Exploring the super-relaxed state of myosin in myofibrils from fast-twitch,slow-twitch,and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s⁻¹) and super-relaxed states (SRX, 0.005 s⁻¹) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969–1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C–30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.     

Kinetic Mechanism of Ca2+-controlled Changes of Skeletal Troponin I in Psoas Myofibrils

Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca²⁺] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca²⁺-activation kinetics. Whereas the fast phase (k_obs∼1000 s⁻¹) is only observed in this study, where kinetics were induced by Ca²⁺, the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692–2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca²⁺ ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca²⁺ binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca²⁺ sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation.     

E-1020, a water soluble imidazopyridine,has direct effects on Ca2+-dependent force and ATP hydrolysis of canine and bovine cardiac myofilaments

E-1020 is a cardiotonic agent that acts as a cyclic-AMP phosphodiesterase inhibitor but also may have actions which alter myofilament response to Ca²⁺. To identify direct actions of E-1020 on cardiac contractile proteins, effects of E-1020 on myofibrillar Ca²⁺ dependent MgATPase and force generation in chemically skinned fiber bundles were measured. In bovine cardiac myofibrils, E-1020 (100 M) significantly increased myofilament Ca²⁺ sensitivity and Ca²⁺-dependent ATPase activity at submaximal pCa values. At pCa 6.75, E-1020 significantly increased ATPase activity in bovine (10–100 pM) and canine (1–100 pM) cardiac myofibrils but had no effect on rat cardiac myofibrils. Moreover, in one population of canine ventricular fiber bundles, E-1020 (0.0–10 M) significantly increased isometric tension at pCa 6.5 and 6.0, whereas in another population of bundles E-1020 had no effect on tension. In no case was resting (pCa 8.0) or maximal tension (pCa 4.5) increased by E-1020. Measurements of Ca²⁺ binding to canine ventricular skinned fiber preparations demonstrated that E-1020 does not alter the affinity of myofilament troponin C for Ca²⁺. We conclude that part of the mechanism by which E-1020 acts as an inotropic agent may involve alterations in the responsiveness of contractile proteins to Ca²⁺. The lack of effect of E-1020 on some preparations may be dependent on isoform populations of myofilament proteins.     

Effects of MCC-135 on Ca2+ uptake by sarcoplasmic reticulum and myofilament sensitivity to Ca2+ in isolated ventricular muscles of rats with diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by delayed cardiac relaxation. Delayed relaxation is suggested to be associated with sarcoplasmic reticulum (SR) dysfunction and/or increase in myofilament sensitivity to Ca²⁺. Although MCC-135, an intracellular Ca²⁺-handling modulator, accelerates the delayed relaxation without inotropic effect in the ventricular muscle isolated from rats with diabetic cardiomyopathy, the underlying mechanism has not been fully understood. We tested the hypotheses that MCC-135 modulates Ca²⁺ uptake by SR and myofilament sensitivity to Ca²⁺. Wistar rats were made diabetic by a single injection of streptozotocin (40 mg/kg i.v.). Seven months later, the left ventricular papillary muscle was isolated and skinned fibers with and without functional SR were prepared by treatment of the papillary muscle with saponin to study SR Ca²⁺ uptake and myofilament sensitivity to Ca²⁺, respectively. In diabetic rats, SR Ca²⁺ uptake was decreased, which was related to decrease in protein level of SR Ca²⁺-ATPase determined by western blot analysis. MCC-135 enhanced SR Ca²⁺ uptake in diabetic rats, but not in normal rats. In diabetic rats, maximum force was decreased but force at diastolic level of Ca²⁺ was increased, without significant change in myofilament sensitivity to Ca²⁺ compared with normal rats. MCC-135 decreased force at any pCa tested (pCa 7.0-4.4), but had no significant effect on myofilament sensitivity to Ca²⁺ in diabetic rats. These results suggest that MCC-135 enhances SR Ca²⁺ uptake and shifts force-pCa curve downward without modulating myofilament sensitivity to Ca²⁺. These effects may contribute to positive lusitropic effect without inotropic effect of MCC-135 observed in the ventricular muscle of diabetic cardiomyopathy.     

Mechanisms of calcium release in sarcoplasmic reticulum.

The involvement of Sarcoplasmic Reticulum (SR) in relaxation of skeletal muscle has been studied extensively since vesicular fragments of SR membrane were found in the microsomal fraction of muscle homogenates (1,2). It was shown that the isolated SR vesicles exhibit ATP dependent calcium transport in vitro, reducing the Ca²⁺ concentration in the medium to levels (3) and at rates (4,5) compatible with relaxation of myofibrils in physiological conditions (6).The question of calcium release, however, has been elusive for a long time. In this regard it is known that skeletal muscle SR is able to store an amount of calcium which is sufficient for activation of myofibrils. Therefore, it is simply assumed that upon membrane excitation calcium is released from SR, thereby raising the Ca²⁺ concentration in the myoplasm and initiating contraction.Recently various experiments were performed demonstrating that calcium release from SR can occur by different mechanisms of great interest and possibly of physiological relevance. These mechanisms will be discussed here.     

The role of extracellular ions in the pathogenesis of myonecrosis induced by a myotoxin isolated from Broad-Banded Copperhead (Agkistrodon contortrix laticinctus) venom

Pathalogical changes in murine skeletal muscle cells induced by ACL (Agkistrodon contortix laticinctus, Broad-Banded Copperhead) myotoxin in vivo were compared to pathological changes induced by an influx of Ca²⁺ and other ions into cut skeletal muscle cells in vitro in the absence of myotoxin. In vivo, ACL myotoxin induced a rapid myonecrosis characterized by densely clumped myofibrils in the cytoplasm. In vitro, this pathological change was not produced by incubating skeletal muscle cells in Ca²⁺ concentrations as high as 200 mM, whereas skeletal muscle cells incubated in concentrations of 150 mM and 300 mM NaCl contained densely clumped myofibrils similar in morphology to muscle cells damaged by ACL myotoxin in vivo. Treatments of 300 mM KCl did not produce densely clumped myofibrils in muscle cells. These results suggest that an influx of Na⁺, possibly through disrupted regions of sarcolemma, be may primarily responsible for the pathological changes, including clumped myofibrils, induced by ACL myotoxin in vivo. However, an influx of extracellular Ca²⁺ which has been proposed to produce densely clumped myofibrils in muscle cells damaged by other snake venom myotoxins, may not be responsible for this pathological change since extracellular Ca²⁺ concentrations much higher than physiological levels did not produce this change in skeletal muscle cells in vitro.     

The exchange of Ca2+-receptive protein complex (troponin) in the myofibrils of fast and slow skeletal muscles

In order to compare the role of the Ca²⁺-receptive protein (troponin), in the characteristic myofibrillar contractile response of chicken fast and slow skeletal muscles, the troponin in both kinds of myofibrils were partially exchanged, under slightly acidic conditions. The Ca²⁺- or Sr²⁺-activation of the ATPase of fast (or slow) skeletal myofibrils hybridized with slow (or fast) skeletal troponin profiles were also investigated. The results indicated that the Ca²⁺- or Sr²⁺-affinity of the myofibrillar ATPase activity were related to the species of troponin. This procedure for replacing troponin in myofibrils under physiological conditions in thus considered to be useful for the study of the Ca²⁺-regulatory mechanism in myofibrillar contraction.     

History-dependent properties of skeletal muscle myofibrils contracting along the ascending limb of the force–length relationship

There is a history dependence of skeletal muscle contraction: stretching activated muscles induces a long-lasting force enhancement, while shortening activated muscles induces a long-lasting force depression. These history-dependent properties cannot be explained by the current model of muscle contraction, and its mechanism is unknown. The purposes of this study were (i) to evaluate if force enhancement and force depression are present at short lengths (the ascending limb of the force–length (FL) relationship), (ii) to evaluate if the history-dependent properties are associated with sarcomere length (SL) non-uniformity and (iii) to determine the effects of cross-bridge (de)activation on force depression. Rabbit psoas myofibrils were isolated and attached between two microneedles for force measurements. Images of the myofibrils were projected onto a linear photodiode array for measurements of SL. Myofibrils were activated by either Ca²⁺ or MgADP; the latter induces cross-bridge attachment to actin independently of Ca²⁺. Activated myofibrils were subjected to three stretches or shortenings (approx. 4% SL at approx. 0.07 µm s⁻¹ sarcomere⁻¹) along the ascending limb of the FL relationship separated by periods (approx. 5 s) of isometric contraction. Force after stretch was higher than force after shortening at similar SLs. The differences in force could not be explained by SL non-uniformity. The FL relationship produced by Ca²⁺- and MgADP-activated myofibrils were similar in stretch experiments, but after shortening MgADP activation produced forces that were higher than Ca²⁺ activation. Since MgADP induces the formation of strongly bound cross-bridges, this result suggests that force depression following shortening is associated with cross-bridge deactivation.     

Protein kinase A–dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin

Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during β-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca²⁺ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca²⁺ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca²⁺ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca²⁺ sensitivity, but also abolished the Ca²⁺-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca²⁺ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca²⁺ sensitivity, and PKA further decreased Ca²⁺ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca²⁺ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.     

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

The Dilated Cardiomyopathy-Causing Mutation ACTC E361G in Cardiac Muscle Myofibrils Specifically Abolishes Modulation of Ca2+ Regulation by Phosphorylation of Troponin I

Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca²⁺ sensitivity and increases the rate of Ca²⁺ release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca²⁺-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca²⁺ regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca²⁺ sensitivity of force was increased, the fast relaxation phase rate constant, k_REL, was reduced, and the length of the slow linear phase, t_LIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC₅₀ P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca²⁺ sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca²⁺ sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC₅₀ P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca²⁺ sensitivity of ACTC E361G myofibrils by sarcomere length or {"type":"entrez-protein","attrs":{"text":"EMD57033","term_id":"451702631","term_text":"EMD57033"}}EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC₅₀ measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by t_LIN and k_REL, was correlated with EC₅₀ but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca²⁺ sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to {"type":"entrez-protein","attrs":{"text":"EMD57033","term_id":"451702631","term_text":"EMD57033"}}EMD57033.     

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, “free” and “blocked”, formed an asymmetric structure named the “interacting-heads motif” (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca²⁺-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.     

Evidence from insect fibrillar muscle about the elementary contractile process

Bundles of myofibrils prepared from the dorsal longitudinal flight muscles of giant water bugs show oscillatory contractile activity in solutions of low ionic strength containing ATP and 10^-8-10^-7 M Ca²⁺. This is due to delay between changes of length and changes of tension under activating conditions. The peculiarities of insect fibrillar muscle which give rise to this behavior are (1) the high elasticity of relaxed myofibrils, (2) a smaller degree of Ca²⁺ activation of ATPase activity in unstretched myofibrils and extracted actomyosin, and (3) a direct effect of stretch on ATPase activity. It is shown that the cross-bridges of striated muscle are probably formed from the heads of three myosin molecules and that in insect fibrillar muscle the cycles of mechanochemical energy conversion in the cross-bridges can be synchronized by imposed changes of length. This material is more suitable than vertebrate striated muscle for a study of the nature of the elementary contractile process.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.     

The Location of Muscle Calcium with Respect to the Myofibrils

Autoradiographs have been prepared from frog toe muscles soaked in Ca⁴⁵ and fixed with an osmium-oxalate solution. A majority of the grains over the A bands were over the myofibrils. The grain density over the I bands was greatest over the space between the myofibrils. The significance of this distribution is discussed in the light of previous information about the longitudinal distribution of Ca⁴⁵ in skeletal muscle.     

Sarcoplasmic reticulum calsequestrins: Structural and functional properties

Calsequestrin is the major Ca²⁺-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca²⁺. Calsequestrin binds Ca²⁺ with high-capacity and with moderate affinity and it functions as a Ca²⁺ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca²⁺ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Expression and purification of human cardiac troponin subunits and their functional incorporation into isolated cardiac mouse myofibrils

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca(2+) concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca(2+) regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca(2+)-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca(2+)-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca(2+). Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.     

Mitsugumin 53 attenuates the activity of sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the roles of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca²⁺-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca²⁺-uptake through SERCA1a (more than 35%) at micromolar Ca²⁺ but not at nanomolar Ca²⁺, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.