sarcoplasmic reticulum (SR) storage organelles. RyR2-dependent Ca²⁺ release is essential for myocardial contraction and relaxation which control systolic and diastolic heart function. The function of the Ca²⁺ release channel depends on four identical RyR2 subunits each associated with a specific set of enzymes which modulate cardiac function. Both genetic and acquired forms of heart disease result in unstable RyR2 channel closure in diastole and detrimental SR Ca²⁺ leak. The acute form of SR Ca²⁺ leak leads to afterdepolarizations of the membrane potential which may trigger deadly arrhythmias, whereas the chronic form of SR Ca²⁺ leak depletes intracellular Ca²⁺ stores and contributes to heart failure. Leaky RyR2 channels are the pharmacological target of novel RyR-selective 1,4-benzothiazepin derivatives, which were found to stabilize the channel closed state through increased calstabin2 binding, to inhibit SR Ca²⁺ leak, and to exert therapeutic in vivo effects against arrhythmias and heart failure progression.     

Functional nonequality of the cardiac and skeletal ryanodine receptors

Dihydropyridine receptors (DHPRs), which are voltage-gated Ca²⁺ channels, and ryanodine receptors (RyRs), which are intracellular Ca²⁺ release channels, are expressed in diverse cell types, including skeletal and cardiac muscle. In skeletal muscle, there appears to be reciprocal signaling between the skeletal isoforms of both the DHPR and the RyR (RyR-1), such that Ca²⁺ release activity of RyR-1 is controlled by the DHPR and Ca²⁺ channel activity of the DHPR is controlled by RyR-1. Dyspedic skeletal muscle cells, which do not express RyR-1, lack excitation–contraction coupling and have an ≈30-fold reduction in L-type Ca²⁺ current density. Here we have examined the ability of the predominant cardiac and brain RyR isoform, RyR-2, to substitute for RyR-1 in interacting with the skeletal DHPR. When RyR-2 is expressed in dyspedic muscle cells, it gives rise to spontaneous intracellular Ca²⁺ oscillations and supports Ca²⁺ entry-induced Ca²⁺ release. However, unlike RyR-1, the expressed RyR-2 does not increase the Ca²⁺ channel activity of the DHPR, nor is the gating of RyR-2 controlled by the skeletal DHPR. Thus, the ability to participate in skeletal-type reciprocal signaling appears to be a unique feature of RyR-1.     

Isolation from porcine cardiac muscle of a Ca2+-activated protease that partially degrades myofibrils

A protein fraction displaying Ca²⁺-activated proteolytic activity has been isolated from porcine cardiac muscle. The crude enzyme was purified approximately 2000 fold by isoelectric precipitation followed by gel permeation chromatography and by ion exchange chromatography. The partially purified enzyme exhibited optimal activity against either cardiac myofibril or casein substrates between pH 7.5 and 8.0, and in the presence of 1 mm Ca²⁺ and at least 2 mm 2-mercaptoethanol. The enzyme removes Z-discs from skeletal and cardiac myofibrils and also removes the density from intercalated discs of cardiac myofibrils. The enzyme hydrolyzes troponin-T and troponin-I of both cardiac and skeletal muscle myofibrils in vitro. In its proteolytic effect on either cardiac or skeletal myofibrils and in all other properties examined, the Ca²⁺-activated, cardiac protease is similar to a Ca²⁺-activated protease (CAF) recently purified from porcine skeletal muscle (Dayton, W. R., Reville, W. J., Goll, D. E. and Stromer, M. H. (1976) Biochemistry15, 2159–2167). It is possible that the Ca²⁺-activated, cardiac protease plays a role in degradation of myofibrils in injured myocardial cells.     

Altered interaction of FKBP12.6 with ryanodine receptor as a cause of abnormal Ca(2+) release in heart failure

OBJECTIVE: Little information is available as to the Ca(2+) release function of the sarcoplasmic reticulum (SR) in heart failure. We assessed whether the alteration in this function in heart failure is related to a change in the role of FK binding protein (FKBP), which is tightly coupled with the cardiac ryanodine receptor (RyR) and recently identified as a modulatory protein acting to stabilize the gating function of RyR. METHODS: SR vesicles were isolated from dog LV muscles [normal (N), n=6; heart failure induced by 3-weeks pacing (HF), n=6]. The time course of the SR Ca(2+) release was continuously monitored using a stopped-flow apparatus, and [3H]ryanodine-binding and [3H]dihydro-FK506-binding assays were also performed. RESULTS: FK506, which specifically binds to FKBP12.6 and dissociates it from RyR, decreased the polylysine-induced enhancement of [3H]ryanodine-binding by 38% in N (P<0.05) but it had no effect in HF. In HF, the rate constant for the polylysine-induced Ca(2+) release from the SR was 61% smaller than in N. FK506 decreased the rate constant for the polylysine-induced Ca(2+) release by 67% in N (P<0.05) but had no effect in HF. The [3H]dihydro-FK506-binding assay revealed that the number (B(max)) of FKBPs was decreased by 83% in HF (P<0.05), while the K(d) value was unchanged. FK506 did not significantly change SR Ca(2+.)-ATPase activity in either N or HF. CONCLUSIONS: In HF, the number of FKBPs showed a tremendous decrease; this may underlie the RyR-channel instability and the impairment of the Ca(2+) release function of RyR seen in the failing heart.     

Expression and regulation of ryanodine receptor/calcium release channels

Intracellular calcium release channels on the endoplasmic or sarcoplasmic reticula (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP(3)R) comprise a unique family of molecules that are structurally and functionally distinct from all other known ion channels. These channels play crucial roles in many cellular signaling pathways including excitation-contraction coupling, oocyte fertilization, hormone secretion, neurotransmitter release, and T lymphocyte activation. Three forms of RyR have been identified: RyR1 expressed predominantly in skeletal muscle, RyR2 in cardiac muscle, and RyR3 in the brain. The tetrameric structures of RyR1 and RyR2 are stabilized by a channel-associated protein, FKBP12. The immunosuppressant drugs FK506 and rapamycin inhibit the prolyl isomerase activity of FKBP12 and could cause cardiac dysfunction by inducing a Ca(2+) leak from the sarcoplasmic reticulum. RyR2 is downregulated and IP(3)R is upregulated during severe end-stage heart failure secondary to dilated cardiomyopathies in humans, suggesting that these channels may contribute to abnormalities in Ca(2+) homeostasis.