Background Oxidative stress-mediated Ca2+/calmodulin-dependent protein kinase II (Ca MKII) phosphorylation of cardiac ion channels has emerged as a critical contributor to arrhythmogenesis in cardiac pathology. However, the link between mitochondrial-derived reactive oxygen species (md ROS ) and increased Ca MKII activity in the context of cardiac arrhythmias has not been fully elucidated and is difficult to establish experimentally. Methods and Results We hypothesize that pathological md ROS can cause erratic action potentials through the oxidation-dependent Ca MKII activation pathway. We further propose that Ca MKII -dependent phosphorylation of sarcolemmal slow Na+ channels alone is sufficient to elicit early afterdepolarizations. To test the hypotheses, we expanded our well-established guinea pig cardiomyocyte excitation- contraction coupling, mitochondrial energetics, and ROS - induced- ROS - release model by incorporating oxidative Ca MKII activation and Ca MKII -dependent Na+ channel phosphorylation in silico. Simulations show that md ROS mediated-Ca MKII activation elicits early afterdepolarizations by augmenting the late Na+ currents, which can be suppressed by blocking L-type Ca2+ channels or Na+/Ca2+ exchangers. Interestingly, we found that oxidative Ca MKII activation-induced early afterdepolarizations are sustained even after md ROS has returned to its physiological levels. Moreover, mitochondrial-targeting antioxidant treatment can suppress the early afterdepolarizations, but only if given in an appropriate time window. Incorporating concurrent md ROS -induced ryanodine receptors activation further exacerbates the proarrhythmogenic effect of oxidative Ca MKII activation. Conclusions We conclude that oxidative Ca MKII activation-dependent Na channel phosphorylation is a critical pathway in mitochondria-mediated cardiac arrhythmogenesis.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration due to mutations in the dystrophin gene. In spite of great advances in the design of curative treatments, most patients currently receive palliative therapies with steroid molecules such as prednisone or deflazacort thought to act through their immunosuppressive properties. These molecules only slightly slow down the progression of the disease and lead to severe side effects. Fundamental research is still needed to reveal the mechanisms involved in the disease that could be exploited as therapeutic targets. By studying a Caenorhabditis elegans model for DMD, we show here that dystrophin-dependent muscle degeneration is likely to be cell autonomous and affects the muscle cells the most involved in locomotion. We demonstrate that muscle degeneration is dependent on exercise and force production. Exhaustive studies by electron microscopy allowed establishing for the first time the chronology of subcellular events occurring during the entire process of muscle degeneration. This chronology highlighted the crucial role for dystrophin in stabilizing sarcomeric anchoring structures and the sarcolemma. Our results suggest that the disruption of sarcomeric anchoring structures and sarcolemma integrity, observed at the onset of the muscle degeneration process, triggers subcellular consequences that lead to muscle cell death. An ultra-structural analysis of muscle biopsies from DMD patients suggested that the chronology of subcellular events established in C. elegans models the pathogenesis in human. Finally, we found that the loss of sarcolemma integrity was greatly reduced after prednisone treatment suggesting a role for this molecule in plasma membrane stabilization.

We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca(2+) efflux pathways (SERCA, Na(+)/Ca(2+) exchange, and sarcolemmal Ca(2+) ATPase) between the dyad and bulk cytoplasm and the effect of adding exogenous Ca(2+) buffers (BAPTA or EGTA), which are used experimentally to differentially buffer Ca(2+) in the dyad and bulk cytoplasm, on cellular Ca(2+) cycling. Increasing the dyadic fraction of a particular Ca(2+) efflux pathway increases the amount of Ca(2+) removed by that pathway, with corresponding changes in Ca(2+) efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca(2+) removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca(2+)-dependent inactivation of the L-type Ca(2+) current, resulted from the buffers acting as slow and fast \"shuttles,\" respectively, removing Ca(2+) from the dyadic space. The data suggest that complex changes in dyadic Ca(2+) and cellular Ca(2+) cycling occur as a result of changes in the location of Ca(2+) removal pathways or the presence of exogenous Ca(2+) buffers, although changing the distribution of Ca(2+) efflux pathways has relatively small effects on the systolic Ca(2+) transient.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.

The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus/gracilis graft were included. Semitendinosus and gracilis tendons were stripped as grafts for the ACL reconstruction. The MTJ was isolated from the grafts and prepared for transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy. It was possible to isolate recognizable MTJ tissue from all 14 patients. TEM images displayed similarities to observations in animals: Sarcolemmal evaginations observed as finger-like processes from the tendon and endomysium surrounding the muscle fibers, with myofilaments extending from the final Z-line of the muscle fiber merging with the tendon tissue. The 3D reconstruction revealed that tendon made ridge-like protrusions, which interdigitiated with groove-like indentations in the muscle cell.

OBJECTIVE: The present study is undertaken to study the ultra structural features of muscle tissue in moderate and advanced stages of oral submucous fibrosis along with retrospective analysis of 80 cases of oral submucous fibrosis ( osmf) 0 during the period of year 2002 to 2005.
METHODS: Five patients with moderate and advanced stages of osmf0 were screened from outpatients department of oral diagnosis, sri Ramachandra dental college and hospital. After a detailed case history, they were subjected to incisional biopsy from an area of buccal mucosa with maximum palpable fibrotic bands.the specimens were cut into two halves, one half was fixed in 10% formalin for routine processing. Second half was fixed in 2.5% glutaraldehyde for electron microscopic examination.
RESULTS: Prospective study of muscle fibres from moderate and advanced stages of osmf0 revealed varying changes in high proportion of muscle fibres which includes, irregularity of surface of fibre,sarcolemmal foldings, reduplicated basement membrane, loss and alterations in the myofilaments, hypercontraction of myofibrils, Z line abnormalities, internal nucleus, autophagic vacuoles. These features are suggestive of muscle atrophy and necrosis.
CONCLUSIONS: Within the limitations of this study, it can be concluded that the ultra structural features In moderate and advanced stages of osmf0 were best studied. These muscle changes can be manifestation of disease, atrophy being secondary to the limited functional activity of the muscles which is brought about by fibrosis or it could be essential part of the disease process itself.

Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology.

Prospects of the use of flow cytometry analysis for investigation of forming K(+)- equilibrium membrane potential on the experimental model of myometrium plasma membrane vesicles in the presence of valinomycine using potential-sensitive probe of DiOC6(3). Transmembrane potential magnitude corresponds to magnitude by Nernst\'s equation. H2O2 and NO2-, probably, increase permeability of membrane for K+ and result in potential dissipation. Given effect is not shown for sodium nitroprusside. The obtained results confirm an assumption as to enhancing the passive transport for K+ through sarcolemma under the action of these substances, that can lead to membrane repolarisation and decline of the level of its excitability.

BACKGROUND: Sarcopenia, the age-related loss of muscle mass, may not be an isolated process but is associated with an increase in fat mass. The aim of this study was to estimate the mortality risk of sarcopenia in the presence or absence of obesity.
METHODS: Data are from 934 participants aged 65 years or older, enrolled in the \"Invecchiare in Chianti\" study, and followed for 6 years. At baseline, a peripheral quantitative computerized tomography (pQCT) scan was performed on all participants to evaluate the muscle density, and the muscular and fat cross-sectional areas of the calf. Walking speed was measured on a 7-m track. Cox proportional hazard models were performed to estimate the association of pQCT measures (per 1 standard deviation increase) with mortality.
RESULTS: Unadjusted analyses showed significant associations of muscle density (hazard ratio [HR] 0.78, 95% confidence interval [CI] 0.69-0.88), muscle area (HR 0.75, 95% CI 0.66-0.86), and fat area (HR 0.82, 95% CI 0.73-0.92) with mortality. After adjustment for potential confounders, no body composition parameter was significantly associated with mortality. Walking speed (used as a reference measure to verify whether the negative results were due to peculiarities of the study sample) confirmed its well-established association with mortality risk (HR 0.73, 95% CI 0.60-0.88). These results did not change after the analyses were stratified according to sarcopenia and body mass index groups, and restricted to participants with frailty or a high inflammatory profile.
CONCLUSIONS: Calf skeletal muscle and fat mass are not significant risk factors for mortality in community-dwelling older adults. Walking speed confirmed to be a powerful predictor of health-related events.

Patients with muscular dystrophy have abnormal cardiac function and decreased high-energy phosphate metabolism. Here, we have determined whether the 8 month old mdx mouse, an animal model of muscular dystrophy, also has abnormal cardiac function and energetics. In vivo cardiac MRI revealed 33% and 104% larger right ventricular end-diastolic and end-systolic volumes, respectively, and 17% lower right ventricular ejection fractions in mdx mice compared with controls. Evidence of left ventricular diastolic dysfunction included 18% lower peak filling rates in mdx mouse hearts. Abnormal cardiac function was accompanied by necrosis and lower citrate synthase activity in the mdx mouse heart, suggesting decreased mitochondrial content. Decreased mitochondrial numbers were associated with 38% lower phosphocreatine concentration, 22% lower total creatine, 36% higher cytosolic free ADP concentration and 1.3 kJ/mol lower free-energy available from ATP hydrolysis in whole isolated, perfused mdx mouse hearts than in controls. Transsarcolemmal creatine uptake was 12% lower in mdx mouse hearts. We conclude that the absence of dystrophin in adult mdx mouse heart, as in the heart of human patient, is associated with right ventricular dilatation, left ventricular diastolic dysfunction and abnormal energy metabolism.