Muscle Cell Death Research Articles

Modulating the MicroRNArchitecture of an Aging Aorta

See related article, pages 1115–1119 The 17th century British physician Thomas Sydenham is credited with saying, “A man is as old as his arteries.” Indeed, aging is associated with myriad structural and functional changes in the cardiovascular system.1 In particular, thoracic aortic aneurysms are commonly found in people during the sixth or seventh decade of life and at a frequency of nearly 1 in 16 000 people per year.2 An aneurysm is a balloon-like bulge that is characterized by thinning and weakness of the vascular wall. Enlarged aortic aneurysms are prone to rupture and often result in death. Maintaining the structural integrity of the aorta is critical to its function and is dictated by the biomechanical properties of the 3 different wall layers: the adventitia, media, and intima. Smooth muscle and extracellular matrix within the media are the largest components of the aortic vascular wall. Smooth muscle cell death or dysfunction and perturbations in extracellular matrix deposition are often associated with aging and aneurysms.3 Advances in surgical procedures to treat aortic aneurysms increase life expectancy and reduce morbidity in the young population but are more risky in the elderly because of cardiovascular comorbidity and surgery-related lethality. Therefore, a comprehensive understanding of the changes in molecular signaling events during aortic aging is necessary to develop novel therapeutics for preventing and treating aneurysm formation. Recent studies have pointed to microRNAs (miRNAs) as key regulators of a variety of cardiovascular disorders and biomarkers of disease progression.4,5 miRNAs negatively regulate target mRNAs through Watson-Crick base pairing with complementary sequences in 3′ untranslated regions of …

Protective effects of ginger supercritical extract against oxidative damage in L6 muscle cells

Antioxidative effect of ginger supercritical extracts in L6 muscle cells was assessed. Ginger supercritical extract potently suppressed hydrogen peroxide-induced L6 muscle cell death and ATP reduction as compared to ginger ethanol extract. Results suggest the intake of ginger supercritical extracts may effectively protect muscle cells against damage induced by radically increased reactive oxygen species due to strenuous exercise.

Effects of Coffee on Physical Performance in Mice

In this study, coffee was shown to effectively inhibit L6 muscle cell death and ATP reduction induced by hydrogen peroxide damage. Additionally, two weeks of oral administration of 7 mg/kg coffee extracts to mice resulted in a 33% increase in treadmill running time relative to that seen in the distilled water administered group.<BR> Blood analysis showed decreased lactate content, which was increased by exercise. Thus, these data suggest that coffee intake may enhance exercise capacity and inhibit damage due to excessive exercise.<BR>

P4.50 Mitochondrial therapy with Cyclosporine A in patients with Ullrich Congenital Muscular Dystrophy

We are reporting the results of a prospective long-term open therapeutic trial to test the effect on muscle strength and respiratory function of CsA in six patients with UCMD. Three of these patients have been originally enrolled in the pilot trial(Merlini L et al. Proc Natl Acad Sci USA 2008;105:5225–9) with a biological endpoint in which treatment with CsA favorably affected mitochondrial function in ColVI myopathies in vivo, and dramatically decreased the frequency of muscle cell death in the patients.

An Abiotic Fluorescent Probe for Cardiac Troponin I

The first ratiometric fluorescent reporter was designed for the detection of cardiac troponin I (cTnI), a key protein elicited during cardiac muscle cell death. In designing this abiotic fluorescent probe, docking simulation studies were performed to predict the probe/protein interactions along the solvent exposed regions of cTnI. Simple cuvette titration experiments in aqueous buffered solution indicate remarkable selectivity for cardiac troponin in the clinically relevant nM region versus skeletal troponin.

Higher thyroid hormone receptor expression correlates with short larval periods in spadefoot toads and increases metamorphic rate

Spadefoot toad species display extreme variation in larval period duration, due in part to evolution of thyroid hormone (TH) physiology. Specifically, desert species with short larval periods have higher tail tissue content of TH and exhibit increased responsiveness to TH. To address the molecular basis of larval period differences, we examined TH receptor (TR) expression across species. Based on the dual function model for the role of TR in development, we hypothesized that desert spadefoot species with short larval periods would have (1) late onset of TR expression prior to the production of endogenous TH and (2) higher TR levels when endogenous TH becomes available. To test these hypotheses, we cloned fragments of TRα and TRβ genes from the desert spadefoot toads Scaphiopus couchii and Spea multiplicata and their non-desert relative Pelobates cultripes and measured their mRNA levels in tails using quantitative PCR in the absence (premetamorphosis) or presence (natural metamorphosis) of TH. All species express TRα and TRβ from the earliest stages measured (from just after hatching), but S. couchii, which has the shortest larval period, had more TRα throughout development compared to P. cultripes, which has the longest larval period. TRβ mRNA levels were similar across species. Exogenous T3 treatment induced faster TH-response gene expression kinetics in S. couchii compared to the other species, consistent with its higher TRα mRNA expression and indicative of a functional consequence of more TRα activity at the molecular level. To directly test whether higher TRα expression may contribute to shorter larval periods, we overexpressed TRα via plasmid injection into tail muscle cells of the model frog Xenopus laevis and found an increased rate of muscle cell death in response to TH. These results suggest that increased TRα expression evolved in S. couchii and contribute to its higher metamorphic rates.

Labedipinedilol-A prevents lysophosphatidylcholine-induced vascular smooth muscle cell death through reducing reactive oxygen species production and anti-apoptosis

Objective : Labedipinedilol-A, a novel calcium antagonist, has been previously demonstrated to have pleiotropic protective effects in the cardiovascular system. This study aimed to investigate its cytoprotective effects in rat vascular smooth muscle cells (VSMCs) treated with lysophosphatidylcholine (lysoPC), a key lipid component mediating atherogenesis. Methods and results : VSMCs were incubated with lysoPC with or without labedipinedilol-A pretreatment to determine its effects on lysoPC-induced cell death, Ca 2+ influx, oxidative stress, MAPK signaling and apoptosis. Labedipinedilol-A attenuated lysoPC-induced cell death and Ca 2+ influx. It also reduced reactive oxygen species (ROS) production evoked by lysoPC and down-regulated expressions of NAD(P)H oxidase subunits, Nox1 and Rac1. Moreover, it inhibited lysoPC-induced phosphorylation of MAPK including ERK1/2, JNK, and p38. It mitigated the dissipation of mitochondrial transmembrane potential induced by lysoPC. Lastly, labedipinedilol-A inhibited lysoPC-induced apoptosis with attenuation of caspase-3/-9 activations and modulation of Bax/Bcl-2 protein expressions. Conclusion : Labedipinedilol-A can suppress lysoPC-induced VSMCs death via reducing ROS production and anti-apoptosis. These protective effects are potentially mediated through the inhibition of Ca 2+ influx, down-regulation of the NAD(P)H oxidase subunits (Nox1/Rac1) and MAPK signaling, and attenuation of mitochondrial depolarization. Thus, labedipinedilol-A may have a valuable role in the preventing atherosclerosis associated with hyperlipidemia.

A link between smooth muscle cell death and extracellular matrix degradation during vascular atrophy

High blood flow induces neointimal atrophy in polytetrafluoroethylene (PTFE) aortoiliac grafts and a tight external PTFE wrap of the iliac artery induces medial atrophy. In both nonhuman primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins at ≤4 days. We hypothesized that matrix loss would be linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy. DNA microarray analysis (Sentrix Human Ref 8; Illumina, San Diego, Calif; ∼23,000 genes) was performed on arterial tissue from the wrap model (n = 9) and graft neointima from the graft model (n = 5) 1 day after wrapping or the switch to high flow, respectively. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied after Fas ligand-induced cell death in cultured smooth muscle cells and organ cultured arteries. Microarray analysis showed 15 genes were regulated in the same direction in both atrophy models: 9 upregulated and 6 downregulated. Seven of nine upregulated genes were confirmed by qRT-PCR in both models. Upregulated genes included the ECM-degrading enzymes ADAMTS4, tissue plasminogen activator (PLAT), and hyaluronidase 2; possible growth regulatory factors, including chromosome 8 open reading frame 4 and leucine-rich repeat family containing 8; a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1); a dead cell removal factor (ficolin 3); and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five downregulated genes were confirmed but only in one or the other model. Of the seven upregulated genes, ADAMTS4, PLAT, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 were also upregulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with Z-VAD inhibited FasL-mediated cell death, but not gene induction. Seven gene products were upregulated in two distinctly different in vivo nonhuman primate vascular atrophy models. Induction of cell death by FasL in vitro induced six of these genes, including the ECM-degrading factors ADAMTS4, hyaluronidase 2, and PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

Targeting Inflammation in Cardiovascular Diseases. Still a Neglected field?

Prevention and treatment of atherosclerosis is still a clinical challenge in the cardiovascular medicine. The classical belief that atherosclerotic lesion development solely depends on lipid deposition has been replaced by the current concept that activation of immune and inflammatory responses plays a central role in plaque initiation and progression. In this review we summarize studies on human and genetically modified animals describing a finite number of cellular and molecular mechanisms that underlie immunoinflammation in atherosclerotic plaques. We focus on the pro- and antiinflammatory mediators activated during atherogenesis and the intracellular signaling pathways regulating these events. Besides the advances on established pharmacological agents, we propose potential strategies for reduction/stabilization of atherosclerotic plaques based on the clinical data in inflammatory-associated pathologies and on the encouraging studies in experimental models of atherosclerosis. We emphasize the potential of such novel inhibitors comprising receptor antagonists, neutralizing antibodies, kinase inhibitors, peptide-based technologies, and chemicals as emerging antiinflammatory strategies for the treatment of atherosclerotic disease complications.

Foxo1 nuclear‐cytoplasmic movement in living skeletal muscle

The transcription factor Foxo1 is integral to the regulation of expression of proteins which promote muscle atrophy. Phosphorylation of Foxo1 causes its translocation to the cytoplasm and thus prevents Foxo1-DNA binding and consequent transcription of genes that cause muscle atrophy and cell death. Thus, phosphorylation of Foxo1 leads to cell survival and muscle hypertrophy. Maintenance of Foxo1 phosphorylation and its resulting cytoplasmic retention could be used to suppress muscle atrophy and thereby shift the atrophy/hypertrophy balance in favor of hypertrophy. Akt and serum- and glucocorticoid-inducible kinase (SGK) are important regulators of the phosphorylation status of Foxo1. These pathways have been well characterized and the effects on Foxo1 localization have been reported. However, the mechanisms which regulate nuclear influx and nuclear efflux have not been separately evaluated. Here, we determine the effects of Akt kinase activity specifically on nuclear influx of Foxo1. To accomplish th/is goal, we quantified nuclear and cytoplasmic levels of adenovirally expressed Foxo1-GFP in cultured flexor digitorum brevis. The nuclear influx during treatment with kinase inhibitor Akt IV alone and in combination with the nuclear efflux inhibitor lemptomycin B provides insight into the activity of Akt as a Foxo1 kinase. Surprisingly, Akt inhibition reveals Akt to have little effect on the rate of nuclear influx of Foxo1. To evaluate translocation of endogenous Foxo1 in a similar manner immunocytochemistry and western blotting techniques were used. These results indicate that Akt either phosphorylates Foxo1 primarily in the nucleus and not in the cytoplasm, or that there is a mechanism for cytoplasmic phosphorylation of Foxo1 other than via Akt. Supported by training grants T32 AR0075592 and T32 HL072751 and research grant R01 AR056477.

Foxo1 Nuclear-Cytoplasmic Movement in Live Skeletal Muscle Fiber

The transcription factor Foxo1 is integral to the regulation of expression of proteins which promote muscle atrophy. Phosphorylation of Foxo1 causes its translocation to the cytoplasm and thus prevents Foxo1-DNA binding and consequent transcription of genes that cause muscle atrophy and cell death. Thus, phosphorylation of Foxo1 leads to cell survival and muscle hypertrophy. Maintenance of Foxo1 phosphorylation and its resulting cytoplasmic retention could be used to suppress muscle atrophy and thereby shift the atrophy/hypertrophy balance in favor of hypertrophy. This in turn may be utilized to develop therapeutic avenues in treatment of muscle wasting as seen in patients with denervation, age-related muscle wasting, and AIDS. Akt and serum- and glucocorticoid-inducible kinase (SGK) are important regulators of the phosphorylation status of Foxo1. These pathways have been well characterized and the effects on Foxo1 localization have been reported. However, the mechanisms which regulate nuclear influx and nuclear efflux have not been separately evaluated. Here, we determine the effects of Akt kinase activity specifically on nuclear influx of Foxo1. To accomplish this goal, we quantified nuclear and cytoplasmic levels of adenovirally expressed Foxo1-GFP in cultured flexor digitorum brevis. The nuclear influx during treatment with kinase inhibitor Akt IV alone and in combination with the nuclear efflux inhibitor lemptomycin B provides insight into the activity of Akt as a Foxo1 kinase. Surprisingly, Akt inhibition reveals Akt to have little effect on the rate of nuclear influx of Foxo1. To evaluate translocation of endogenous Foxo1 in a similar manner immunocytochemistry and western blotting techniques were used. These results indicate that Akt either phosphorylates Foxo1 primarily in the nucleus and not in the cytoplasm, or that there is a mechanism for cytoplasmic phosphorylation of Foxo1 other than via Akt. Supported by NIH-NIAMS Grants R01-AR056477 and T32-AR007592.

Myotube vs myoblast sensitivity to apoptosis induction by chemical triggers

Apoptosis is necessary for skeletal muscle tissue development and homeostasis, where it plays a multifaceted role. Muscle cell death increase is associated to disuse, denervation and to several muscle myopathies (Siu et al., Life Sci, 2009), while apoptosis resistance is typical of muscle differentiation (Xiao et al., Apoptosis, 2011) The aim of this study is to evaluate in vitro apoptotic response in differentiated myotubes exposed to chemical triggers and to compare apoptosis susceptibility to that of proliferant myoblasts. (Salucci et al., Micron, 2010). C2C12 myotubes, differentiated as previously described (Curci et al, Micron, 2008), were exposed to etoposide (ETO), cisplatin (CP), hydrogen peroxide (H2O2) and staurosporine (SP) and apoptosis was evaluated by flow cytometry and transmission electron microscopy. Flow cytometry analysis revealed a certain subdiploid peak after ETO and CP exposure, lower, however, than that evidenced in myoblasts. Differently, DNA cleavage after H2O2 or SP treatments, was absent, in contrast with undifferentiated cells. At ultrastructural analysis, characteristic chromatin condensation was observed after ETO and CP treatments, but few apoptotic cells were detected, differently from what evidenced at undifferentiated stage. Moreover, autophagic cell death appeared. Cells exposed to ETO presented a particular behaviour: myonuclei with condensed chromatin coexisted with normal nuclei, in the same myotube, as previously demonstrated in UVB-induced apoptosis (D’Emilio et al., Histol Histopathol, 2010). On the contrary, and differently from myoblasts, apoptosis was absent in myotubes after H2O2 exposure, being necrosis and autophagy the most common cell death processes. After SP treatment all cells showed features of secondary necrosis, a response present also in myoblasts. In addition, autophagic vacuoles were observed, exclusively in differentiated cells. These results show a certain resistance to apoptosis in myotubes, if compared to myoblasts, that could be associated to an increase in autophagic cell death and in a probable upregulation of anti-apoptotic pathways. Particularly intriguing appears the response to ETO treatment where apoptosis could be correlated to a localized gene expression within each myonuclear domain, suggesting that this process works to selectively eliminate the designated nuclei in multinucleated skeletal muscle fibers.

Effect of Metformin Treatment on Skeletal Muscle Cell Death Pathways in Huntington's Disease

Effect of Metformin Treatment on Skeletal Muscle Cell Death Pathways in Huntington's Disease

Autophagy plays a protective role in free cholesterol overload-induced death of smooth muscle cells

Smooth muscle cells (SMC) make up most of the vascular system. In advanced atherosclerotic plaques, dying SMCs undergo a complex death mode. In the present study, we examined the activation of autophagy in SMCs overloaded with excess free cholesterol (FC) and investigated the possible role which autophagy plays during the FC-induced cell death. After incubation with excess FC, a robust expression of autophagic vacuoles (AV) was detected using both fluorescence microscopy and transmission electron microscopy (TEM). The results revealed that FC induced a time-dependent upregulation of microtubule-associated protein-1 light chain 3-II (LC3-II). Inhibition of autophagy by 3-methyladenine (3-MA) enhanced both cell apoptosis and necrosis, while on the contrary, rapamycin inhibited cell death following cholesterol application. Furthermore, the impact of the colocalization of fragmented mitochondria with AVs was observed after cholesterol treatment. Our results also revealed that the modulation of autophagy directly influenced the cellular organellar stress. In conclusion, our findings demonstrated that excess FC induced the activation of autophagy in SMCs as a cellular defense mechanism, possibly through the degradation of dysfunctional organelles such as mitochondria and endoplasmic reticulum.

MOLECULAR AND BIOLOGICAL FACTORS AFFECTING SKELETAL MUSCLE CELLS AFTER SLAUGHTERING AND THEIR IMPACT ON MEAT QUALITY: A MINI REVIEW

ABSTRACT The postmortem factors like rate of glycolysis and muscle shortening have a major role on meat characteristics and these have been efficiently manipulated by methods like chilling regime, electrical stimulation and different hanging. These methods have been optimized with time leaving a little scope for further improvement. During the harvesting process of meat there is a generalized cell death often referred to as necrosis or apoptosis. This process of cell death has been well studied with respect to various physiological functions in vivo but its role in postmortem meat quality has received little attention. It is mainly regulated by caspases and also probably by calpains, cathepsins, proteasomes, lysosomal enzymes and other molecules, antioxidants and stress proteins. The term apoptosis appears to be more appropriate as evident by involvement of the enzyme systems and cell shrinkage. Further, examination of postmortem events effecting meat quality and correlate them to understand the cell death process may reveal the possible involvement of both apoptosis and necrosis processes, one preceding the other. This article is an attempt to review the apoptotic process of various cell lines and possible implication for muscle cells after slaughter and meat quality control.PRACTICAL APPLICATIONSUnderstanding the intricate biochemical mechanisms governing cell death processes after slaughter may help us provide better solutions for preslaughter animal handling and postslaughter interventions to manage meat toughness. Investigations on these tracks would distinguish muscle cell death processes after slaughter from apoptosis or necrosis, and consequently may lead to enhanced technological meat quality as available literature suggests that a number of molecules such as caspase family are likely to be involved in cell death process after slaughter and also in meat tenderization.

Satellite cell characterization from aging human muscle

Objectives: Satellite cells (SCs) are skeletal muscle progenitor cells located between the basal lamina and the sarcolemma of muscle fibers. They are responsible for muscle growth and repair. In humans, aging results in the depletion of the SC population and in its proliferative activity, but not in its function. It has not yet been determined whether under conditions of massive muscle fiber death in vivo, the regenerative potential of SCs is totally or partially compromised in old muscle. No studies have yet tested whether advanced age is a factor that restrains the response of SCs to muscle denervation in humans; this is also due to difficulties in the isolation and in the culture of SCs from a small human surgery fragment. The aim of this study was to study in depth muscle regeneration analysing the SC ability of SCs to proliferate and differentiate in aging human patients.Methods: In order to study in more detail the molecular mechanism, the proliferative and differentiative ability of aging SCs, we isolated SCs from aging human muscle biopsies and analysed their morphology by transmission electron microscopy and immunocytochemical analysis (antibodies against desmin, N-CAM and M-cadherin) and their capacity to grow and to expand in vitro. Moreover, in order to evaluate gene expression of myogenic regulatory factors Myf5, MyoD and myogenin (Myf4), RT-PCR was performed.Results and discussion: SCs isolated from aging human muscle biopsies and plated into favorable proliferation and differentiation conditions were able to proceed through the myogenic program and actively form myotubes, although taking longer than the young control sample. The RT-PCR analysis together with the ultrastructural SC features showed that the myogenic potential seemed to be compromised during the aging human muscle proliferation in vitro.

Osteo-Progenitors in Vascular Calcification

Vascular calcification has been associated with the incidence of cardiovascular events and thus there has been interest in better understanding its pathogenesis. Early theories considered vascular calcification to be a passive process which occurred as a non-specific response to tissue injury or necrosis. More recent theories propose vascular calcification results from loss of molecular inhibitors or via an active cell mediated process. The origin of the cells responsible for vascular calcification is controversial and may vary in different sites and patients. Calcification has been reported as result of apoptosis or death of vascular smooth muscle cells for example. One novel source of cells controlling vascular calcification is from the bone marrow. A circulating immature bone marrow derived population has been identified and a small subset of this bone marrow population has been reported to possess bone forming properties in vitro and hence termed osteo-progenitors. This article reviews evidence supporting the contribution of these naive bone marrow derived circulating osteo-progenitor cells in vascular calcification.

Naringenin Inhibits Neointimal Hyperplasia Following Arterial Reconstruction With Interpositional Vein Graft

Vessels respond to injury by a healing process that includes the development of neointima. Stenosis secondary to neointima formation is the main cause of failure following arterial reconstructions. Vessel wall homeostasis is regulated by proinflammatory cytokines that affect smooth muscle cell proliferation, growth, migration, and death. We assessed the hypothesis that naringenin, a flavinoid possessing anti-inflammatory, antioxidant, and antiproliferative activities, reduces neointimal hyperplasia (NIH) following vascular injury.Arterial injury was created by interposition grafting of autologous right superficial epigastric vein graft into the right femoral artery (FA) in 48 male Sprague-Dawley rats. Following injury, the rats were divided into 4 groups (n = 12). Two groups were treated with naringenin (100 mg/kg intraperitoneal q daily) for 2 and 4 weeks each while 2 control groups received normal saline for the same durations. For Sham group (n = 10), the FA and vein were isolated without any additional procedure. Rats were killed at the end of treatment regimen in all groups, and FAs were harvested. Thickness of intima was measured in histologic sections, and levels of platelet derived growth factor (PDGF)-BB, TNFalpha, and Ki67 labeling index (Ki67 LI) were quantified in immunohistochemical analyses to assess the amount of NIH and mechanisms underlying its formation.Although there was no significant difference between the groups at 2 weeks, neointima thickness was lower in the naringenin treated group at 4 weeks (23.7 +/- 2.3 vs. 35.6 +/- 2.6 microm in control group; P < 0.001). The levels of PDGF-BB, and TNFalpha were lower in naringenin treated groups at both 2 weeks (PDGF-BB [0.21% +/- 0.03% versus 0.39% +/- 0.05% in control group, P < 0.001), TNFalpha (21.2% +/- 0.8% vs. 36.1% +/- 1.9% in control group, P < 0.001]) and 4 weeks (PDGF-BB [0.25% +/- 0.03% vs. 0.57% +/- 0.09% in control group, P < 0.001], TNFalpha [25.5% +/- 1.8% vs. 45.0% +/- 2.9% in control group, P < 0.001]). Ki67 LI was lower in naringenin treated groups at 2 weeks (13.9% +/- 2.8% vs. 18.7% +/- 3.7% in control group, P < 0.05), and at 4 weeks (17.5% +/- 2.6% vs. 31.1% +/- 4.7% in control group, P < 0.001), indicating a lower level of cellular proliferation.Naringenin reduces NIH following arterial reconstruction. This may be mediated by a decrease in PDGF-BB and TNFalpha levels and the resulting down-regulation of smooth muscle cells' migration and proliferation.

Morphological and biochemical patterns in skeletal muscle apoptosis.

Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.

Impaired Skeletal Muscle Repair after Ischemia-Reperfusion Injury in Mice

Ischemia/reperfusion (IR) injury can induce skeletal muscle fibre death and subsequent regeneration. By 14 days, absolute and specific maximal forces and fatigue resistance in ischemic/reperfused soleus muscles were still reduced (−89%, −81%, and −75%, resp.) as compared to control muscles (P < .05). The decrease of these parameters in ischemic/reperfused muscle was much greater than that of myotoxic injured muscles (−12%, −11%, and −19%; P < .05). In addition, at 14 days ischemic/reperfused muscle structure was still abnormal, showing small muscle fibres expressing neonatal myosin heavy chain and large necrotic muscle fibres that were not observed in myotoxin treated muscles. By 56 days, in contrast to myotoxin treated muscles, specific maximal force and muscle weight of the ischemic/reperfused muscles did not fully recover (P < .05). This differential recovery between ischemic/reperfused and myotoxin treated muscles was not related to the differences in the initial cell death, loss of satellite cells after injury, expression of growth factors (IGF1, IGF2..), or capillary density in regenerating muscles. In conclusion, our results demonstrate that IR injury in mice induces long term detrimental effects in skeletal muscles and that the recovery following IR injury was delayed for yet unknown reasons as compared to myotoxic injury.