Store-operated Ca(2+) entry (SOCE) contributes to normal skeletal muscle contractility in young but not in aged skeletal muscle.

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility.

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.

Functional and biochemical modifications in skeletal muscles from malarial mice.

Although it is well established that patients suffering from malaria experience skeletal muscle problems (contracture, aches, fatigue, weakness), detailed studies have not been performed to investigate changes in the contractile function and biochemical properties of intact and skinned skeletal muscles of mammals infected with malaria. To this end, we investigated such features in the extensor digitorium longus (EDL, fast-twitch, glyocolytic) and in the soleus (SOL, slow-twitch, oxidative) muscles from mice infected with Plasmodium berghei. We first studied maximal tetanic force (T(max)) produced by intact control and malaria-infected muscles before, during and after fatigue. Triton-skinned muscle fibres were isolated from these muscles and used to determine isometric contractile features as well as a basic biochemical profile as analysed by silver-enhanced SDS-PAGE. We found that the T(max) of intact muscles and the maximal Ca2+-activated force (F(max)) of Triton-skinned muscle fibres were reduced by approximately 50% in malarial muscles. In addition, the contractile proteins of Triton-skinned muscle fibres from malarial muscles were significantly less sensitive to Ca2+. Biochemical analysis revealed that there was a significant loss of essential contractile proteins (e.g. troponins and myosin) in Triton-skinned muscle fibres from malarial muscles as compared to controls. The biochemical alterations (i.e., reduction of essential contractile proteins) seem to explain well the functional modifications resolved in both intact muscles and Triton-skinned muscle fibres and may provide a suitable paradigm for the aetiology of muscle symptoms associated with malaria.

Defective maintenance of intracellular Ca2+ homeostasis is linked to increased muscle fatigability in the MG29 null mice.

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of mg29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca2+-uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and mg29 knockout mice. Compared with the control muscle, submaximal Ca2+-uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca2+ inside the SR was less in the mutant muscle, due to increased leakage process of Ca2+ movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca2+/caffeine -induced Ca2+ release to myoplasmic Ca2+. Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca2+ uptake, modification of Ca2+-induced Ca2+ release (CICR), and a reduction in total SR Ca2+ content.

Truncation by Glu180 nonsense mutation results in complete loss of slow skeletal muscle troponin T in a lethal nemaline myopathy.

A lethal form of nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), is linked to a nonsense mutation at codon Glu180 in the slow skeletal muscle troponin T (TnT) gene. We found that neither the intact nor the truncated slow TnT protein was present in the muscle of patients with ANM. The complete loss of slow TnT is consistent with the observed recessive pattern of inheritance of the disease and indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils. Expression of slow and fast isoforms of TnT is fiber-type specific. The lack of slow TnT results in selective atrophy of type 1 fibers. Slow TnT confers a higher Ca2+ sensitivity than does fast TnT in single fiber contractility assays. Despite the lack of slow TnT, individuals with ANM have normal muscle power at birth. The postnatal onset and infantile progression of ANM correspond to a down-regulation of cardiac and embryonic splice variants of fast TnT in normal developing human skeletal muscle, suggesting that the fetal TnT isoforms complement slow TnT. These results lay the foundation for understanding the molecular pathophysiology and the potential targeted therapy of ANM.