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

Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/β-catenin pathway

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ∼25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1. We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt/ β-Catenin pathway

Store-Operated Ca2+ 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 Ca2+ to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca2+ 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 Ca2+ to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca2+ release channel-mediated Ca2+ 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 Ca2+ entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle

Hyperthermia Induces Functional and Molecular Modifications in Cardiac, Smooth and Skeletal Muscle Cells

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionHyperthermia is used for the treatment of a number of diseases, including muscle injuries, inflammations, tendinitis, and osteoarticular disorder. More recently, hyperthermia has been used as an adjuvant in cancer treatment. Only two studies have shown that hyperthermia leads to hypertrophy in in-vitro models of cardiac and skeletal muscle cells. Functional, biochemical and molecular mechanisms of hyperthermia-induced hypertrophy in muscles remain largely undiscovered. We investigated the effects of mild heat shock (HS) on C2C12 skeletal, HL-1 cardiac and AR-75 smooth muscle cells. Mild HS (20 min 43ºC) induced increases in the cell area in all muscle cells tested. C2C12 cells are a well-accepted model of skeletal muscle fibers, and were selected for complementary studies. First, to biochemically confirm an increase in protein synthesis we measured and found an increase of ~6% in total protein content 24 hrs after HS. Second, we examined potential modifications in calcium (Ca) homeostasis regulation by measuring intracellular Ca. We detected a lower resting level of intracellular Ca and smaller and longer caffeine-induced Ca transients in C2C12 muscle cells 24 hrs after HS. Next, to search for molecular mechanisms involved with HS-induced hypertrophy and calcium homeostasis modifications, mRNA from C2C12 muscle cells was analyzed at different time points after HS (0, 1, 2, and 24 hrs). We used an ABI Step One Plus RT2 PCR Array System and a custom-built 96 gene array. We report for the first time that the expression of key heat-shock, hypertrophy/ metabolic, and Ca+2 signaling genes were altered after HS. Hsp70 and Hsp72 genes were highly expressed (211-1829 fold change) after HS. Also, Myh7 (MHC-I), Myh6, Srf, Ppp3r1 and Pck1 were up-regulated by 2-6 fold change compared with control cells.. Furthermore, a reduction in the expression of RyR and Trdn genes was observed (2- 3.6 fold change) with an associated increase in the expression of IP3R genes (2-4 fold change). These results indicate that hyperthermia modulates not only heat-shock related and hypertrophy genes, but also genes involved with metabolism, apoptosis repression, calcium homeostasis and signaling, and cell homeostasis. Our studies offer an initial exploration of the functional, biochemical and molecular mechanisms that may help explain the beneficially adaptive effects of hyperthermia on muscle function. Our studies shall also prove useful for the refinement of a specific device (EM-Stim) to be employed for the treatment of muscle and bone diseases (See poster by Hatem et al). Importantly, our studies have potential translational applications. By learning how to more precisely use hyperthermia to control specific genes that can improve or treat muscle injuries, musculoskeletal, and cardiovascular diseases, the ensuing benefits shall be unmistakable. Our short and long-term goals are: i) optimize our protocols; ii) test HS in animal models; iii) manipulate expression of promising genes of interest in vitro and in in-vivo animal models; iv) initiate clinical studies to fully translate from the bench to the bed-side

Silicon Ions Enhance Myogenic Differentiation in C2C12 Skeletal Muscle Cells

The regeneration of bone and muscle tissue following musculoskeletal injuries is essential in sports medicine in order to restore function and prevent chronic musculoskeletal disorders related to physical inactivity. According to data collected by the American Academy of Orthopaedic Surgeons from 2012 to 2014, 8.3% (approximately 2.6 million people) of the adult population in the United States received treatment for musculoskeletal injuries, costing an aggregate total of $213 billion dollars. Recent regenerative musculoskeletal research suggests that the restoration of function and structure for normal physical activity is dependent on the synergy of regeneration processes found in bone and muscle tissue. Current treatments for severe musculoskeletal defects lack biocompatibility and rarely restore full function, so focus has shifted to regenerative biomaterials. Recent evidence indicates that bioactive gels and implants incorporating silicon (e.g. silicon ion, orthosilicate acid, amorphous silica) markedly increased osteogenesis in vitro, but little research has been conducted over the effect of these biomaterials on myogenesis. Establishing the existence of myogenic properties in silicon could lead to the development of a biomaterial that enhances the synergistic capacities of bone and muscle regeneration. PURPOSE: The purpose of this study was to investigate the effect of silicon ions on C2C12 skeletal muscle cells in vitro, in order to determine the regenerative viability of musculoskeletal gels and implants incorporating silicon. METHODS: In order to evaluate the effect of Silicon ions on myogenesis, in vitro cell culture studies were performed using C2C12 mouse myoblast cell lines. Cells were differentiated for four and seven days in media containing three different concentrations of silicon ions (0.1, 0.5, and 1.0 mM) and a silicon free control. Samples were immunohistochemically stained and imaged using a Zeiss fluorescent microscope. Cellsens software was used to determine total nuclei count and ImageJ was used to count the number of fused nuclei within myotubules. Rates of myogenic differentiation were determined based on fusion index, the percent of nuclei found within myotubes relative to the total nuclei. RESULTS: After four days there was a significant increase in fusion index (p\u3c0.001) in the 0.1mM group (41±3.4) compared to the control (31±4.2) . After seven days all three silicon groups exhibited significantly higher fusion indices (0.1mM 60±3.2, p\u3c0.001; 0.5mM 57.8±2.9, p\u3c0.01; 1.0mM 54.8±3.6 p\u3c0.05) compared to the control (50±2.4). Both four and seven day studies confirmed that the 0.1 Mm group had a markedly higher fusion index, indicating the highest rate of myogenic differentiation. CONCLUSION: Based on these results it can be concluded that silicon ions enhance myogenic differentiation. The myogenic potential of silicon ions exhibited by these results, combined with previously reported osteogenic effects, prompt further investigation into the potential of silicon-containing biomaterials to accelerate musculoskeletal regeneration, and decrease the risk of acute and chronic complications of injury

Cellular and Physiological Effects of Dietary Supplementation with β-Hydroxy-β-Methylbutyrate (HMB) and β-Alanine in Late Middle-Aged Mice.

There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C2C12 myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid β-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C2C12 myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C2C12 myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an ex vivo muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with β-alanine, or diet with HMB and β-alanine. In β-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus β-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm2, P = 0.021). Also in EDL muscle, β-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and β-alanine supplementation on skeletal muscle function in aging mice