Thomas M. Nosek

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation–contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.

Deficiency of MIP/MTMR14 phosphatase induces a muscle disorder by disrupting Ca 2+ homeostasis

The intracellular Ca2+ concentration ([Ca2+]i) in skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process. However, the signalling components involved in such rapid Ca2+ movement are not fully understood. Here we report that mice deficient in the newly identified PtdInsP (phosphatidylinositol phosphate) phosphatase MIP/MTMR14 (muscle-specific inositol phosphatase) show muscle weakness and fatigue. Muscles isolated from MIP/MTMR14−/− mice produced less contractile force, had markedly prolonged relaxation and showed exacerbated fatigue relative to normal muscles. Further analyses revealed that MIP/MTMR14 deficiency resulted in spontaneous Ca2+ leakage from the internal store — the sarcoplasmic reticulum. This was attributed to decreased metabolism (dephosphorylation) and the subsequent accumulation of MIP/MTMR14 substrates, especially PtdIns(3,5)P2 and PtdIns (3,4)P2. Furthermore, we found that PtdIns(3,5)P2 and PtdIns(3,4)P2 bound to, and directly activated, the Ca2+ release channel (ryanodine receptor 1, RyR1) of the sarcoplasmic reticulum. These studies provide the first evidence that finely controlled PtdInsP levels in muscle cells are essential for maintaining Ca2+ homeostasis and muscle performance.

Kruppel-like factor 15 regulates skeletal muscle lipid flux and exercise adaptation

The ability of skeletal muscle to enhance lipid utilization during exercise is a form of metabolic plasticity essential for survival. Conversely, metabolic inflexibility in muscle can cause organ dysfunction and disease. Although the transcription factor Kruppel-like factor 15 (KLF15) is an important regulator of glucose and amino acid metabolism, its endogenous role in lipid homeostasis and muscle physiology is unknown. Here we demonstrate that KLF15 is essential for skeletal muscle lipid utilization and physiologic performance. KLF15 directly regulates a broad transcriptional program spanning all major segments of the lipid-flux pathway in muscle. Consequently, Klf15-deficient mice have abnormal lipid and energy flux, excessive reliance on carbohydrate fuels, exaggerated muscle fatigue, and impaired endurance exercise capacity. Elucidation of this heretofore unrecognized role for KLF15 now implicates this factor as a central component of the transcriptional circuitry that coordinates physiologic flux of all three basic cellular nutrients: glucose, amino acids, and lipids.

Hypoxia/fatigue-induced degradation of troponin I and troponin C: new insights into physiologic muscle fatigue

Abstract. Conditions such as respiratory failure and cardiopulmonary arrest can expose the diaphragm to hypoxemia. In skeletal muscles, fatiguing stimulation renders muscles hypoxic, which has long been known to dramatically reduce muscle function. We have previously demonstrated that fatiguing stimulation under hypoxic conditions disrupts both the excitation-contraction coupling (ECC) process and the isometric contractile properties (ICP) in intact diaphragm muscle strips and the contractile properties of skinned fibers isolated from these muscles. Here we have analyzed the effects of intermittent fatiguing stimulation on specific muscle proteins in muscle strips from mouse diaphragms that have been exposed to hypoxia. We report for the first time that the effects of hypoxia-fatigue, namely to decrease maximal tetanic force, maximal calcium-activated force and calcium sensitivity of the mouse diaphragm muscle, are associated with the degradation of troponins TnI and TnC (Western blot analysis). The concentrations of TnT and actin did not change under these same conditions. Because troponins are integrally involved in regulating the interaction between actin and myosin during the cross-bridge cycle, the degradation of TnI and TnC may explain the effects of hypoxia-fatigue on the ICP. This interpretation is supported by the observations that extraction of troponins from control skinned fibers mimics the effects of hypoxia-fatigue on contractile function and that incorporation of native troponins into fibers isolated from hypoxic-fatigued muscles partially restores function.

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

ABSTRACTMitsugumin 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.

Hypoxia and fatigue-induced modification of function and proteins in intact and skinned murine diaphragm muscle

Fatigue studies of isolated, intact muscles typically utilize solutions saturated with O2. However, under in vivo fatiguing conditions, less oxygen is delivered to the muscles and they actually experience hypoxia. No studies to date have correlated the effects of acute hypoxia on the isometric contractile properties of intact muscles, skinned fibers isolated from the same muscles, and the cellular content of specific muscle proteins. Therefore, we have studied the effects of in vitro acute hypoxia on the fatigability of intact diaphragm muscle strips and on the isometric contractile properties of single Triton-skinned fibers isolated from control and hypoxic diaphragm muscles. We found that hypoxia and fatiguing stimulation per se affect the tetanic force of intact muscle strips without exhibiting any significant deleterious effects on the calcium-activated force of skinned muscle fibers dissected from the intact muscles. In contrast, fatiguing stimulation under hypoxic conditions decreased both the tetanic force of muscle strips and the calcium-activated force of skinned muscle fibers. Gel electrophoresis of muscles subjected to hypoxia and hypoxic-fatigue revealed that there is a significant reduction in three protein bands when compared to control muscles. Protein modification may be the underlying mechanism of muscle fatigue under physiologic conditions.

Influence of ageing on the fatigability of isolated mouse skeletal muscles from mature and aged mice

We investigated the influence of ageing on the fatiguing characteristics of the mouse extensor digitorum longus (EDL) muscle as compared to those of the soleus muscle. Fatigue was produced by an intermittent stimulation protocol. We report for mature and aged animals the effects of fatigue on force produced during stimulation patterns that in non‐fatigued muscle gave maximum force (Tmax, high frequency stimulation) and approximately half‐maximum force (1/2Tmax, low frequency stimulation). In 15‐month‐old (mature) mice, fatiguing stimulation decreased Tmax in EDL and soleus muscle to 10.3 ± 1.0% and 33.4 ± 3.0% of control, respectively. In 30‐month‐old (aged) mice, the decrease in Tmax in EDL and soleus was statistically equal to that of the younger animals. Fatiguing stimulation decreased 1/2Tmax in EDL and soleus from 15‐month‐old animals to 22.5 ± 2.9% and 45.7 ± 0.3% of control, respectively. In 30‐month‐old animals, the 1/2Tmax in EDL and soleus muscle decreased to 18.2 ± 1.3% and 35.0 ± 3.6% of control, respectively. Under all conditions, the soleus fatigued significantly less. Contractile recovery from fatiguing stimulation was complete for the soleus in both age groups after 30 min, but incomplete for the EDL. The 1/2Tmax/Tmax ratio significantly increased in EDL and soleus muscle from 15‐month‐old animals after fatiguing stimulation. This increase was less significant in EDL, and absent in soleus muscle, from 30‐month‐old animals. These results indicate that fatiguing stimulation induces a leftward shift in the force‐frequency relationship in the young animals; this shift is either significantly less (EDL) or absent (soleus) in the older animals. We speculate that the leftward shift of the force‐frequency relationship may reflect a protective mechanism in younger animals against some of the damaging effects of fatiguing stimulation (i.e. oxidative stress).

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 (Tmax) 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 Tmax of intact muscles and the maximal Ca2+‐activated force (Fmax) of Triton‐skinned muscle fibres were reduced by ∼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.

Temporal Adaptive Changes in Contractility and Fatigability of Diaphragm Muscles from Streptozotocin-Diabetic Rats

Diabetes is characterized by ventilatory depression due to decreased diaphragm (DPH) function. This study investigated the changes in contractile properties of rat DPH muscles over a time interval encompassing from 4 days to 14 weeks after the onset of streptozotocin-induced diabetes, with and without insulin treatment for 2 weeks. Maximum tetanic force in intact DPH muscle strips and recovery from fatiguing stimulation were measured. An early (4-day) depression in contractile function in diabetic DPH was followed by gradual improvement in muscle function and fatigue recovery (8 weeks). DPH contractile function deteriorated again at 14 weeks, a process that was completely reversed by insulin treatment. Maximal contractile force and calcium sensitivity assessed in Triton-skinned DPH fibers showed a similar bimodal pattern and the same beneficial effect of insulin treatment. While an extensive analysis of the isoforms of the contractile and regulatory proteins was not conducted, Western blot analysis of tropomyosin suggests that the changes in diabetic DPH response depended, at least in part, on a switch in fiber type.

Excessive microtubules are not responsible for depressed force per cross-bridge in cardiac neural-crest-ablated embryonic chick hearts.

Abstract Ablation of the cardiac neural crest (CNCA) in embryonic chicks results in a high incidence of persistent truncus arteriosus, a congenital heart defect associated with decreased myocardial contractility. Using left ventricular trabeculae from chicks at embryonic day (ED) 15, we have previously shown that the twitch force of intact preparations is significantly reduced whereas the maximal calcium-activated force of skinned preparations is not significantly different in CNCA and sham-operated animals. We also previously found that the ventricular content of myosin, as well as of actin and tropomyosin, was nearly doubled in ED 15 hearts after CNCA. Since the number of cross-bridges is proportional to the myosin concentration, these data suggest that the force exerted per cross-bridge is decreased in CNCA hearts. We investigated the possibility that the decrease in force per cross-bridge is caused by inhibition of the contractile apparatus by excessive microtubules. To the contrary, we found that the total β-tubulin content and the fraction of β-tubulin polymerized in microtubules measured by Western blotting was the same in ventricular muscle strips from CNCA and sham-operated embryos. Furthermore, exposure to microtubule-destabilizing agents did not improve the force-producing capability of the contractile apparatus in CNCA embryos. We conclude that depression of force per cross-bridge in hearts from CNCA embryos is not due to an excess of microtubules.