Transverse Tubule Membranes Research Articles

Calcium ATPase (PMCA) and GLUT-4 Upregulation in the Transverse Tubule Membrane of Skeletal Muscle from a Rat Model of Chronic Heart Failure.

Intolerance to exercise is a symptom associated with chronic heart failure (CHF) resulting in SM waste and weakness in humans. The effect of CHF on skeletal muscle (SM) arose from experimental evidence in rat models to explain the underlying mechanism. We investigated SM mechanical and metabolic properties in sham rats and with coronary ligation-induced CHF. After twelve weeks of CHF, rats were catheterized to measure right auricular pressure, SM mechanical properties, SERCA-ATPase activity and plasma membrane Ca2+-ATPase (PMCA) hydrolytic activity in isolated sarcoplasmic reticulum (SR) and transverse tubule (TT membrane), respectively, in the sham and CHF. The right auricular pressure and plasma nitrite concentration in CHF increased two-fold with respect to the sham. Pleural effusion and ascites were detected in CHF, confirming CHF. SERCA activity was conserved in CHF. In TT membranes from CHF, the glucose transporter GLUT4 increased seven-fold, and the PMCA hydrolytic activity increased five-fold, but in isolated muscle, the mechanical properties were unaffected. The absence of a deleterious effect of coronary ligation-induced CHF in the rat model on SM could be explained by the increased activity of PMCA and increased presence of GLUT-4 on the TT membrane, which may be involved in the mechanical outcome of the EDL.

In situ structural insights into the excitation-contraction coupling mechanism of skeletal muscle.

Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.

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.

A mouse model of Huntington's disease shows altered ultrastructure of transverse tubules in skeletal muscle fibers.

Huntington's disease (HD) is a fatal and progressive condition with severe debilitating motor defects and muscle weakness. Although classically recognized as a neurodegenerative disorder, there is increasing evidence of cell autonomous toxicity in skeletal muscle. We recently demonstrated that skeletal muscle fibers from the R6/2 model mouse of HD have a decrease in specific membrane capacitance, suggesting a loss of transverse tubule (t-tubule) membrane in R6/2 muscle. A previous report also indicated that Cav1.1 current was reduced in R6/2 skeletal muscle, suggesting defects in excitation-contraction (EC) coupling. Thus, we hypothesized that a loss and/or disruption of the skeletal muscle t-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle. We used live-cell imaging with multiphoton confocal microscopy and transmission electron microscopy to assess the t-tubule architecture in late-stage R6/2 muscle and found no significant differences in the t-tubule system density, regularity, or integrity. However, electron microscopy images revealed that the cross-sectional area of t-tubules at the triad were 25% smaller in R6/2 compared with age-matched control skeletal muscle. Computer simulation revealed that the resulting decrease in the R6/2 t-tubule luminal conductance contributed to, but did not fully explain, the reduced R6/2 membrane capacitance. Analyses of bridging integrator-1 (Bin1), which plays a primary role in t-tubule formation, revealed decreased Bin1 protein levels and aberrant splicing of Bin1 mRNA in R6/2 muscle. Additionally, the distance between the t-tubule and sarcoplasmic reticulum was wider in R6/2 compared with control muscle, which was associated with a decrease in junctophilin 1 and 2 mRNA levels. Altogether, these findings can help explain dysregulated EC coupling and motor impairment in Huntington's disease.

Beat-by-Beat Cardiomyocyte T-Tubule Deformation Drives Tubular Content Exchange.

The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.

PEGylated-Ligands as tools to study β-adrenergic receptor function on surface and T-tubule membranes

Adult rat ventricular myocytes (ARVMs) membrane contains invaginations (transverse tubule membrane, TTM). TTM is continuous with the outer surface membrane (OSM) to allow rapid electrical coupling during the action potential. β-adrenergic receptors (βAR) are located in both OSM and TTM, but at different densities. TT provides the development of dyadic junctions with the sarcoplasmic reticulum, which is essential for excitation-contraction coupling (ECC). Yet, how β-AR compartmentation modulates ECC remains unknown. To selectively activate βARs in OSM and not in TTM using a steric hindrance approach. We covalently linked a 5 kDa PolyEthylene-Glycol (PEG) on βAR agonist (Isoprenaline, Iso) or antagonist (Alprenolol, Alp) to increase the size of the βAR ligand and prevent its access to TTM. Chemical grafting and PEG-ligand purification were optimized. A fluorescent PEG (PEG-FITC) was used to visualize its distribution in ARVMs. Pharmacological characterization of the PEG-ligands was performed using radioligand binding assays on β1 or β2AR, and cAMP measurement by FRET in ARVMs. The effects of PEG-ligands on ECC were examined in ARVMs using the Ionoptix system. (1) PEG-FITC does not penetrate in the TT network. (2) The chemical grafting was validated. (3) PEG-ligands bind to β1 and β2AR with Ki values of, respectively: 1300 and 430 μM ( n = 5) for PEG-Iso, and 20.0 and 5.6 μM ( n = 5) for PEG-Alp. (4) PEG-Iso produces a dose-dependent increase in cytosolic cAMP. The maximal response is comparable to free Iso. (5) PEG-Alp antagonizes the cytosolic cAMP response to free Iso, with a maximal effect close to full inhibition. (6) Activation of βAR at the OSM, using PEG-Iso, has smaller impact on ECC than global activation using free Iso. Our results indicate that (1) activation of βARs at the OSM is sufficient to produce maximal elevation of cytosolic cAMP, but (2) βARs in the TTM are more efficiently coupled to contractility and Ca2+ regulation.

Exogenous Cardiac Bridging Integrator 1 Benefits Mouse Hearts With Pre-existing Pressure Overload-Induced Heart Failure.

Background: Cardiac bridging integrator 1 (cBIN1) organizes transverse tubule (t-tubule) membrane calcium handling microdomains required for normal beat-to-beat contractility. cBIN1 is transcriptionally reduced in heart failure (HF). We recently found that cBIN1 pretreatment can limit HF development in stressed mice. Here, we aim to explore whether cBIN1 replacement therapy can improve myocardial function in continuously stressed hearts with pre-existing HF.Methods: Adult male mice were subjected to sham or transverse aortic constriction (TAC) surgery at the age of 8–10 weeks old. Adeno-associated virus 9 (AAV9) transducing cBIN1-V5 or GFP-V5 (3 × 1010 vg) was administered through retro-orbital injection at 5 weeks post-TAC. Mice were followed by echocardiography to monitor cardiac function until 20 weeks after TAC. Overall survival, heart and lung weight (LW), and HF incidence were determined. In a second set of animals in which AAV9-cBIN1 pretreatment prevents HF, we recorded cardiac pressure-volume (PV) loops and obtained myocardial immunofluorescence imaging.Results: The overall Kaplan-Meir survival of AAV9-cBIN1 mice was 77.8%, indicating a significant partial rescue between AAV9-GFP (58.8%) and sham (100%) treated mice. In mice with ejection fraction (EF) ≥30% prior to AAV9 injection at 5 weeks post-TAC, AAV9-cBIN1 significantly increased survival to 93.3%, compared to 62.5% survival for AAV9-GFP treated mice. The effect of exogenous cBIN1 was to attenuate TAC-induced left ventricular (LV) dilation and prevent further HF development. Recovery of EF also occurs in AAV9-cBIN1-treated mice. We found that EF increases to a peak at 6–8 weeks post-viral injection. Furthermore, PV loop analysis identified that AAV9-cBIN1 increases both systolic and diastolic function of the post-TAC hearts. At the myocyte level, AAV9-cBIN1 normalizes cBIN1 expression, t-tubule membrane intensity, and intracellular distribution of Cav1.2 and ryanodine receptors (RyRs).Conclusions: In mice with pre-existing HF, exogenous cBIN1 can normalize t-tubule calcium handling microdomains, limit HF progression, rescue cardiac function, and improve survival.

Modeling and simulation of excitation- contraction coupling of fast-twitch skeletal muscle fibers.

BACKGROUND: The current excitation-contraction coupling model of fast-twitch skeletal muscle fibers cannot completely simulate the excitation-contraction process.OBJECTIVE: To solve this problem, this study proposes an excitation-contraction model of fast-twitch skeletal muscle fibers based on the physiological structure and contractile properties of half-sarcomeres.METHODS: The model includes the action potential model of fast-twitch fiber membranes and transverse tubule membranes, the cycle model of in myofibril, the cross-bridge cycle model, and the fatigue model of metabolism.RESULTS: Finally, detailed analyses of the results from the simulation are conducted using the Simulink toolbox in MATLAB. Two conditions, non-coincidence and coincidence, are analyzed for both the thick and thin myofilaments.CONCLUSIONS: The simulation results of two groups of models are the same as the previous research results, which validates the accuracy of models.

Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers

In adult skeletal muscles, 2 junctophilin isoforms (JPH1 and JPH2) tether the sarcoplasmic reticulum (SR) to transverse tubule (T-tubule) membranes, generating stable membrane contact sites known as triads. JPHs are anchored to the membrane of the SR by a C-terminal transmembrane domain (TMD) and bind the T-tubule membrane through their cytosolic N-terminal region, which contains 8 lipid-binding (MORN) motifs. By combining expression of GFP-JPH1 deletion mutants in skeletal muscle fibers with in vitro biochemical experiments, we investigated the molecular determinants of JPH1 recruitment at triads in adult skeletal muscle fibers. We found that MORN motifs bind PI(4,5)P2 in the sarcolemma, but do not mediate the selective localization of JPH1 at the T-tubule compartment of triads. On the contrary, fusion proteins containing only the TMD of JPH1 were able to localize at the junctional SR compartment of the triad. Bimolecular fluorescence complementation experiments indicated that the TMD of JPH1 can form dimers, suggesting that the observed localization at triads may result from dimerization with the TMDs of resident JPH1. A second domain, capable of mediating homo- and heterodimeric interactions between JPH1 and JPH2 was identified in the cytosolic region. FRAP experiments revealed that removal of either one of these 2 domains in JPH1 decreases the association of the resulting mutant proteins with triads. Altogether, these results suggest that the ability to establish homo- and heterodimeric interactions with resident JPHs may support the recruitment and stability of newly synthesized JPHs at triads in adult skeletal muscle fibers.

Excess membrane cholesterol is an early contributing reversible aspect of skeletal muscle insulin resistance in C57BL/6NJ mice fed a Western-style high-fat diet.

Skeletal muscle insulin resistance manifests shortly after high-fat feeding, yet mechanisms are not known. Here we set out to determine whether excess skeletal muscle membrane cholesterol and cytoskeletal derangement known to compromise glucose transporter (GLUT)4 regulation occurs early after high-fat feeding. We fed 6-wk-old male C57BL/6NJ mice either a low-fat (LF, 10% kcal) or a high-fat (HF, 45% kcal) diet for 1 wk. This HF feeding challenge was associated with an increase, albeit slight, in body mass, glucose intolerance, and hyperinsulinemia. Liver analyses did not reveal signs of hepatic insulin resistance; however, skeletal muscle immunoblots of triad-enriched regions containing transverse tubule membrane showed a marked loss of stimulated GLUT4 recruitment. An increase in cholesterol was also found in these fractions from HF-fed mice. These derangements were associated with a marked loss of cortical filamentous actin (F-actin) that is essential for GLUT4 regulation and known to be compromised by increases in membrane cholesterol. Both the withdrawal of the HF diet and two subcutaneous injections of the cholesterol-lowering agent methyl-β-cyclodextrin at 3 and 6 days during the 1-wk HF feeding intervention completely mitigated cholesterol accumulation, cortical F-actin loss, and GLUT4 dysregulation. Moreover, these beneficial membrane/cytoskeletal changes occurred concomitant with a full restoration of metabolic responses. These results identify skeletal muscle membrane cholesterol accumulation as an early, reversible, feature of insulin resistance and suggest cortical F-actin loss as an early derangement of skeletal muscle insulin resistance.

Ligand-grafted nanoparticles as tools to study L-type calcium channel and β-adrenergic receptor function on surface and T-tubule membranes

The cell membrane of adult ventricular myocytes (AVMs) is characterized by many invaginations, constituting the transverse tubule (TT) system. TT membrane (TTM) is continuous with the outer surface membrane (OSM) to allow the rapid electrical coupling during the action potential and represents about 30% of the total cell membrane. Most membrane proteins, such as L-type Ca 2+ channels (LTCC) and β-adrenergic receptors (β- AR) are located in both OSM and TTM, but at different densities. However, how the location of these proteins in OSM vs. TTM impacts on their function remains poorly understood, mainly because of the impossibility to selectively activate or inhibit a protein in one compartment without acting on its counterpart in the other compartment. For example, an important function of the TT system is to provide proximity between LTCCs in TTM and ryanodine receptor type 2 in the sarcoplasmic reticulum membrane. However, the role of their counterparts located in OSM is unknown. The goal of our study is to meet this challenge using an innovative nanotechnology approach. We designed nanoparticles (NPs) with immobilized ligands on their surface in order to prevent their access to TTM. The first step was to graft ligands on 5kDa PEG (polyethylene glycol) to favour ligand flexibility. PEG- and NPs-ligand affinity were determined by radioligand binding assays. PEG- and NPs-ligand potency were evaluated by FRET experiments. Confocal microscopy has been used to visualize the membrane distribution of the fluorescent PEGs and NPs after incubation of AVMs. PEG-ligand affinity and potency are lower than affinity and potency of free ligand. The confocal microscopy assay shows that neither NPs nor PEGs penetrate in the TT network. PEG-ligands seem to act on proteins located on the surface membrane. After this validation, functional experiments will be realized to discriminate the respective role of β-AR and LTCC subpopulations.

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

Microparticles (MPs) are cell–cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

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

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

Membrane Cholesterol in Skeletal Muscle: A Novel Player in Excitation-Contraction Coupling and Insulin Resistance.

Membrane cholesterol is critical for signaling processes in a variety of tissues. We will address here current evidence supporting an emerging role of cholesterol on excitation-contraction coupling and glucose transport in skeletal muscle. We have centered our review on the transverse tubule system, a complex network of narrow plasma membrane invaginations that propagate membrane depolarization into the fiber interior and allow nutrient delivery into the fibers. We will discuss current evidence showing that transverse tubule membranes have remarkably high cholesterol levels and we will address how modifications of cholesterol content influence excitation-contraction coupling. In addition, we will discuss how membrane cholesterol levels affect glucose transport by modulating the insertion into the membrane of the main insulin-sensitive glucose transporter GLUT4. Finally, we will address how the increased membrane cholesterol levels displayed by obese animals, which also present insulin resistance, affect these two particular skeletal muscle functions.

Skeletal muscle: from birth to old age, routes to mechanical and metabolic failure.

Skeletal muscle (SM) is the most abundant tissue and the largest reservoir of protein in the body. It transports glucose in an insulin dependent manner by the glucose transporter type 4 (GLUT4) and contributes in the maintenance of serum amino acids concentration. By its mass and energetic requirements, it is fundamental for the systemic metabolic balance. In the present work, we present the effect of gestational undernourishment (GU) on the mechanical and metabolic properties of SM at birth and in old age in an animal model. Mechanical studies were performed on isolated muscles, while the GLUT4, amino acid transporters LAT2, SNAT2 and insulin receptors (IR) determination were performed on isolated transverse-tubule membranes (TT). The GU in offspring at birth, results in low muscle mass with increased contraction force and resistance to fatigue. However, in two-years old rats, there was muscle hypotrophy and sarcopenia, the force decreased between 50 and 70% in control rats and rats with GU respectively, accompanied by a lower expression of LAT2, SNAT2 and IR in TT. In conclusion, GU irreversibly affects the SM, an effect that could be similar in humans, which help us to understand the events that associate the GU with the metabolic debacle of SM and the metabolic diseases of human adulthood.

Core skeletal muscle ryanodine receptor calcium release complex.

The core skeletal muscle ryanodine receptor (RyR1) calcium release complex extends through three compartments of the muscle fibre, linking the extracellular environment through the cytoplasmic junctional gap to the lumen of the internal sarcoplasmic reticulum (SR) calcium store. The protein complex is essential for skeletal excitation-contraction (EC)-coupling and skeletal muscle function. Its importance is highlighted by perinatal death if any one of the EC-coupling components are missing and by myopathies associated with mutation of any of the proteins. The proteins essential for EC-coupling include the DHPR α1S subunit in the transverse tubule membrane, the DHPR β1a subunit in the cytosol and the RyR1 ion channel in the SR membrane. The other core proteins are triadin and junctin and calsequestrin, associated mainly with SR. These SR proteins are not essential for survival but exert structural and functional influences that modify the gain of EC-coupling and maintain normal muscle function. This review summarises our current knowledge of the individual protein/protein interactions within the core complex and their overall contribution to EC-coupling. We highlight significant areas that provide a continuing challenge for the field. Additional important components of the Ca2+ release complex, such as FKBP12, calmodulin, S100A1 and Stac3 are identified and reviewed elsewhere.

Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3

Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+ These findings define critical roles for Stac3 in EC coupling and human disease.

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

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

Dysferlin deficiency blunts β-adrenergic-dependent lusitropic function of mouse heart.

Dysferlin is a cell membrane bound protein with a role in the repair of skeletal and cardiac muscle cells. Deficiency of dysferlin leads to limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. In cardiac muscle, dysferlin is located at the intercalated disc and transverse tubule membranes. Loss of dysferlin causes death of cardiomyocytes, notably in ageing hearts, leading to dilated cardiomyopathy and heart failure in LGM2B patients. To understand the primary pathogenesis and pathophysiology of dysferlin cardiomyopathy, we studied cardiac phenotypes of young adult dysferlin knockout mice and found early myocardial hypertrophy with largely compensated baseline cardiac function. Cardiomyocytes isolated from dysferlin-deficient mice showed normal shortening and re-lengthening velocities in the absence of external load with normal peak systolic Ca(2+) but slower Ca(2+) re-sequestration than wild-type controls. The effects of isoproterenol on relaxation velocity, left ventricular systolic pressure and stroke volume were blunted in dysferlin-deficient mouse hearts compared with that in wild-type hearts. Young dysferlin-deficient mouse hearts expressed normal isoforms of myofilament proteins whereas the phosphorylation of ventricular myosin light chain 2 was significantly increased, implying a molecular response to the impaired lusitropic function. These early phenotypes of diastolic cardiac dysfunction and blunted lusitropic response of cardiac muscle to β-adrenergic stimulation indicate a novel pathogenic mechanism of dysferlin cardiomyopathy.

Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase.

Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in Ca(2+) homeostasis and excitation-contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein-tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca(2+) transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca(2+) release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP-expressing fibers challenged by 5-s-long depolarizing pulses, the Ca(2+) level late in the pulse (3 s after initiation) was significantly lower at 120 mV than at 20 mV. Furthermore, Ci-VSP-expressing fibers showed a reversible depression of Ca(2+) release during trains, with the peak Ca(2+) transient being reduced by ∼30% after the application of 10 200-ms-long pulses to 100 mV. A similar depression was observed in Dr-VSP-expressing fibers. Cav1.1 Ca(2+) channel-mediated current was unaffected by Ci-VSP activation. In fibers expressing Ci-VSP and a pleckstrin homology domain fused with monomeric red fluorescent protein (PLCδ1PH-mRFP), depolarizing pulses elicited transient changes in mRFP fluorescence consistent with release of transverse tubule-bound PLCδ1PH domain into the cytosol; the voltage sensitivity of these changes was consistent with that of Ci-VSP activation, and recovery occurred with a time constant in the 10-s range. Our results indicate that the PtdIns(4,5)P2 level is tightly maintained in the transverse tubule membrane of the muscle fibers, and that VSP-induced depletion of PtdIns(4,5)P2 impairs voltage-activated Ca(2+) release from the SR. Because Ca(2+) release is thought to be independent from InsP3 signaling, the effect likely results from an interaction between PtdIns(4,5)P2 and a protein partner of the E-C coupling machinery.