FDB Muscle Research Articles

Role of the ubiquitin-proteasome system in the sarcopenic-like phenotype induced by CCL5/RANTES.

Sarcopenia is characterized by reduced muscle strength and mass and a decline in muscle fiber diameter and amount of sarcomeric proteins. Sarcopenia involves the activation of the ubiquitin-proteasome system (UPS). MuRF-1 and atrogin-1 are E3 ubiquitin ligases belonging to UPS, leading to proteolysis mediated by the PSMB 5, 6, and 7 subunits of 20S proteasome. CCL5/RANTES induces a sarcopenic-like effect in muscle cells. The present work explored the impact of CCL5 on UPS components and the influence of UPS on its sarcopenic-like effect. We demonstrated that CCL5 increased MuRF-1 and atrogin-1 protein levels and mRNA levels of subunits PSMB 5, 6, and 7. We used the MG132 inhibitor to elucidate the role of the 20S proteasome in the CCL5-induced sarcopenic-like effect. This inhibitor prevented the decrease in troponin and MHC protein levels and partially prevented the reduction in the diameter of single-isolated FDB muscle fibers induced by CCL5. These findings indicate that CCL5 actively modulates the UPS. Moreover, our results show the direct participation of UPS in the sarcopenic-like phenotype induced by CCL5.

P.149 Differential expression of intermediate filament proteins: lamins A/C and desmin within and between adult skeletal muscles

The cell cytoskeleton is composed of three main components: microfilaments, microtubules, and intermediate filaments. Intermediate filaments consist of intermediate filament proteins that are widely distributed in cells and tissues, either in the cytoplasm or the nucleus. Desmin is a tissue-specific cytoplasmic intermediate filament protein, while A-type lamins including lamin A/C are ubiquitously expressed nuclear intermediate filament proteins. Both play major roles in skeletal muscle functions. Mutations in the <i>DES</i> gene, encoding Desmin, and <i>LMNA</i> gene, encoding A-type lamins, lead to a group of rare disorders called desminopathies and laminopathies, respectively. The most frequent pathologies are those involving skeletal muscles. Despite the universal expression of lamins A/C in almost all cells and tissues, different phenotypes associated with laminopathies reflect a high degree of tissue-specificity, the reasons for which are not completely understood. It has also been shown that specific muscles/muscle groups are affected earlier than others in these myopathies. We hypothesized that desmin and lamins A/C are not uniformly expressed in different skeletal muscles and or muscle parts. Accordingly, we have investigated the relative expression of desmin, lamin A & lamin C in 9 skeletal muscles, of which 7 were divided into belly and myotendinous junction (MTJ). Our preliminary results revealed that there is no significant difference in the relative expression of desmin and lamins A/C between bellies and MTJs within a single muscle. We also observed that the relative expression of lamins A & C is correlated with myogenic markers; MyoD and myogenin, while desmin is not. Strikingly, FDB muscle showed the highest expression of lamins A/C compared to other muscles. We are currently investigating desmin, lamin A and lamin C protein levels by western blot and immunohistochemistry and the effect of age and exercise on the expression of these intermediate filament proteins.

Specific effects of individual C2 domains on recovery of voltage-induced SR calcium release after osmotic shock in muscle fibers from dysferlin-null mice

Our earlier studies used osmotic shock injury (OSI) to show that dysferlin modulates the coupling of excitation to Ca2+ release in skeletal myofibers (Lukyanenko et al., 2017). As the C2A domain plays an important role in Ca2+ signaling, we expressed it in dysferlin-null A/J mouse FDB myofibers to assess its ability to support normal Ca2+ signaling. cDNAs encoding C2 domains with N-terminal Venus (Venus-C2A, -C2A-C2A, -C2A-C2A-C2A-C2A, -C2A-C2B, -C2C, and -C2E) with or without mutations (pathogenic: W52R, V67D; polymorphisms: V68L; A84V), and with or without dysferlin's C-terminal transmembrane domain (TM), were electroporated into A/J FDB muscles.

Lifetime of autofluorescence: A novel method to determine murine skeletal muscle fiber types

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes derived from a two-exponential fit (τ1 and τ2) in freshly dissected mouse FDB and soleus muscles. While τ1 values did not change between muscles, the distribution of τ2 shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes in cryosections of both muscles and paired them with immunofluorescence images of myosin heavy chain isoforms (MHC), which allow identification of fiber types. In soleus, τ2 was 3.1 ns for type I (SEM = 0.009, n = 49), 3.4 ns for type IIA (SEM = 0.01, n = 30), and 3.3 ns for type IIX (SEM = 0.01, n = 21). In FDB muscle, τ2 was 3.17 ns for type I (SEM = 0.04, n = 18), 3.5 ns for type IIA (SEM = 0.03, n = 27), and 3.62 ns for type IIX (SEM = 0.03, n = 22). From the distribution of measures, it follows that an FDB fiber with τ2 >3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first- and second-class errors estimated at 0.06 and 0.27, respectively. Studies in progress aim at further elucidating the reasons for the different lifetimes, not just among fiber types but between fibers of the same type in the two muscles. Preliminary results point at differences in both the oxidation-reduction and protein-bound versus free states of flavins as causes for the observed divergence of fluorescence lifetimes. Lifetime maps of autofluorescence therefore constitute a tool to identify fiber type that, being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.

PO-033 Mitochondria-associated Ampk mediates mitochondrial quality control in skeletal muscle

Objective Mitochondria exist as a complex, interconnected reticulum with the degree of complexity at homeostasis varying by cell type. Declines in mitochondrial quality is a hallmark of numerous diseases and is partly maintained through the targeted removal and degradation of damaged/dysfunctional regions of the reticulum known as mitophagy. We have demonstrated that acute exercise causes targeted degradation of a subset (&lt;1%) of the mitochondrial reticulum through mitophagy in an AMPK-dependent manner. However, how such spatial regulation is achieved is unclear. Given that AMPK is a well-known bioenergetics sensor critical for maintenance of energetic homoeostasis, and numerous studies have linked AMPK-signaling to mitochondrial remodeling and functional adaptations under physiological and pathological conditions, we hypothesized that AMPK regulates mitophagy by being physically associated with the mitochondria. &#x0D; Methods C57BL/6 mice (3 or 20 months of age) were kept in standard conditions (12:12 light, dark cycle) on normal chow. Enriched mitochondrial fractions from skeletal muscle were obtained via differential centrifugation and percoll gradient isolation. Skeletal muscle energetic stress was induced via acute treadmill running, direct electrical stimulation, or hindlimb ischemia-reperfusion. Somatic gene transfer into skeletal muscle was done via electraporation. Confocal microscopy of whole mount skeletal muscle was performed at least 10 days post-electraporation to assess MitoTimer fluorescence Red:Green Ratio and Mitophagy (i.e. presence of pure red puncta).&#x0D; Results We have discovered that AMPK is enriched in isolated mitochondria from both mouse and human skeletal muscle (mitoAMPK) and that, in mice, mitoAMPK consists exclusively of ⍺1/β2/γ1 isoforms. Furthermore, skeletal muscle mitoAMPK Thr172 phosphorylation, indicative of activation, is increased following electrical stimulation, ischemia-reperfusion, and acute treadmill running. By co-transfecting mouse FDB muscle with pMitoTimer and a mitochondrially-targeted AMPK inhibitor peptide (mitoAIP), we show that mitoAMPK activity is required to maintain mitochondrial quality (as evidenced by increased MitoTimer Red:Green ratio) and exercise-induced mitophagy (MitoTimer pure red puncta). Interestingly, association of mitoAMPK with mitochondria declines by ~50% in skeletal muscle in old mice and coincides with poor mitochondrial quality (increased MitoTimer Red:Green ratio), which may explain the loss of mitochondrial quality and metabolic flexibility with aging.&#x0D; Conclusions Our current working hypothesis is that activation of mitoAMPK by exercise plays an instrumental role in promoting mitochondrial remodeling, hence contractile and metabolic adaptations, by spatially recognizing damaged regions of the mitochondrial reticulum. Future research on mitoAMPK will significantly improve the mechanistic understanding of exercise training-induced adaptation in skeletal muscle and AMPK biology.

ER stress disturbs SR/ER-mitochondria Ca2 + transfer: Implications in Duchenne muscular dystrophy

Besides its role in calcium (Ca2+) homeostasis, the sarco-endoplamic reticulum (SR/ER) controls protein folding and is tethered to mitochondria. Under pathophysiological conditions the unfolded protein response (UPR) is associated with disturbance in SR/ER-mitochondria crosstalk. Here, we investigated whether ER stress altered SR/ER-mitochondria links, Ca2+ handling and muscle damage in WT (Wild Type) and mdx mice, the murine model of Duchenne Muscular Dystrophy (DMD). In WT mice, the SR/ER-mitochondria links were decreased in isolated FDB muscle fibers after injection of ER stress activator tunicamycin (TM). Ca2+ imaging revealed an increase of cytosolic Ca2+ transient and a decrease of mitochondrial Ca2+ uptake. The force generating capacity of muscle dropped after TM. This impaired contractile function was accompanied by an increase in autophagy markers and calpain-1 activation. Conversely, ER stress inhibitors restored SR/ER-mitochondria links, mitochondrial Ca2+ uptake and improved diaphragm contractility in mdx mice. Our findings demonstrated that ER stress-altered SR/ER-mitochondria links, disturbed Ca2+ handling and muscle function in WT and mdx mice. Thus, ER stress may open up a prospect of new therapeutic targets in DMD.

150. Further Development of an Allele-Specific Gene Silencing Strategy to Correct a Dominant-Negative Mutation Causing Collagen VI-Related Muscular Dystrophy

Collagen VI-related congenital muscular dystrophies (COL6-RD), caused by mutations in any of the three genes coding for collagen type VI (COL6A1, COL6A2, COL6A3), underlie a spectrum of disorders ranging from severe life-threatening early onset Ullrich muscular dystrophy via intermediate phenotypes to the milder Bethlem type, typically presenting with generalized muscle weakness, proximal joint contractures, and respiratory failure. At present, there are no treatment options available for individuals affected with these diseases. The fact that the majority of COL6-RD cases are carriers of inherited or de novo dominant mutations, acting as dominant-negative, poses a challenge for the development of targeted therapies. In contrast to gene replacement, allele-specific silencing has the potential to treat these disorders, as it would convert the dominant-negative state into a clinically asymptomatic haploinsufficient state. Therefore, in the laboratory we aim at exploring targeted RNA interference (RNAi) approaches as a potential therapeutic approach for dominant COL6-RD. We have previously demonstrated the allele-specificity and efficacy of siRNA oligos to downregulate the expression of a mutant COL6A3 transcript in vitro in patient-derived fibroblasts. In preparation of in vivo testing we have now extended our study to cells isolated from a mouse model of the disorder, which carries the most frequent dominant-negative mutation, a deletion of exon 16 on Col6a3. We transfected a series of small interfering RNA (siRNA) oligonucleotides into Col6a3del16+/- fibroblast cells, isolated from skin and muscle. To assay efficacy we used outcome measures such as unsaturated PCR, quantitative RT-PCR, and immunofluorescence. In addition, we performed in vivo electroporation of siRNA oligonucleotides in FDB muscles of Col6a3del16+/- mice. We found that siRNA oligonucleotides were effective in Col6a3del16+/- cells, to specifically knockdown the expression from the mutant allele and to restore the production of a collagen VI matrix in culture. This study provides further proof-of-concept of the use of RNAi as a potential treatment for COL6-RD.

IP3 Receptor Determines Increased Basal Mitochondrial Calcium And Autophagy Regulation In Mdx Skeletal Muscle

Several reports describe that IP3 receptor (IP3R) is essential for efficient mitochondrial respiration and maintenance of cellular bioenergetics. This is due to the participation of this receptor in calcium transfer from the ER to the mitochondria. Changes in IP3R function can compromise mitochondrial function, changing ATP production and finally altering autophagy. Nevertheless until now there is no detailed study of the IP3R/calcium/autophagy axis in DMD. Our aim was to investigate the participation of IP3R in the regulation of mitochondrial calcium and autophagy in fibers from a DMD animal model, mdx. We analyzed protein levels of LC3, p62, atg5, beclin and bcl‐2 in FDB muscle of mdx (5–7 weeks), electroporated with a short‐hairpin for IP3R. We also evaluated the calcium transfer with mitochondria‐targeted probes that can be electroporated and also mitochondrial potential with TMRE in skeletal muscle fibers. The basal levels of expression of LC3II are diminished in mdx fibers compared with controls. This result correlates with an increased expression of p62 in mdx fibers. We also found differences in other autophagy proteins like atg5, beclin1 and Bcl‐2. When we performed the knockdown for IP3R we observed differences in the expression of almost all of the proteins analyzed. The expression of LC3II was increased in the electroporated mdx fibers with a decrease in p62 expression. We also confirmed that basal mitochondrial calcium level was increased in mdx fibers. Moreover, when we decreased the expression of IP3R, the basal mitochondrial calcium levels returned to normal. Similar result was obtained when we measured the mitochondrial potential in mdx fibers. Collectively, these results suggest that IP3R regulates mitochondrial calcium flux in adult skeletal muscle and is responsible for the increased mitochondrial calcium in mdx fibers. Moreover, knockdown of IP3R can regulate the expression levels of proteins associated with autophagy. Finally, we propose that some inhibitors of the IP3 pathway can be used as a new treatment for the muscle weakness observed in DMD patientsSupport or Funding InformationFONDECYT 3140491(DV), 3140532(FW), 1151293(EJ), ACT‐111 (EJ, SL), FONDAP 15130011(SL)

Modulation of Skeletal Muscle Fatigue Kinetics by Extracellular Glucose and Glycogen Content

Increasing activity and the demand on skeletal muscle invariably leads to a depletion of energy reserves and a reduction in the ability of the muscle to produce force, known as muscle fatigue. Studies have shown glycogen to be of integral importance to this process and to play a role that is more complex than as a simple glucose reservoir. Here, using mouse FDB muscle bundles, we investigated the respective influence of glycogen and extracellular glucose on muscle fatigue. During a first bout of fatigue (FAT1) glycogen usage and force production was similar between control and insulin-exposed muscles. Glycogen replenishment 60 min after FAT1 was such that glycogen content prior to a second fatigue bout (FAT2) and the amount used during FAT2 was greater than for FAT1. This corresponded to an increase in time to fatigue. For a low glycogen model, FAT1 was followed by a 60 min recovery period in 0 glucose, resulting in a decline in pre-FAT2 glycogen content. When FAT2 was elicited also in 0 glucose, a marked decrease in time to fatigue was observed, which was partially recovered when if glucose was reintroduced 10 min before FAT2, to spite very low glycogen content and usage. A correlation was revealed when pre-FAT glycogen content and time to fatigue were compared for FAT2 bouts. From these results, we support that glycogen is a consistently critical factor in muscle fatigue, and suggest that extracellular glucose cannot fully compensate for force loss after excessive glycogen depletion.

Changes in myoplasmic Ca2+ during fatigue differ between FDB fibers, between glibenclamide-exposed and Kir6.2-/- fibers and are further modulated by verapamil.

One objective of this study was to document how individual FDB muscle fibers depend on the myoprotection of KATP channels during fatigue. Verapamil, a CaV1.1 channel blocker, prevents large increases in unstimulated force during fatigue in KATP-channel-deficient muscles. A second objective was to determine if verapamil reduces unstimulated [Ca2+]i in KATP-channel-deficient fibers. We measured changes in myoplasmic [Ca2+] ([Ca2+]i) using two KATP-channel-deficient models: (1) a pharmacological approach exposing fibers to glibenclamide, a channel blocker, and (2) a genetic approach using fibers from null mice for the Kir6.2 gene. Fatigue was elicited with one tetanic contraction every sec for 3 min. For all conditions, large differences in fatigue kinetics were observed from fibers which had greater tetanic [Ca2+]i at the end than at the beginning of fatigue to fibers which eventually completely failed to release Ca2+ upon stimulation. Compared to control conditions, KATP-channel-deficient fibers had a greater proportion of fiber with large decreases in tetanic [Ca2+]i, fade and complete failure to release Ca2+ upon stimulation. There was, however, a group of KATP-channel-deficient fibers that had similar fatigue kinetics to those of the most fatigue-resistant control fibers. For the first time, differences in fatigue kinetics were observed between Kir6.2-/- and glibenclamide-exposed muscle fibers. Verapamil significantly reduced unstimulated and tetanic [Ca2+]i. It is concluded that not all fibers are dependent on the myoprotection of KATP channels and that the decrease in unstimulated force by verapamil reported in a previous studies in glibenclamide-exposed fibers is due to a reduction in Ca2+ load by reducing Ca2+ influx through CaV1.1 channels between and during contractions.

Sarcolemmal Biomechanics and Excitability in Malformed Muscle Fibers of Dystrophic Mice

Background: Duchenne muscular dystrophy (DMD), the most common and severe dystrophy, is caused by the absence of dystrophin. Muscle weakness and fragility (i.e. increased susceptibility to damage) are presumably due to structural weakness of the myofiber cytoskeleton, but recent studies suggest that malformed/split myofibers in dystrophic muscle may also play a role. We have previously studied the biomechanical properties of the sarcolemma in single myofibers isolated mechanically from extensor digitorum longus (EDL) muscles in wild-type (WT) and dystrophic (mdx, mouse model for DMD) mice. PURPOSE: We use similar biomechanical methods on enzymatically-dissociated myofibers (both normal and malformed) from the flexor digitorum brevis muscle (FDB) of WT and mdx mice.Methods: FDB muscles were enzymatically-dissociated and plated on specialized coverslips. Suction pressures (P) applied through a pipette to the membrane generated a bleb, which increased in height with increasing P. Larger increases in P ruptured the connections between the sarcolemma and myofibrils (speration P, or SP) and eventually caused the sarcolemma to burst. We also examined excitability using high-speed confocal microscopy and the voltage-sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate to assess the action potential (AP).Results: The mechanical results from dissociated FDB myofibers match findings from dissected EDL myofibers, but SP was up to 14-fold higher in the FDB than EDL. SP was 27% lower in mdx myofibers and 50% less in branches of split mdx fibers compared to the trunk. AP amplitude was not altered in between groups, but this work is ongoing.Conclusions: Data indicate a reduction in muscle stiffness, increased sarcolemmal deformability and instability in mdx muscle. This approach corroborates the labor-intensive data obtained from single fiber dissection and allows a facile high throughput model. Findings suggest mechanical differences due to altered morphology, despite comparable excitability.

A Novel MitoTimer Reporter Gene for Mitochondrial Content, Structure, Stress, and Damage in Vivo

Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.

Not all skeletal muscle fibers depend on the myoprotection of KATP channels during fatigue (1102.15)

During fatigue, KATP channels are crucial in reducing action potential amplitude to eventually lower Ca2+ release by sarcoplasmic reticulum, which helps preserve ATP during a metabolic stress by decreasing Ca2+ ATPase and myosin ATPase activity. In the absence of KATP channel activity, skeletal muscle muscles suffer major contractile dysfunctions, which lead to faster fatigue rate because of fiber damage. In our studies at 37°C, we have observed a tremendous variability in the fatigue kinetics among mouse single FDB muscle fibers. Under control conditions, such variability is expected as fatigue resistance is in the order of type I&gt;IIA&gt;IIX&gt;IIB (note IIB fibers are not present in FDB). However, we also observed a large variability in fatigue kinetics when KATP channels are completely blocked with 10 µM glibenclamide. Our results suggest that the importance of the KATP channel for myoprotection during fatigue is in the order of type IIX &gt; IIA &gt; I types, an order similar to the difference in KATP channel content between fiber types; i.e., the KATP channels are the most important in glycolytic fibers and the least important in oxidative fibers.Grant Funding Source: NSERC

Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca2+signals and an IL-6 autocrine loop

Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop.

Temperature Effects on the Myoplasmic Free Ca2+ Transients in FDB Muscle Fibers of the Mouse

The fast, low affinity, Ca2+ dye Mag-Fluo-4 loaded in its AM moiety, in mouse FDB fast-twitch fibers has allowed to study, with high temporal resolution, the decay phase of single or tetanic (350 ms-100 Hz), Ca2+ transients (Calderon et al. JMRCM 2009;30). At 23°C, a double exponential function with time constants (always expressed in ms), τ1=1.4±0.1, τ2=15.3±1.3 (n=20), and τ1=23.4±2.5, τ2=594±102.4 (n=13), respectively, describe the decay phase of single and tetanic transients. These time constants have been associated with Ca2+ binding to parvalbumin, and SERCA, respectively. We tested for differential effect of tetanic Ca2+ loads and temperature on the putative Ca2+ removal mechanisms. Increasing temperature, from 23 to 33°C, reversibly diminishes the single transient amplitude (ΔF/F), by 31%, and increases the base line by 32% (n=25). At 33°C, in 15 out of 20 fibers, the decay of single transients followed a single exponential, τ=4.7±0.7, while in the other 5 fibers, two exponentials provided a better fit, τ1=0.6± 0.1 and τ2=5.1±0.4; two exponentials τ1=10.1±1.8 and τ2=394.9±51.2 (n=13), provided the best fit for tetanic decay. The results show that the two time constants, that describe Ca2+ single and tetanic responses decay, are differentially affected by temperature and by tetani imposed Ca2+ loads. The fast decay components of single and tetanic responses, represented by τ1, and associated with parvalbumin Ca2+ binding, are affected similarly by Ca2+ load (17x increase) or by temperature (2.3x increase for 10°C increase). The slower components of the single and tetanic responses, represented by τ2, and associated with SERCA operation, are differentially affected by a 10°C temperature raise (3.1x vs 1.5x) and by the tetanic Ca2+ load (39x vs 77x) respectively.

Optogenetic Control of Skeletal Muscle Excitability

We assessed the feasibility of using light-activated channels and pumps to modulate the excitability of adult skeletal muscle fibers. Fibers were enzymatically isolated from FDB muscles of the mouse and studied under current- or voltage-clamp conditions using a two-microelectrode amplifier. Fibers were kept in Tyrode at a resting (or holding) potential of −90mV. Contraction was arrested by adding 40mM EGTA to the pipettes' solution. Plasmids coding channelrhodopsin-2-EYFP [CHR2; Addgene] and archaerhodopsin-3-ECFP [ARCH; subcloned from pFCK-ARCH-CFP (Addgene) into pECFPN1], were transfected into FDB fibers via in vivo electroporation. Expression was evaluated 7-10 days later. CHR2 and ARCH were excited with the 488nm and 514nm laser lines from an argon laser. Light power was controlled with an acousto-optic modulator, and focused onto selected areas (10-20μm) of the fibers via optic fibers. TPLSM imaging showed that CHR2 and ARCH were targeted to t-tubules and surface membranes. In fibers expressing CHR2, 1-3ms light pulses of increasing power elicited graded transient depolarizations until an action potential (AP) was triggered. The amplitude and FDHM of light-triggered APs were similar to those in response to current pulses. At all powers used, illuminating non-transfected fibers had no effect. Repetitive (<10Hz) pulsed illuminations elicited trains of APs with no failures. Failures at higher frequencies could be reduced by increasing the muscle fiber's input resistance (e.g. by blocking the chloride conductance with 9-AC), and/or increasing the illumination power. In voltage-clamped fibers, 40ms light pulses elicited slowly decaying inward currents with amplitudes graded with the applied illumination power. When fibers expressing ARCH were illuminated (at 514nm) otherwise supra-threshold current pulses became ineffective to trigger APs. Our results demonstrate that optogenetic tools can be used to excite or depress the excitability of adult skeletal muscle fibers.Supported by NIH/NIAMS grants AR047664, AR041802, and AR054816.

P.5.1 Sarcolemmal repair and reorganization of microtubule

Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and LGMD2B, and is involved in sarcolemmal repair. Proteomics analyses have identified multiple dysferlin-binding partners including cytoskeletal proteins. Affixin, a binding partner of dysferlin, regulates cytoskeletal actin. We reported that affixin, β- and γ-actin accumulated at the sarcolemmal injury site of wild-type mice in response to membrane rapture. α-tubulin and microtubule-associated proteins have been reported as dysferlin binding partners, however, their involvement in sarcolemmal repair remains unclear. The purpose of this study is to examine involvement of microtubule in sarcolemmal repair. To clarify molecular behavior of α-tubulin in sarcolemmal repair, GFP2-tagged human α-tubulin was expressed in mouse FDB muscle (C57BL/6J and dysferlin-deficient SJL) by electroporation. Membrane wound-repair assay of single myofiber was performed using confocal microscope equipped two-photon laser. GFP2-α-tubulin expressed in wild-type mice was localized at sarcolemma and T-tubule, and observed as a striated pattern. This striation pattern of GFP2-α-tubulin was slightly disorganized in SJL mice. After sarcolemmal injury, GFP2-α-tubulin in wild-type mice was disassembled and accumulates slower compared to GFP-β- and γ -actin. In SJL mice, GFP2-α-tubulin around the injury site was depolymerized, however, accumulation of GFP2-α-tubulin was not observed. α-Tubulin may also have a role in sarcolemmal repair.

Methyl‐β‐cyclodextrin increases GLUT4‐mediated glucose transport in skeletal muscle fibers from insulin resistant mice

Insulin stimulates glucose uptake in skeletal muscle fibers by promoting the translocation of Glut4 glucose transporters to the transverse tubule (TT) system. Cholesterol is highly enriched in TT, raising the possibility that modifying TT cholesterol content may affect insulin‐induced glucose transport. By feeding mice a high fat diet (HFD), we increased TT cholesterol content and modified insulin‐induced glucose uptake. Male C57BL/6J mice were fed a normal or a HFD for 8 weeks. Cholesterol levels in triads from HFD‐fed mice were 30% higher than in controls whereas insulin‐stimulated glucose uptake in fibers from HFD‐fed mice was lower than in controls. In FDB muscle, pre‐incubation with low doses of Methyl‐β‐cyclodextrin (MβCD), reported to extract membrane cholesterol, decreased Akt phosphorylation without altering the activity of AMPK, and increased both basal and insulin‐induced glucose uptake in muscle fibers from controls or HFD‐fed mice. MβCD restored insulin sensitivity in HFD‐fed mice, even after Akt or CaMKII inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, inhibited the effect of MβCD. Our findings suggest that TT cholesterol content has a critical role in GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may be a strategy to improve insulin resistance. Supported by ACT1111 and FONDECYT 3110105.

PI3‐kinase is involved in plasticity of adult myofibers acting as a nodal step that originates divergent pathways for gene expression

In skeletal myofibers, transcription of a specific group of genes is regulated by different stimulation frequencies, process known as muscle plasticity. We described IP3‐dependent calcium signals after slow frequency stimulation which were dependent on Cav1.1 and ATP release through pannexin channels. Phosphatidyl inositol 3 kinase (PI3K) is one of the downstream proteins involved in activation of this signaling pathway. Here we present a central role for PI3K in muscle plasticity. We studied expression of sarcomeric (slow and fast troponin I) and metabolic (citrate synthase and enolase) genes, ATP release and IP3 production in adult myofibers from murine FDB muscle. We found a biphasic ATP release and intracellular IP3 increments after 20 Hz stimulation, as well as an increase in TnI‐slow and a decrease in TnI‐fast mRNA levels. In metabolic genes, we observed an increase in citrate synthase and a decrease in enolase mRNA levels. These events were dependent on Cav1.1, pannexin and PI3K. Cav1.1 and Pannexin co‐localize at the T‐tubule membrane. Nevertheless, when fibers were treated with an IP3R blocker (5 μM xestospongin B), we observed an inhibition of TnI gene expression but not of metabolic genes. At 90 Hz, gene expression appears to be independent of IP3‐driven calcium movements. These results show PI3K as a nodal step for plasticity‐related gene expression where signals originated by low frequency stimulation diverge to different routes that control the expression of some sarcomeric genes on one hand and of some metabolic genes on the other.FONDECYT‐1110467/ACT‐1111, AT‐24110054

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.