SUMMARY1. Maximal calcium-activated force (Fmax) and calcium sensitivity were markedly decreased in detergent-skinned fibres from skeletal and cardiac muscle by solutions that mimicked the total milieu changes associated with fatigue and hypoxia. Further experiments determined the relative contribution of each of the individual changes in milieu.2. Both Ca21 sensitivity and Fmax of skeletal and cardiac fibres were decreased with increased [H+] or inorganic phosphate (Pi). These effects were greater in cardiac muscle.3. Decreasing MgATP over the range observed with fatigue and hypoxia (6-18-4-7 mM) had no effect on Fmax or Ca2" sensitivity of either muscle type. 4. Decreasing phosphocreatine (PCr: 15-1 mM) increased Fmax but had little effect on Ca2" sensitivity in both muscle types. In cardiac fibres, the effect on Fmax could be mimicked by inhibition of endogenous creatine kinase.5. ADP (0-7 mm) increased Fmax and Ca21 sensitivity, while AMP (0-06 mM) slightly increased Fmax but had no effect on Ca2" sensitivity of either skeletal or cardiac fibres.6. Creatine (25 mM) had no significant effect on either Ca2" sensitivity or Fmax of skeletal and cardiac muscle fibres. At higher levels (50 mM), however, creatine depressed Fmax and slightly altered Ca2" sensitivity.7. Thiophosphorylation of myosin P light chains (phosphorylatable light chains of myosin) in rabbit psoas fibres had no effect on Ca21 sensitivity, yet slightly but significantly increased Fmax under fatigue conditions. 8. Reducing the affinity for ATP hydrolysis (by adding ADP, AMP and creatine) over the range calculated for fatigue/hypoxia (60-45 kJ/mol) produced the enhancement in Fmax expected from added ADP and AMP in cardiac but not skeletal muscle, indicating that changes in affinity influence Fmax of skeletal muscle. Reducing affinity produced little change in Ca21 sensitivity of skeletal muscle. In contrast, the change produced in cardiac muscle was greater than that expected from addition of ADP and AMP; i.e. decreasing affinity increases calcium sensitivity of the heart. 9. Simple summation of all significant changes expected from each constituent R. E. GODT AND T. M. NOSEK altered by fatigue/hypoxia adequately predicted the observed changes in F... and Ca2+ sensitivity in both cardiac and skeletal muscle fibres with but one exception (the change in Ca2+ sensitivity of skeletal muscle at pH 7 was slightly overestimated).

The store-operated calcium channel (SOC) located in the plasma membrane (PM) mediates capacitative entry of extracellular calcium after depletion of intracellular calcium stores in the endoplasmic or sarcoplasmic reticulum (ER/SR). An intimate interaction between the PM and the ER/SR is essential for the operation of this calcium signalling pathway. Mitsugumin 29 (MG29) is a synaptophysin-family-related protein located in the junction between the PM and SR of skeletal muscle. Here, we identify SOC in skeletal muscle and characterise its regulation by MG29 and the ryanodine receptor (RyR) located in the SR. Targeted deletion of mg29 alters the junctional membrane structure, causes severe dysfunction of SOC and SR calcium homeostasis and increases the susceptibility of muscle to fatigue stimulation. Severe dysfunction of SOC is also identified in muscle cells lacking both type 1 and type 3 RyRs, indicating that SOC activation requires an intact interaction between the PM and the SR, and is linked to conformational changes of RyRs. Whereas defective SOC seems to be inconsequential to short-term excitation-contraction coupling, the slow cumulative calcium entry through SOC is crucial for long-term calcium homeostasis, such that reduced SOC activity exaggerates muscle fatigue under conditions of intensive exercise.

The intracellular Ca 2+ ([Ca 2+ ] i ) level of skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process 1 . However, the signaling components involved in such rapid Ca 2+ movement are not fully understood. Here, we report that mice deficient in the novel phosphatidylinositol phosphate (PIP) phosphatase MIP displayed muscle weakness and fatigue. Muscles isolated from MIP −/− mice produced less contractile force, markedly prolonged relaxation, and exhibited exacerbated fatigue. Further analyses revealed that MIP deficiency resulted in spontaneous Ca 2+ leak from the internal store -the sarcoplasmic reticulum (SR). This was attributed to the decreased metabolism/dephosphorylation and the subsequent accumulation of MIP substrates, especially PI(3,5)P 2 and PI(3,4)P 2 . Furthermore, we found that PI(3,5)P 2 and PI(3,4)P 2 bound to and directly activated the Ca 2+ release channel/ryanodine receptor (RyR1) of the SR. These studies provide the first evidence that finely controlled PIP levels in muscle cells are essential for maintaining Ca 2+ homeostasis and muscle performance.During our systematic genome-wide survey for tyrosine/dual specificity phosphatases (unpublished work), we discovered a novel phosphatase by hidden Markov database mining using the conserved catalytic motif ([V/I][V/I]HCXXGXXR[T/S]) as the bait sequence. Both human (BC035690) and mouse (BC018294) homologies were identified. They share 90% identity in amino acid sequences ( Supplementary Information, Fig. S1). Northern blotting analyses illustrated that this phosphatase was predominantly expressed in skeletal muscle and heart (Fig. 1a). Immunostaining indicates that it is primarily localized in the cytoplasm (data not shown). To verify its phosphatase property, we generated a GST fusion protein and tested its catalytic activity using pNPP (p-Nitrophenyl Phosphate), a widely used non-specific 7Correspondence should be addressed to: C.K.Q. (e-mail: E-mail: cxq6@case.edu). 6 These authors contributed equally to this work. AUTHOR CONTRIBUTIONSJ.S., W.M. Y., M.B., J.A.S., and C.S. conducted the research and summarized the data. C.K.Q., M.B., H.H.V., T.M.N., and C.G. designed the experiments and wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. (Fig. 1b). Instead, it dephosphorylated a variety of PIPs, especially PI(3,5) P 2 (Fig. 1c), similar to PTEN and myotubularin and myopathy related (MTMR) phosphatases that also favor PIPs as substrates despite containing tyrosine phosphatase domains 2 . As this new phosphatase is mainly expressed in skeletal muscle and heart, we named it MIP (musclespecific inositol phosphatase). While our gene knockout work on MIP was ongoing, the Mustalin group also identified this phosphatase (FLJ20133) in their comprehensive collection of tyrosine phosphatases from the human genome and listed it as the 14 th member of the MTMR family (MTMR14) based on the homology of its catalytic motif to myotubularin 3 . More recently, ina...

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.

The increases in the intracellular concentrations of inorganic phosphate and hydrogen ion accompanying fatigue of skeletal muscle appear to be the most important metabolic changes associated with the decrease in contractile force. Experiments on chemically skinned single fibers from rabbit psoas muscle with pH ranging between 6 and 7.25 demonstrate that the depression of maximal calcium-activated force by inorganic phosphate correlates nicely with the concentration of the acidic (diprotonated) species. Therefore, in addition to the well-known depressant effect on the contractile machinery of lowering pH per se, any decrease of intracellular pH associated with fatigue further depresses force production by converting more of the total inorganic phosphate within the cell to the inhibitory diprotonated form.