T-tubules Research Articles

Direct proteomic and high-resolution microscopy biopsy analysis identifies distinct ventricular fates in severe aortic stenosis

The incidence of aortic valve stenosis (AS), the most common reason for aortic valve replacement (AVR), increases with population ageing. While untreated AS is associated with high mortality, different hemodynamic subtypes range from normal left-ventricular function to severe heart failure. However, the molecular nature underlying four different AS subclasses, suggesting vastly different myocardial fates, is unknown. Here, we used direct proteomic analysis of small left-ventricular biopsies to identify unique protein expression profiles and subtype-specific AS mechanisms. Left-ventricular endomyocardial biopsies were harvested from patients during transcatheter AVR, and inclusion criteria were based on echocardiographic diagnosis of severe AS and guideline-defined AS-subtype classification: 1) normal ejection fraction (EF)/high-gradient; 2) low EF/high-gradient; 3) low EF/low-gradient; and 4) paradoxical low-flow/low-gradient AS. Samples from non-failing donor hearts served as control. We analyzed 25 individual left-ventricular biopsies by data-independent acquisition mass spectrometry (DIA-MS), and 26 biopsies by histomorphology and cardiomyocytes by STimulated Emission Depletion (STED) superresolution microscopy. Notably, DIA-MS reliably detected 2273 proteins throughout each individual left-ventricular biopsy, of which 160 proteins showed significant abundance changes between AS-subtype and non-failing samples including the cardiac ryanodine receptor (RyR2). Hierarchical clustering segregated unique proteotypes that identified three hemodynamic AS-subtypes. Additionally, distinct proteotypes were linked with AS-subtype specific differences in cardiomyocyte hypertrophy. Furthermore, superresolution microscopy of immunolabeled biopsy sections showed subcellular RyR2-cluster fragmentation and disruption of the functionally important association with transverse tubules, which occurred specifically in patients with systolic dysfunction and may hence contribute to depressed left-ventricular function in AS.

Skeletal Muscle is a Source of α-Synuclein with a Sarcolemmal Non-Lipid Raft Distribution.

Alpha synuclein (αSN) is a widely distributed protein in vertebrates whose physiological significance in many tissues remains unclear, being a key protein present in neurodegenerative disease such as Parkinson's Disease, Lewy Body Dementia, and in Sporadic-Inclusion Body Myositis. We search for αSN in skeletal muscle (SM) and neuronal plasma membrane isolated from brain (BR) from young and old rats. In isolated Sarcolemma from SM and from myelin-free neuronal plasma membrane isolated from BR, we determine by Western blot with anti-αSN (2B2D1) and anti-P-αSN (EP1536Y) the αSN membrane distribution, and the SM αSN intra and extracellular localization. In SM and BR, αSN is present in cytosol (CYT) as monomer and oligomer structures mainly tetramers (TM) and in plasma membranes as oligomers (TM and PM). All αSN oligomers were localized in non-lipid rafts and their distribution was unaffected by cholesterol-depletion with Methyl-β-Cyclodextrin. Membranes with natively high cholesterol content such as Transverse Tubules in SM and myelin in BR, reduce the presence of αSN. Under the same experimental conditions, aged SM and BR plasma membranes show ≈2 folds more αSN. In SM, αSN is extruded without cell damage in young and old rats. We conclude that oligomeric αSN are regularly present in SM and BR plasma membranes of healthy young and old rats. Interestingly, low-cholesterol content membranes promote αSN interaction. SM, the largest tissue in vertebrate body is a source of αSN and may contribute to the presence of αSN in extracellular fluids.

Early transverse tubule involvement in cardiomyocytes in hereditary transthyretin amyloidosis: a possible cause of cardiac events

BackgroundCardiac involvement is one of the most frequent and fatal manifestations of hereditary transthyretin (ATTRv) amyloidosis. This study sought to clarify the pathogenesis of ATTRv amyloidosis, specifically, how transthyretin (TTR) amyloid begins to deposit in cardiomyocytes and how this deposition progresses in these cells. MethodsWe analyzed autopsy cardiac tissues from five patients with ATTRv amyloidosis by using confocal microscopy with thioflavin S staining and immunofluorescence and electron microscopy to demonstrate the pattern of TTR amyloid deposition in cardiomyocytes. ResultsWe demonstrated predominant amyloid deposition in the transverse tubules (t-tubules) of cardiomyocytes at the early stage of TTR amyloid deposition. Also, a pattern of the progression of amyloid deposition from deeply invaginated extracellular matrix, that is, t-tubules, to cell surface extracellular matrix, that is, basement membrane, was noted. Three-dimensional confocal microscopic images revealed the abnormal architecture of the t-tubules with nodular swelling, branching, and confluence in the cardiomyocytes with amyloid deposition. Double immunofluorescence staining with anti-TTR antibody and CACNA1C antibody demonstrated reduced voltage-dependent calcium channels around amyloid deposition. ConclusionsOur pathological study demonstrated that t-tubule involvement is an early event in cardiomyocytes in the pathogenesis of ATTRv amyloidosis. This finding may indicate that disruption of t-tubules in cardiomyocytes may contribute to the pathogenesis of cardiac events including heart failure and arrhythmia.

The distal C terminus of the dihydropyridine receptor β1a subunit is essential for tetrad formation in skeletal muscle

The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.

Neddylation is critical for cardiomyocyte maturation by promoting perinatal metabolic transition

The heart undergoes significant structural, metabolic, gene expression and functional alterations during the perinatal to postnatal transition. Although recent studies have delineated several novel epigenetic and transcriptional regulators of cardiac maturation, post‐transcriptional mechanisms regulating cardiomyocyte (CM) maturation remain poorly defined. We hypothesized that neddylation, a protein modification that conjugates a small ubiquitin‐like protein NEDD8 to protein substrates via an E1‐E2‐E3 cascade, plays a critical role in driving cardiac maturation program. Taking advantage of an AAV9‐cTnT‐Cre system and the mTmG reporter, we achieved CM‐specific, mosaic knockout of NAE1, a critical neddylation E1 enzyme, in postnatal hearts. Mice with high levels (>90%) of NAE1 deletion in CMs exhibited a cardiomyopathy as evidenced by significant chamber dilatation and greatly compromised cardiac contractility. While mice with modest levels (30%) of NAE1 deletion in CMs had preserved cardiac function, inhibition of neddylation caused severe disruption of transverse tubules (T‐tubules) network and a disturbance in binucleation and cell enlargement in NAE1‐null CMs, as well as upregulation of fetal genes and downregulation of their adult forms, indicating a key, cell‐autonomous role for neddylation in CM maturation. Transcriptome analysis revealed an upregulation of glycolytic genes and a downregulation of oxidative metabolic genes in NAE1‐deficient hearts. Biochemical analyses confirmed defects in fatty acid oxidative metabolism and mitochondrial dysfunction in NAE1‐deficient hearts and neonatal CMs treated with a neddylation inhibitor. Mechanistically, we found that HIF1α, a transcription factor known to promote glycolysis and suppress oxidative metabolism, is a putative NEDD8 target. Inhibition of neddylation stabilized HIF1α and led to abnormal accumulation of HIF1α in NAE1‐deficient hearts and neonatal CMs treated with neddylation inhibitor, which may contribute to the observed metabolic dysfunction. Together, our findings suggest that neddylation serves as a novel post‐translational mechanism regulating CM maturation through sustaining the glycolytic to oxidative metabolic switch in postnatal hearts.

CMYA5 establishes cardiac dyad architecture and positioning

Cardiac excitation-contraction coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse tubules and junctional sarcoplasmic reticulum. Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac development, homeostasis, and disease. Here, we use proximity proteomics in intact, living hearts to identify proteins enriched near dyads. Among these proteins is CMYA5, an under-studied striated muscle protein that co-localizes with Z-lines, junctional sarcoplasmic reticulum proteins, and transverse tubules in mature cardiomyocytes. During cardiac development, CMYA5 positioning adjacent to Z-lines precedes junctional sarcoplasmic reticulum positioning or transverse tubule formation. CMYA5 ablation disrupts dyad architecture, dyad positioning at Z-lines, and junctional sarcoplasmic reticulum Ca2+ release, leading to cardiac dysfunction and inability to tolerate pressure overload. These data provide mechanistic insights into cardiomyopathy pathogenesis by demonstrating that CMYA5 anchors junctional sarcoplasmic reticulum to Z-lines, establishes dyad architecture, and regulates dyad Ca2+ release.

The C2 domains of dysferlin: roles in membrane localization, Ca2+ signalling and sarcolemmal repair.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.

Possible mechanisms underlying the dynamic assembly of calcium entry units: the role of temperature and pH

Skeletal muscle function is regulated by intracellular Ca2+ levels. Two main mechanisms control movements of Ca2+ ions from intracellular stores (i.e. the sarcoplasmic reticulum, SR) and from extracellular space: i) excitation-contraction (EC) coupling; and ii) store-operated Ca2+ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of Calcium Entry Units (CEUs), intracellular junctions located at the I band that are formed by two distinct elements: SR-stacks and transverse tubules (TTs).

High-throughput platform of reconstituted skeletal muscle depolarization-induced Ca2+release in HEK293 cells

In skeletal muscle excitation-contraction coupling, depolarization of transverse tubule membrane causes conformational change in dihydropyridine receptor (DHPR), which in turn opens type 1 ryanodine receptor (RyR1) to release Ca2+ from sarcoplasmic reticulum. This 'depolarization-induced Ca2+ release' (DICR) occurs through physical interaction between DHPR and RyR1 and requires additional components (β1a, Stac3, junctophilin-2). However, it remains so far unclear about molecular mechanism of DICR, especially conformational change in RyR1.

Ca2+ leak through ryanodine receptor 1 regulates thermogenesis in resting skeletal muscle

Mammals rely on nonshivering thermogenesis (NST) from skeletal muscle so that cold temperatures can be tolerated. NST results from activity of the sarcoplasmic reticulum (SR) Ca2+ pump in skeletal muscle, but the mechanisms that regulate this activity are unknown. Here, we develop a single-fiber assay to investigate the role of Ca2+ leak through ryanodine receptor 1 (RyR1) to generate heat at the SR Ca2+ pump in resting muscle. By inhibiting a subpopulation of RyR1s in a single-fiber preparation via targeted delivery of ryanodine through transverse tubules, we achieve in-preparation isolation of RyR1 Ca2+ leak. This maneuver provided a critical increase in signal-to-noise of the SR-temperature-sensitive dye ER thermoyellow fluorescence signal from the fiber to allow detection of SR temperature changes as either RyR1 or SR Ca2+ pump activity was altered. We found that RyR1 Ca2+ leak raises cytosolic [Ca2+] in the local vicinity of the SR Ca2+ pump to amplify thermogenesis. Furthermore, gene-dose-dependent increases in RyR1 leak in RYR1 mutant mice result in progressive rises in leak-dependent heat, consistent with raised local [Ca2+] at the SR Ca2+ pump via RyR1 Ca2+ leak. We also show that basal RyR Ca2+ leak and the heat generated by the SR Ca2+ pump in the absence of RyR Ca2+ leak is greater in fibers from mice than from toads. The distinct function of RyRs and SR Ca2+ pump in endothermic mammals compared to ectothermic amphibians provides insights into the mechanisms by which mammalian skeletal muscle achieves thermogenesis at rest.

Phosphatidylinositol-4,5-Bisphosphate Binding to Amphiphysin-II Modulates T-Tubule Remodeling: Implications for Heart Failure.

BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cβ1 (PLCβ1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.

Abstract 12880: Neddylation Regulates Murine Cardiomyocyte Maturation

From birth to adulthood cardiomyocytes (CMs) undergo a complex maturation process characterized by drastic changes in cell size, ultrastructure, metabolism and gene expression. The molecular mechanisms regulating CM maturation remain poorly defined. Neddylation is a novel post-translational modification that covalently attaches a small ubiquitin-like protein, NEDD8, to target proteins. We found that deletion of NAE1, a critical NEDD8 E1 enzyme that triggers neddylation, in embryonic and perinatal mouse hearts via Nkx2.5 Cre and aMHC Cre , respectively, led to myocardial hypoplasia, heart failure and early lethality. The severe cardiac phenotypes are accompanied by disorganized sarcomere structure and defective fetal-to-adult gene transition, suggesting a possible role for neddylation in CM maturation. To rule out the confounding effect of secondary changes caused by perturbed CM differentiation and cardiac stress response in the mutant hearts, we used AAV9-cTnT-Cre to achieve mosaic knockout of NAE1 in postnatal hearts. High-dose AAV9 deleted NAE1 in 80% CMs and caused heart failure, whereas low-dose AVV9 deleted NAE1 in 30% CMs and preserved heart function. In the context of preserved cardiac function, CMs lacking NAE1 showed severe disrupted transverse tubules (T-tubules) network, which was further exacerbated in hearts with modest dysfunction. In contrast, T-tubule morphology was largely maintained in CMs with intact NAE1 expression from low-dose AAV9 infected hearts, as well as in CMs with deletion of one allele of NAE1, indicating a key, cell-autonomous role of neddylation in T-tubule maturation. Biochemical and histological analyses further revealed dysregulated metabolic and sarcomeric gene expression and disrupted sarcomere ultrastructure in high-dose AAV9 infected hearts, supporting defective CM maturation. In vitro, pharmacological inhibition of neddylation also impaired myofibril assembly in isolated neonatal and adult CMs. Together, our data reveal an important role of neddylation in CM maturation in the developing heart, warranting further investigations of detailed posttranslational mechanisms underlying CM maturation.

Cardiac Transverse Tubules in Physiology and Heart Failure.

In mammalian cardiac myocytes, the plasma membrane includes the surface sarcolemma but also a network of membrane invaginations called transverse (t-) tubules. These structures carry the action potential deep into the cell interior, allowing efficient triggering of Ca2+ release and initiation of contraction. Once thought to serve as rather static enablers of excitation-contraction coupling, recent work has provided a newfound appreciation of the plasticity of the t-tubule network's structure and function. Indeed, t-tubules are now understood to support dynamic regulation of the heartbeat across a range of timescales, during all stages of life, in both health and disease. This review article aims to summarize these concepts, with consideration given to emerging t-tubule regulators and their targeting in future therapies.

Possible mechanisms underlying the dynamic assembly of calcium entry units: The role of temperature and pH

Skeletal muscle function is regulated by intracellular Ca2+ levels. Two main mechanisms control movements of Ca2+ ions from intracellular stores (i.e., the sarcoplasmic reticulum; SR) and from extracellular space: (1) excitation-contraction (EC) coupling and (2) store-operated Ca2+ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of calcium entry units (CEUs), intracellular junctions located at the I band that are formed by two distinct elements: SR stacks and transverse tubules (TTs). Assembly of CEUs during exercise promotes the interaction between STIM1 and Orai1, the two main proteins that mediate SOCE, and increases muscle resistance to fatigue in the presence of extracellular Ca2+. The molecular mechanisms underlying the exercise-dependent remodeling of SR and TT leading to CEU assembly remain to be fully elucidated. Here, we first verified whether CEUs can assemble ex vivo (in the absence of blood supply and innervation), subjecting excised EDL muscles from mice to an ex vivo incremental fatigue protocol (80 Hz tetanus stimulation lasting 45 min): the data collected demonstrate that CEUs can assemble ex vivo in isolated EDL muscles. We then evaluated if intracellular parameters that are affected by exercise, such as temperature and pH, may influence the assembly of CEUs. We found that higher temperature (36°C versus 25°C) and lower pH (7.2 versus 7.4) promotes formation of CEUs increasing the percentage of fibers containing SR stacks, the number of SR stacks/area, and the elongation of TTs at the I band. Importantly, increased assembly of CEUs at higher temperature (36°C) or at lower pH (7.2) correlated with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+, suggesting that CEUs assembled ex vivo are functional.

Voltage sensor movements of CaV1.1 during an action potential in skeletal muscle fibers

In excitation-contraction coupling (ECC), when the skeletal muscle action potential (AP) propagates into the transverse tubules, it modifies the conformational state of the voltage-gated calcium channels (CaV1.1). CaV1.1 serves as the voltage sensor for activation of calcium release from the sarcoplasmic reticulum (SR); however, many questions about this function persist. CaV1.1 α1 subunits contain four distinct homologous domains (I-IV). Each repeat includes six transmembranal helical segments; the voltage-sensing domain (VSD) is formed by S1-S4 segments, and the pore domain is formed by helices S5-S6. Because, in other voltage-gated channels, individual VSDs appear to be differentially involved in specific aspects of channel gating, here we thus hypothesized that not all the VSDs in CaV1.1 contribute equally to calcium-release activation. Yet, the voltage-sensor movements during an AP (the physiological stimulus for the muscle fiber) have not been previously measured in muscle. Reorientation of VSDs I-IV in CaV1.1 during an AP should generate a small but measurable electrical current. Still, neither the voltage-sensor charge movement during the AP nor the contribution of the individual VSDs to voltage-gated calcium release have been previously monitored. Here, we electrically monitor VSD movements using an AP voltage-clamp technique applied to muscle fibers. We introduce AP-fluorometry, a variant of the functional site-directed fluorescence, to track the movement of each VSD via a cysteine substitution on the extracellular region of S4 of each VSD and its labeling with a cysteine-reacting fluorescent probe, which served as an optical reporter of local rearrangements. Independent optical recordings of AP and calcium transients were performed to establish the temporal correlation between AP, AP-elicited charge movement, VSDs conformational changes, and calcium release flux. Our results support the hypothesis that not all VSDs in CaV1.1 contribute to ECC.

Effects of dihydropyridine receptor signal on sarcoplasmic reticulum Ca2+ leakage after muscle contractions in rats

The purpose of this study is to investigate the mechanism underlying sarcoplasmic reticulum (SR) Ca2+ leakage at recovery phase after in vivo contractions. Rat gastrocnemius muscles were electrically stimulated in vivo, and then mechanically skinned fibers were prepared from the muscles excised 30 min after repeated high-intensity contractions. SR Ca2+ leakage was increased in the skinned fibers from stimulated muscles. Thereafter, SR Ca2+ leakage in skinned fibers was measured (1) under a continuously depolarized condition and (2) in the presence of nifedipine in the sealed transverse tubular system. In either of the two conditions, SR Ca2+ leakage in the rested fibers reached a level similar to that in the stimulated fibers. Furthermore, 1 mM tetracaine (Tet) treatment, but not 3 mM Mg2+ (3 Mg) treatment, lessened SR Ca2+ leakage in stimulated fibers. Depolarization-induced force in skinned fibers was more greatly decreased by Tet treatment than by 3 Mg treatment (92% reduction in Tet versus 31% reduction in 3 Mg), whereas caffeine-induced force in skinned fibers was similarly decreased by either treatment (73% reduction in Tet versus 75% reduction in 3 Mg). This difference indicates that Tet exerts a greater inhibitory effect on the dihydropyridine receptor (DHPR) signal to ryanodine receptor (RYR) than 3 Mg, although their inhibitory effects on RYR are almost similar. These results suggest that the increased Ca2+ leakage after muscle contractions is mainly caused by the orthograde signal of DHPRs to RYRs.

Couplons between transverse tubules and sarcoplasmic reticulum: roles in calcium signaling

Couplons between transverse tubules and sarcoplasmic reticulum: roles in calcium signaling

Alteration of STIM1/Orai1-Mediated SOCE in Skeletal Muscle: Impact in Genetic Muscle Diseases and Beyond.

Intracellular Ca2+ ions represent a signaling mediator that plays a critical role in regulating different muscular cellular processes. Ca2+ homeostasis preservation is essential for maintaining skeletal muscle structure and function. Store-operated Ca2+ entry (SOCE), a Ca2+-entry process activated by depletion of intracellular stores contributing to the regulation of various function in many cell types, is pivotal to ensure a proper Ca2+ homeostasis in muscle fibers. It is coordinated by STIM1, the main Ca2+ sensor located in the sarcoplasmic reticulum, and ORAI1 protein, a Ca2+-permeable channel located on transverse tubules. It is commonly accepted that Ca2+ entry via SOCE has the crucial role in short- and long-term muscle function, regulating and adapting many cellular processes including muscle contractility, postnatal development, myofiber phenotype and plasticity. Lack or mutations of STIM1 and/or Orai1 and the consequent SOCE alteration have been associated with serious consequences for muscle function. Importantly, evidence suggests that SOCE alteration can trigger a change of intracellular Ca2+ signaling in skeletal muscle, participating in the pathogenesis of different progressive muscle diseases such as tubular aggregate myopathy, muscular dystrophy, cachexia, and sarcopenia. This review provides a brief overview of the molecular mechanisms underlying STIM1/Orai1-dependent SOCE in skeletal muscle, focusing on how SOCE alteration could contribute to skeletal muscle wasting disorders and on how SOCE components could represent pharmacological targets with high therapeutic potential.

RyR2 and Calcium Release in Heart Failure.

Heart Failure (HF) is defined as the inability of the heart to efficiently pump out enough blood to maintain the body's needs, first at exercise and then also at rest. Alterations in Ca2+ handling contributes to the diminished contraction and relaxation of the failing heart. While most Ca2+ handling protein expression and/or function has been shown to be altered in many models of experimental HF, in this review, we focus in the sarcoplasmic reticulum (SR) Ca2+ release channel, the type 2 ryanodine receptor (RyR2). Various modifications of this channel inducing alterations in its function have been reported. The first was the fact that RyR2 is less responsive to activation by Ca2+ entry through the L-Type calcium channel, which is the functional result of an ultrastructural remodeling of the ventricular cardiomyocyte, with fewer and disorganized transverse (T) tubules. HF is associated with an elevated sympathetic tone and in an oxidant environment. In this line, enhanced RyR2 phosphorylation and oxidation have been shown in human and experimental HF. After several controversies, it is now generally accepted that phosphorylation of RyR2 at the Calmodulin Kinase II site (S2814) is involved in both the depressed contractile function and the enhanced arrhythmic susceptibility of the failing heart. Diminished expression of the FK506 binding protein, FKBP12.6, may also contribute. While these alterations have been mostly studied in the left ventricle of HF with reduced ejection fraction, recent studies are looking at HF with preserved ejection fraction. Moreover, alterations in the RyR2 in HF may also contribute to supraventricular defects associated with HF such as sinus node dysfunction and atrial fibrillation.

Nanoscale Organisation of Ryanodine Receptors and Junctophilin-2 in the Failing Human Heart.

The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 μm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.