Evolutionary isolation of ryanodine receptor isoform 1 for muscle-based thermogenesis in mammals.

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.

Calpain-3 Is Not a Sodium Dependent Protease and Simply Requires Calcium for Activation.

Calpain-3 (CAPN3) is a muscle-specific member of the calpain family of Ca2+-dependent proteases. It has been reported that CAPN3 can also be autolytically activated by Na+ ions in the absence of Ca2+, although this was only shown under non-physiological ionic conditions. Here we confirm that CAPN3 does undergo autolysis in the presence of high [Na+], but this only occurred if all K+ normally present in a muscle cell was absent, and it did not occur even in 36 mM Na+, higher than what would ever be reached in exercising muscle if normal [K+] was present. CAPN3 in human muscle homogenates was autolytically activated by Ca2+, with ~50% CAPN3 autolysing in 60 min in the presence of 2 µM Ca2+. In comparison, autolytic activation of CAPN1 required about 5-fold higher [Ca2+] in the same conditions and tissue. After it was autolysed, CAPN3 unbound from its tight binding on titin and became diffusible, but only if the autolysis led to complete removal of the IS1 inhibitory peptide within CAPN3, reducing the C-terminal fragment to 55 kDa. Contrary to a previous report, activation of CAPN3, either by raised [Ca2+] or Na+ treatment, did not cause proteolysis of the skeletal muscle Ca2+ release channel-ryanodine receptor, RyR1, in physiological ionic conditions. Treatment of human muscle homogenates with high [Ca2+] caused autolytic activation of CAPN1, accompanied by proteolysis of some titin and complete proteolysis of junctophilin (JP1, full length ~95 kDa), generating an equimolar amount of a diffusible ~75 kDa N-terminal JP1 fragment, but without any proteolysis of RyR1.

Muscle fiber type-specific autophagy responses following an overnight fast and mixed meal ingestion in human skeletal muscle.

The aim of the present study was to investigate the fiber type-specific abundance of autophagy-related proteins after an overnight fast and following ingestion of a mixed meal in human skeletal muscle. Twelve overweight, healthy young male volunteers underwent a 3-h mixed meal tolerance test following an overnight fast. Blood samples were collected in the overnight-fasted state and throughout the 180-min postmeal period. Skeletal muscle biopsies were collected in the fasted state, and at 30 and 90 min after meal ingestion. Protein content of key autophagy markers and upstream signaling responses were measured in whole muscle and pooled single fibers using immunoblotting. In the fasted state, type I fibers displayed lower LC3B-I but higher LC3B-II abundance and higher LC3B-II/LC3B-I ratio compared with type II fibers (P < 0.05). However, there were no fiber type differences in p62/SQSTM1, unc-51 like autophagy activating kinase (ULK1), ATG5, or ATG12 (P > 0.05). Compared with the fasted state, there was a reduction in LC3B-II abundance, indicative of lower autophagosome content, in whole muscle and in both type I and type II fibers following meal ingestion (P < 0.05). This reduction in autophagosome content occurred alongside similar increases in p-AktS473 and p-mTORS2448 in both type I and type II muscle fibers (P < 0.05). In human skeletal muscle, type I fibers have a greater autophagosome content than type II fibers in the overnight-fasted state despite comparable abundance of other key upstream autophagy proteins. Autophagy is rapidly inhibited in both fiber types following the ingestion of a mixed meal.NEW & NOTEWORTHY This study examined the fiber type-specific content of key autophagy proteins in human muscle. We showed that markers of autophagosome content are higher in type I fibers in the overnight-fasted state, whereas autophagy is rapidly inhibited in both type I and type II fibers after the ingestion of a mixed meal.

Effects of voluntary wheel running on mitochondrial content and dynamics in rat skeletal muscle.

This study reports that in rat skeletal muscle the proteins specifically responsible for mitochondrial dynamics, mitofusin-2 (MFN2) and mitochondrial dynamics protein 49 (MiD49), are higher (p < 0.05) in oxidative soleus (SOL) muscle compared with predominantly glycolytic extensor digitorum longus (EDL) muscle, but not seen for optic atrophy 1 (OPA1; p = 0.06). Markers of mitochondrial content, complex I component, NADH:Ubiquinone oxidoreductase subunit A9 (NDUFA9) and complex IV protein, cytochrome C oxidase subunit IV (COXIV; p < 0.05) were also higher in SOL compared with EDL muscle; however, there was no difference in mitochondrial content between muscles, as measured using a citrate synthase assay (p > 0.05). SOL and EDL muscles were compared between age-matched sedentary rats that were housed individually with (RUN) or without (SED) free-access to a running wheel for 12 weeks and showed no change in mitochondrial content, as examined by the abundances of NDUFA9 and COXIV proteins, as well as citrate synthase activity, in either muscle (p > 0.05). Compared to SED animals, MiD49 and OPA1 were not different in either EDL or SOL muscles, and MFN2 was higher in SOL muscles from RUN rats (p < 0.05). Overall, these findings reveal that voluntary wheel running is an insufficient stimulus to result in a significantly higher abundance of most markers of mitochondrial content or dynamics, and it is likely that a greater stimulus, such as either adding resistance to the wheel or an increase in running volume by using a treadmill, is required for mitochondrial adaptation in rat skeletal muscle.

Characterization of muscle ankyrin repeat proteins in human skeletal muscle.

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule.

Fiber Type Identification of Human Skeletal Muscle.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy. The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples. These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Nuclei isolation methods fail to accurately assess the subcellular localization and behaviour of proteins in skeletal muscle.

AIM: Subcellular fractionation is often used to determine the subcellular localization of proteins, including whether a protein translocates to the nucleus in response to a given stimulus. Examining nuclear proteins in skeletal muscle is difficult because myonuclear proteins are challenging to isolate unless harsh treatments are used. This study aimed to determine the most effective method for isolating and preserving proteins in their native state in skeletal muscle. METHODS: We compared the ability of detergents, commercially available kit-based and K+ -based physiological methodologies for isolating myonuclear proteins from resting samples of human muscle by determining the presence of marker proteins for each fraction by western blot analyses. RESULTS: We found that following the initial pelleting of nuclei, treatment with 1% Triton-X 100, 1% CHAPS or 0.5% Na-deoxycholate under various ionic conditions resulted in the nuclear proteins being either resistant to isolation or the proteins present behaving aberrantly. The nuclear proteins in brain tissue were also resistant to 1% Triton-X 100 isolation. Here, we demonstrate aberrant behaviour and erroneous localization of proteins using the kit-based method. The aberrant behaviour was the activation of Ca2+ -dependent protease calpain-3, and the erroneous localization was the presence of calpain-3 and troponin I in the nuclear fraction. CONCLUSION: Our findings indicate that it may not be possible to reliably determine the translocation of proteins between subcellular locations and the nucleus using subcellular fractionation techniques. This study highlights the importance of validating subcellular fractionation methodologies using several subcellular-specific markers and solutions that are physiologically relevant to the intracellular milieu.

Expression of titin-linked putative mechanosensing proteins in skeletal muscle after power resistance exercise in resistance-trained men.

Little is known about the molecular responses to power resistance exercise that lead to skeletal muscle remodeling and enhanced athletic performance. We assessed the expression of titin-linked putative mechanosensing proteins implicated in muscle remodeling: muscle ankyrin repeat proteins (Ankrd 1, Ankrd 2, and Ankrd 23), muscle-LIM proteins (MLPs), muscle RING-finger protein-1 (MuRF-1), and associated myogenic proteins (MyoD1, myogenin, and myostatin) in skeletal muscle in response to power resistance exercise with or without a postexercise meal, in fed, resistance-trained men. A muscle sample was obtained from the vastus lateralis of seven healthy men on separate days, 3 h after 90 min of rest (Rest) or power resistance exercise with (Ex + Meal) or without (Ex) a postexercise meal to quantify mRNA and protein levels. The levels of phosphorylated HSP27 (pHSP27-Ser15) and cytoskeletal proteins in muscle and creatine kinase activity in serum were also assessed. The exercise increased (P ≤ 0.05) pHSP27-Ser15 (∼6-fold) and creatine kinase (∼50%), whereas cytoskeletal protein levels were unchanged (P > 0.05). Ankrd 1 (∼15-fold) and MLP (∼2-fold) mRNA increased, whereas Ankrd 2, Ankrd 23, MuRF-1, MyoD1, and myostatin mRNA were unchanged. Ankrd 1 (∼3-fold, Ex) and MLPb (∼20-fold, Ex + Meal) protein increased, but MLPa, Ankrd 2, Ankrd 23, and the myogenic proteins were unchanged. The postexercise meal did not affect the responses observed. Power resistance exercise, as performed in practice, induced subtle early responses in the expression of MLP and Ankrd 1 yet had little effect on the other proteins investigated. These findings suggest possible roles for MLP and Ankrd 1 in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.NEW & NOTEWORTHY This is the first study to assess the early changes in the expression of titin-linked putative mechanosensing proteins and associated myogenic regulatory factors in skeletal muscle after power resistance exercise in fed, resistance-trained men. We report that power resistance exercise induces subtle early responses in the expression of Ankrd 1 and MLP, suggesting these proteins play a role in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.