Sarcoplasmic Reticulum Calcium Release Channel Research Articles

Individuals and Families Affected by RYR1-Related Diseases: The Patient/Caregiver Perspective.

Pathogenic variants of RYR1, the gene encoding the principal sarcoplasmic reticulum calcium release channel (RyR1) with a crucial role in excitation-contraction coupling, are among the most common genetic causes of non-dystrophic neuromuscular disorders. We recently conducted a questionnaire study focusing on functional impairments, fatigue, and quality of life (QoL) in patients with RYR1-related diseases (RYR1-RD) throughout the recognized disease spectrum. In this previous questionnaire study the medical perspective was taken, reflective of a study protocol designed by neurologists and psychologists. With this present study we wanted to specifically address the patient perspective. Together with affected individuals, family members, and advocates concerned with RYR1-RD, we developed an online patient survey that was completed by 227 patients or their parents/other caretakers (143 females and 84 males, 0-85 years). We invited 12 individuals, representing most of the patient group based on age, sex, race, and type and severity of diagnosis, to share their personal experiences on living with a RYR1-RD during an international workshop in July 2022. Data were analyzed through a mixed-methods approach, employing both a quantitative analysis of the survey results and a qualitative analysis of the testimonials. Data obtained from the combined quantitative and qualitative analyses provide important insights on six topics: 1) Diagnosis; 2) Symptoms and impact of the condition; 3) Physical activity; 4) Treatment; 5) Clinical research and studies; and 6) Expectations. Together, this study provides a unique patient perspective on the RYR1-RD spectrum, associated disease impact, suitable physical activities and expectations of future treatments and trials, and thus, offers an essential contribution to future research.

Time course and fibre type-dependent nature of calcium-handling protein responses to sprint interval exercise in human skeletal muscle.

Sprint interval training (SIT) causes fragmentation of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor 1 (RyR1), 24h post-exercise, potentially signalling mitochondrial biogenesis by increasing cytosolic [Ca2+ ]. Yet, the time course and skeletal muscle fibre type-specific patterns of RyR1 fragmentation following a session of SIT remain unknown. Ten participants (n=4 females; n=6 males) performed a session of SIT (6×30s 'all-out' with 4.5min rest after each sprint) with vastus lateralis muscle biopsy samples collected before and 3, 6 and 24h after exercise. In whole muscle, full-length RyR1 protein content was significantly reduced 6h (mean (SD); -38 (38)%; P<0.05) and 24h post-SIT (-30 (48)%; P<0.05) compared to pre-exercise. Examining each participant's largest response in pooled samples, full-length RyR1 protein content was reduced in type II (-26 (30)%; P<0.05) but not type I fibres (-11 (40)%; P>0.05). Three hours post-SIT, there was also a decrease in sarco(endo)plasmic reticulum Ca2+ ATPase 1 in type II fibres (-23 (17)%; P<0.05) and sarco(endo)plasmic reticulum Ca2+ ATPase 2a in type I fibres (-19 (21)%; P<0.05), despite no time effect for either protein in whole muscle samples (P>0.05). PGC1A mRNA content was elevated 3 and 6h post-SIT (5.3- and 3.7-fold change from pre, respectively; P<0.05 for both), but peak PGC1A mRNA expression was not significantly correlated with peak RyR1 fragmentation (r2 =0.10; P>0.05). In summary, altered Ca2+ -handling protein expression, which occurs primarily in type II muscle fibres, may influence signals for mitochondrial biogenesis as early as 3-6h post-SIT in humans. KEY POINTS: Sprint interval training (SIT) has been shown to cause fragmentation of the sarcoplasmic reticulum calcium-release channel, ryanodine receptor 1 (RyR1), 24h post-exercise, which may act as a signal for mitochondrial biogenesis. In this study, the time course was examined of RyR1 fragmentation in human whole muscle and pooled type I and type II skeletal muscle fibres following a single session of SIT. Full-length RyR1 protein content was significantly lower than pre-exercise by 6h post-SIT in whole muscle, and fragmentation was detectable in type II but not type I fibres, though to a lesser extent than in whole muscle. The peak in PGC1A mRNA expression occurred earlier than RyR1 fragmentation. The increased temporal resolution and fibre type-specific responses for RyR1 fragmentation provide insights into its importance to mitochondrial biogenesis in humans.

Inhibition of chymotrypsin-like activity of the proteasome by ixazomib prevents mitochondrial dysfunction during myocardial ischemia.

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM.

A Mechanism for Statin-Induced Susceptibility to Myopathy

This study aimed to identify a mechanism for statin-induced myopathy that explains its prevalence and selectivity for skeletal muscle, and to understand its interaction with moderate exercise. Statin-associated adverse muscle symptoms reduce adherence to statin therapy; this limits the effectiveness of statins in reducing cardiovascular risk. The issue is further compounded by perceived interactions between statin treatment and exercise. This study examined muscles from individuals taking statins and rats treated with statins for 4weeks. In skeletal muscle, statin treatment caused dissociation of the stabilizing protein FK506 binding protein (FKBP12) from the sarcoplasmic reticulum (SR) calcium (Ca2+) release channel, the ryanodine receptor 1, which was associated with pro-apoptotic signaling and reactive nitrogen species/reactive oxygen species (RNS/ROS)-dependent spontaneous SR Ca2+ release events (Ca2+ sparks). Statin treatment had no effect on Ca2+ spark frequency in cardiac myocytes. Despite potentially deleterious effects of statins on skeletal muscle, there was no impact on force production or SR Ca2+ release in electrically stimulated muscle fibers. Statin-treated rats with access to a running wheel ran further than control rats; this exercise normalized FKBP12 binding to ryanodine receptor 1, preventing the increase in Ca2+ sparks and pro-apoptotic signaling. Statin-mediated RNS/ROS-dependent destabilization of SR Ca2+ handling has the potential to initiate skeletal (but not cardiac) myopathy in susceptible individuals. Importantly, although exercise increases RNS/ROS, it did not trigger deleterious statin effects on skeletal muscle. Indeed, our results indicate that moderate exercise might benefit individuals who take statins.

RyR2 Inhibition by Dantrolene Requires both Calmodulin and FKBP12.6

Dantrolene protects cardiac muscle from arrhythmia and heart failure by inhibiting Ca2+ release from the sarcoplasmic reticulum (SR). Single-channel recordings were obtained from SR calcium release channels (RyR2) isolated from sheep and human heart and incorporated into artificial lipid bilayers. Dantrolene inhibition was measured by exposing RyR2 both in the absence and presence of exogenous 100 nM CaM. In diastolic cytoplasmic [Ca2+] (100 nM), dantrolene (50 μM) reduced the mean open probability of RyR2 to 45 ± 6% in the presence of CaM (n = 7, p<0.05, Student's paired t-test) but had no significant effect when CaM was absent (95 ± 9%, n = 20, p = 0.24). Dantrolene exhibited a hyperbolic dose response in the presence of CaM with an IC50 of 0.16 ± 0.03 μM and with a saturating relative Po (Emax) of 52 ± 4%. Emax increased to 1 as cytoplasmic Ca2+ was increased to levels >1 μM. In saponin-permeablised mouse cardiomyocytes supplemented with 100 nM CaM, dantrolene reduced the frequency and amplitude of Ca2+ waves, with an IC50 of 0.3 μM. However, when cells were depleted of CaM, dantrolene had no effect. Thus, CaM is essential for inhibitory action of dantrolene. Incubation of RyR2 to rapamycin prior to single-channel recording, known to dissociate FKBP12.6 from RyR2, removed the inhibitory action of dantrolene. This could be restored by adding FKBP12.6 to the bathing media. We conclude that the dantrolene inhibition required the presence of both CaM and FKBP12.6 in the RyR2 complex.

Relationship between Multi-Scale Cardiomyocyte Organization and Function in Trabeculae of the Failing Human Heart

Invaginations of the plasma membrane within cardiomyocytes form the transverse tubule network (t-tubules), and at “dyadic junctions” these come into close association with regions of the sarcoplasmic reticulum (SR). In addition to many of the key excitation-contraction (EC) coupling proteins, junctions contain junctophilin-2 (JPH2), which is implicated in both the formation and maintenance of junctions. In human heart failure there is a loss of cardiac function which is generally associated with impairment of EC coupling processes, as well as changes to t-tubule structure and protein distribution. The aim of this study was to investigate the link between structural re-organization of cardiomyocytes and the loss of contractile function in trabeculae from failing human hearts.We obtained trabeculae from explanted human hearts in end-stage heart failure and performed force measurements followed by immuno-labelling to determine correlations between protein distribution and functional performance in the failing human heart. Cardiac trabeculae were processed along with samples taken from the free wall to investigate the variability of structure and protein distribution, including t-tubules, JPH2 and ryanodine receptors (the SR calcium release channel) using confocal imaging. Findings from these experiments revealed that there is a high degree of variability of trabecular structure and function, both within and between hearts. Trabeculae exhibiting poor contractile function often had low cardiomyocyte content and remaining myocytes showed disorganization of t-tubules and EC coupling proteins.These findings demonstrate a clear link between trabecula structure and function in the diseased human heart and support the idea that cardiomyocyte organization at cell and tissue levels is correlated with cardiac function. Furthermore, while trabeculae are useful for studying cardiac function, trabecula and cardiomyocyte structure must be considered when interpreting functional results.

Inhibition of the mevalonate pathway ameliorates anoxia-induced down-regulation of FKBP12.6 and intracellular calcium handling dysfunction in H9c2 cells

Statins have beneficial pleiotropic effects beyond lipid lowering on the cardiovascular system. These cardio-protective effects are mediated through inhibition of the intracellular mevalonate pathway, by decreasing isoprenoid intermediate synthesis and the subsequent post-translational modification of small GTPases, such as Ras, Rho, and Rac. Impaired intracellular calcium handling is considered an important pathophysiologic mechanism responsible for cardiac dysfunction. Our study aimed at investigating the influence of mevalonate pathway, including its downstream small GTPases (Ras, RhoA, and Rac1) on anoxia-mediated alterations of calcium handling in H9c2 cardiomyocytes. Cultured H9c2 cardiomyocytes were exposed to acute anoxia after pretreatment with different drugs that specifically antagonize five key components in the mevalonate pathway, including 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, Rho-kinase, Rac1 and Ras farnesyltransferase. Thereafter, we evaluated the effects of the mevalonate pathway on anoxia-induced cell death, expression of the sarcoplasmic reticulum calcium release channel (ryanodine receptor 2) and its regulator FK506-binding protein 12.6, as well as functional calcium release from intracellular calcium stores. Our experiments confirmed the role of prenylated proteins in regulating cardiomyocyte dysfunction, especially via RhoA- and Ras-related signaling pathways. Furthermore, our data demonstrated that inhibition of the mevalonate pathway could ameliorate anoxia-mediated calcium handling dysfunction with the up-regulated expression of FK506-binding protein 12.6 and consequently provided evidence for FK506-binding protein 12.6 as a “stabilizer” of ryanodine receptor 2.

EPR with Rigidly Bound Spin Labels used to Probe the Interaction of Calmodulin with the Ryanodine Receptor

We have used site-directed spin labeling and EPR spectroscopy to probe the structural dynamics of calmodulin bound to a ryanodine receptor target peptide. Calmodulin binds to a conserved 30-amino acid sequence on the sarcoplasmic reticulum calcium release channel (ryanodine receptor, RyR) and directly modulate its activity in muscle contraction. In order to characterize the structural details of this crucial interaction, a bifunctional spin label was used to rigidly label di-cysteine mutant calmodulin at positions 34 and 38. Solid-phase peptide synthesis was used to create a peptide corresponding to the calmodulin binding site on the ryanodine receptor channel (RyRp, residues 3614-3643 in RyR1) with a TOAC spin label rigidly coupled to the backbone. The intermolecular distance distribution between the two spin probes was determined by EPR, using both continuous wave dipolar broadening and dipolar electron-electron resonance (DEER). The center of the distance distribution was in good agreement with the crystal structure of the complex (18 A), but the width of the distribution was ∼ 1 nm, comparable to that observed previously within calmodulin itself. Thus calmodulin retains significant inter-lobe domain motion, even after RyRp binding. These results are consistent with a model in which calmodulin binds RyRp, bringing its two terminal lobes close together but preventing them from physical contact with each other, allowing flexibility in each of the lobe.

In silico study of potential autoimmune threats from rotavirus infection

Rotavirus, the major cause of infantile nonbacterial diarrhea, was found to be associated with development of diabetes-associated auto-antibodies. In our study we tried to find out further potential autoimmune threats of this virus using bioinformatics approach.We took rotaviral proteins to study similarity with Homo sapiens proteome and found most conserved structural protein VP6 matches at two regions with ryanodine receptor, an autoimmune target associated with myasthenia gravis. Myasthenia gravis, a chronic neurodegenerative autoimmune disorder with no typical known reason, is characterized by fluctuating muscle weakness which is typically enhanced during muscular effort. Affected patients generate auto antibodies against mainly acetyl choline receptor and sarcoplasmic reticulum calcium-release channel protein ryanodine receptor. Further, we observed that two regions which matched with ryanodine receptor remain conserved in all circulating rotaviral strains and showed significant antigenecity with respect to myasthenia gravis associated HLA haplotypes.Overall, our study detected rotaviral VP6 as a potential threat for myasthenia gravis and enlighten an area of virus associated autoimmune research.

Calcium influx and release mechanism(s) in histamine-induced myometrial contraction in buffaloes

The present study was undertaken to characterize the presence of histamine H1R using molecular biology tools and unravel the influx and release mechanism(s) involved in calcium signalling cascades in histamine-induced myometrial contraction in buffaloes. The presence of H1R mRNA transcript and immunoreactive membrane protein in buffalo myometrium was confirmed by RT-PCR and Western blot analysis. Further, histamine produced concentration-dependent (1nM–10μM) contraction in buffalo myometrium with a potency of 7.13±0.11. When myometrial strips were pre-incubated either with Ca2+ free solution or with nifedipine, a L-type Ca2+ channel blocker, dose response curve (DRC) of histamine was significantly (P<0.05) shifted towards right with decline in maximal contraction (Emax). Reduction in Emax of histamine in the presence of nifedipine (55.75±3.10%) was significantly (P<0.001) greater than that in the presence of ruthenium red (93.61±3.43%), a blocker of IP3-gated and RyR-sensitive Ca2+ channels. Moreover, histamine produced only 26.87±1.99% of the maximum contraction in the presence of both nifedipine and CPA (blocker of sarco-endoplasmic reticulum Ca2+-ATPase). Interestingly, following concurrent exposure to U-73122 (a PL-C inhibitor) and nifedipine, the DRC of histamine was significantly (P<0.05) shifted towards left with increase in maximal contraction (126.30±3.36%). Our findings in buffalo uterus thus suggest that influx of extracellular calcium plays a major role in histamine-induced myometrial contraction, while release of intracellular calcium through calcium-release channels of sarcoplasmic reticulum has a minor role. A possible involvement of non-selective cation channels in histamine-induced myometrial contraction cannot be ruled out, and therefore requires further investigations.

Dantrolene

The porcine stress syndrome is a major cause of poor meat quality and death in the pork industry. It is known to be more prevalent in some pig strains than others,1 and susceptible animals can be identified by a challenge with halothane, which results in striking elevation in body temperature. This scenario parallels the clinical entity of familial malignant hyperthermia on exposure to general anesthetics, which was one of the earliest recognized human pharmacogenetic syndromes. We now know that affected pigs and people share the same molecular mechanism, mutations in the sarcoplasmic reticulum (SR) calcium release channel of skeletal muscle encoded by RYR1 .2,3 In the pig world, selective breeding programs have been used to develop strains resistant to malignant hyperthermia. In humans, malignant hyperthermia is an anesthetic emergency and is treated by immediate intravenous administration of dantrolene, which is effective and thought to be safe. Chronic oral dantrolene is also approved to treat severe muscle spasticity, and, in this setting, the limiting toxicity is hepatitis, which can be fulminant and fatal in up to 1% of exposed subjects. Article see p 875 Abrupt membrane depolarization (excitation) in skeletal or in cardiac muscle results in calcium release from SR stores via ryanodine receptor (RyR) calcium release channels, and the ensuing rise in intracellular calcium then activates the contractile apparatus. The details of the way in which excitation couples to contraction differs somewhat in the 2 types of muscle (in the heart, but not in skeletal muscle, calcium influx via voltage-gated Cav1.2 channels is required to activate RyR channels; Figure), and there are different genes encoding the channels, RYR1 in skeletal muscle and RYR2 in cardiac muscle. Early studies of the mechanism of action of dantrolene highlighted its ability to decouple excitation from contraction in skeletal muscle, …

Nanoscale Changes in the Organisation of Junctional Proteins in JPH2 Transgenic Mice

In cardiomyocytes, transverse tubule (t-tubule) and sarcoplasmic reticulum (SR) membranes form close associations, known as diadic junctions, which are critical for excitation-contraction (E-C) coupling. Ryanodine receptors (RyRs), which are the SR calcium release channels, are concentrated in junctions where they play a key role for calcium release. In human and animal models of heart failure junctions and transverse (t-) tubules often become disorganised but the underlying mechanisms remain unclear. The SR membrane protein junctophilin 2 (JPH2) has been implicated in junction formation and maintenance. In this study, transgenic mouse models were used to investigate the influence JPH2 expression on RyR organisation. Cardiomyocytes from adult mice in which JPH2 had been acutely knocked down or over-expressed and control cells labeled for RyR and JPH2. Confocal laser scanning and optical super-resolution microscopy were used to investigate junctional protein organisation and t-tubule architecture.As previously observed in response to JPH2 knockdown t-tubule organisation was disrupted. Regular t-tubules in line with the z-discs were predominant in the control cardiomyocytes, but greatly reduced and irregular in JPH2 knockdown cells. In JPH2 overexpressing myocytes the t-tubules and longitudinal tubules were more abundant.RyR organisation was also altered in response to changes in JPH2 expression. Large RyR superclusters (>200nm diameter) were present in control cells and more abundant in JPH2 over-expressing cardiomyocytes. Such superclusters were mostly absent from JPH2 knockdown cells in which smaller clusters were observed. These changes only became apparent with the resolution provided by optical super-resolution but were difficult to detect with conventional confocal imaging. Our results indicate that cardiomyocyte organisation is severely affected by altered expression of JPH2 and raise questions on the functional role of RyR superclusters and their effect on local and global Ca2+ release.

Exon Skipping as a Therapeutic Strategy Applied to anRYR1Mutation with Pseudo-Exon Inclusion Causing a Severe Core Myopathy

Central core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new in-frame pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level.

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

BackgroundRapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive.Methodology/Principal FindingsTo investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s.Conclusions/SignificanceWe have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.

Mechanical unloading reverses transverse tubule remodelling and normalises local calcium-induced calcium release in a rodent model of heart failure

Calcium-induced calcium release (CICR) is crucial for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for calcium release (L-type calcium channel, dihydropyridine receptors) and the sarcoplasmic reticulum calcium-release channels (ryanodine receptors). Transverse tubule disruption occurs early in heart failure. Clinical studies of left ventricular assist devices in heart failure indicate that mechanical unloading induces reverse remodelling. We hypothesise that unloading of failing hearts normalises t-tubule structure and improves CICR.Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). Heart failure reduced the t-tubule density as measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in heart failure was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type calcium current density, measured with whole-cell patch clamping, was reduced in heart failure but unaffected by unloading. The variance of the time-to-peak of the calcium transient, an index of CICR dyssynchrony, was increased in heart failure and normalised by unloading. The increased calcium spark frequency observed in heart failure was reduced in HF-UN. These results could be explained by the recoupling of orphaned ryanodine receptors in heart failure, as indicated by immunofluorescence.Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR. FundingBritish Heart Foundation.

Ryanodine Receptor Oxidation Causes Intracellular Calcium Leak and Muscle Weakness in Aging

Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (3-6months) adults. This RyR1 channel complex remodeling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca(2+) release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function.

RYR1 mutations are a common cause of congenital myopathies with central nuclei

Centronuclear myopathy (CNM) is a rare congenital myopathy characterized by prominence of central nuclei on muscle biopsy. CNM has been associated with mutations in MTM1, DNM2, and BIN1 but many cases remain genetically unresolved. RYR1 encodes the principal sarcoplasmic reticulum calcium release channel and has been implicated in various congenital myopathies. We investigated whether RYR1 mutations cause CNM. We sequenced the entire RYR1 coding sequence in 24 patients with a diagnosis of CNM from South Africa (n = 14) and Europe (n = 10) and identified mutations in 17 patients. The most common genotypes featured compound heterozygosity for RYR1 missense mutations and mutations resulting in reduced protein expression, including intronic splice site and frameshift mutations. The high incidence in South African patients (n = 12/14) in conjunction with recurrent RYR1 mutations associated with common haplotypes suggested the presence of founder effects. In addition to central nuclei, prominent histopathological findings included (often multiple) internalized nuclei and type 1 fiber predominance and hypotrophy with relative type 2 hypertrophy. Although cores were not typically seen on oxidative stains, electron microscopy revealed subtle abnormalities in most cases. External ophthalmoplegia, proximal weakness, and bulbar involvement were prominent clinical findings. Our findings expand the range of RYR1-related phenotypes and suggest RYR1 mutations as a common cause of congenital myopathies with central nuclei. Corresponding to recent observations in X-linked CNM, these findings indicate disturbed assembly and/or malfunction of the excitation-contraction machinery as a key mechanism in CNM and related myopathies.

Calcium Influx Analysis by TIRF Microscopy on Cultured Primary Myotubes from Patients with RyR1 Mutations

Central core disease (CCD) and malignant hyperthermia (MH) have been linked to point mutations in the gene encoding the skeletal muscle sarcoplasmic reticulum calcium release channel (ryanodine receptor), which is localized on human chromosome 19 (RYR11). Central core disease is a relatively mild, slowly progressive autosomal dominant myopathy, characterized histologically by the presence of centrally located cores running the length of the muscle fibres. MH is a pharmacogenetic induced hypermetabolic disease.

Calcium Gating by the DHPR-RyR1 Pair

In skeletal muscle the L-type voltage-gated calcium channel (DHPR) in the t-tubule coexists in a tight functional interaction with the sarcoplasmic reticulum calcium release channel (RyR1). By means of this interaction a depolarization-induced conformational change in the DHPR is translated into RyR1's opening, and a massive calcium release from intracellular stores. We are performing structural studies on purified DHPRs and RyRs to understand structural details involved in the gating mechanism.The DHPR is a heteropentamer with total molecular weight of ∼450 kDa. Up to now he best structural knowledge has been gained by electron microscopy, although its relative small size has limited the resolution obtained to date. Our new 25A resolution 3D reconstruction shows two distinct parts: a main body shaped like an irregular pentagon with distinct corners, and a hook-shaped feature. Consistent with the considerable conservation of membrane topology among voltage-gated channels, a good part of the main body can be closely fitted with an atomic structure of a full-length potassium channel, and this in turn is helping to locate the RyR1-interacting domains identified using biochemistry and molecular biology techniques.The RyR is a large homotetramer of 2.2 MDa, which has facilitated its structural study by 3D cryo-electron microscopy. Its reproducible 3D structure consists of a large cytoplasmic domain and a smaller transmembrane domain. Our 3D reconstructions of RyR1 in the open and closed states at 10 A resolution show that the ion pathway consists of two right-handed bundles converging into a constriction (putative ion gate) that changes its diameter by ∼4A upon gating. Although the molecular distance between the putative ion gate and the closest site of proximity to the DHPR is very large (>130 A), the conformational changes associated with gating are generalized, suggesting long-range allosteric pathways connecting these distant domains.