Jatin G. Burniston

Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5

Increases in aerobic capacity and intramuscular triglyceride (IMTG) utilization are well‐described adaptations to endurance training (ET) and contribute to improvements in insulin sensitivity. Sprint interval training (SIT) also improves aerobic capacity and insulin sensitivity with a lower time commitment than ET. This study aimed to determine whether SIT also induces improvements in insulin sensitivity and net IMTG breakdown, and to investigate the underlying mechanisms. Six weeks of ET and SIT increased net IMTG breakdown during moderate‐intensity cycling, and improved insulin sensitivity. A greater concentration of lipid droplet‐associated proteins, perilipin 2 and perilipin 5, was observed following both training modes and contributes to the increases in net IMTG breakdown following training. The results suggest a novel mechanism for the training‐induced improvements in IMTG breakdown and insulin sensitivity, and clearly demonstrate that SIT is an alternative, time‐efficient training strategy that induces similar beneficial metabolic adaptations.

Catecholamine‐induced apoptosis and necrosis in cardiac and skeletal myocytes of the rat in vivo: the same or separate death pathways?

High levels of catecholamines are myotoxic but the relative amounts of apoptosis and necrosis have not been established in vivo in cardiac and skeletal muscles. Immunohistochemistry was used to detect and quantify myocyte‐specific necrosis (myosin antibody in vivo) and apoptosis (caspase‐3 antibody in vitro) in the heart and soleus muscles of male Wistar rats that had received single subcutaneous injections of isoprenaline over the range 1 μg to 5 mg [kg body weight (BW)]−1. Peak myocyte apoptosis occurred 3–6 h after, and necrosis 18 h after, a single injection of 5 mg (kg BW)−1 isoprenaline in vivo. In the heart myocyte death was mediated through the β1‐adrenergic receptor whereas myocyte death in the soleus muscle was mediated through the β2‐adrenergic receptor. Cardiomyocyte death was heterogeneously distributed throughout the heart, being greatest in the left ventricle (LV) subendocardium and peaking close to the apex, but with significantly more necrosis than apoptosis. Extensive co‐localization of caspase‐3 and myosin labelling was found in the myocytes of both the heart and the slow‐twitch soleus muscle. This, together with similar spatial distributions and responses to catecholamine doses, suggests that either caspase‐3 activation occurs in necrotic as well as apoptotic myocytes or that a large proportion of apoptotic myocytes progress to secondary necrosis in vivo.

Cardiomyocyte death and the ageing and failing heart

Mammalian cardiomyocytes have limited regenerative capacity, such that cell death can result in a net loss of viable contractile elements and a decrease in cardiac functional reserve, both during normal ageing and after insults to the myocardium leading to heart failure. At least four types of cell death have been described, with apoptosis and necrosis being the most extreme phenotypes and most extensively studied. Many of the classical morphological and biochemical features associated with these forms of cell death have been derived from studies conducted in vitro and these may not always faithfully reflect events occurring in vivo. Before therapeutic interventions can be realistically developed, more studies need to be undertaken in vivo to simultaneously investigate these different death pathways, their control mechanisms and their relative contributions in depleting the pool of viable cardiomyocytes. We recently demonstrated immunohistochemically that a single injection of either a natural or synthetic catecholamine induces both cardiomyocyte apoptosis (identified by an anti‐caspase 3 antibody) and necrosis (identified by an anti‐myosin antibody) in the rat heart in vivo. After optimising the experimental conditions for hormone dose and temporal and spatial peaks of damage, the incidence of necrosis was 4‐10 times greater than the incidence of apoptosis. Myocytes in the soleus muscle were also severely (7‐10%) damaged, involving both apoptosis and necrosis. In both striated muscles high levels of myocyte co‐localisation for apoptosis and necrosis were observed, suggesting that secondary necrosis had occurred in most of the apoptotic myocytes in vivo. The ability of the catecholamines to cause myocyte death suggests that they might play an aetiological role in the progression of heart failure where over‐activation of the sympathetic system results in sustained pathophysiological levels of these catecholamines.

Yap is a novel regulator of C2C12 myogenesis

The expression, regulation and function of mammalian Hippo pathway members in skeletal muscle is largely unknown. The aim of this study was thus to test the hypothesis that core members of the mammalian Hippo pathway are expressed in skeletal muscle and that the transcriptional co-factor Yap, a core member of the Hippo pathway, regulates C2C12 myogenesis. We found that the major components of the mammalian Hippo pathway including Yap are all expressed in skeletal muscles, C2C12 myoblasts and myotubes. In C2C12 myoblasts, Yap Ser127 phosphorylation is low and Yap localises to nuclei. Upon differentiation, Yap Ser127 phosphorylation increases approximately 20-fold and Yap translocates from the nucleus to the cytosol. To test whether the observed increase of Yap Ser127 phosphorylation is required for differentiation we overexpressed hYAP1 S127A, a mutant that can not be phosphorylated at Ser127, in C2C12 myoblasts. We found that overexpression of hYAP S127A prevented myotube formation, whereas the overexpression of wildtype hYAP1 or empty vector had no effect. In addition, more hYAP1 S127A overexpressing cells progressed through the S phase of the cell cycle and the expression of MRFs (myogenin, Myf5), Mef2c and cell cycle regulators (p21, cyclin D1) was significantly changed when compared to wildtype hYAP1 and empty vector overexpressing cells. This data suggests that the phosphorylation of Yap at Ser127 leads to a changed expression of MRFs and cell cycle regulators and is required for C2C12 myoblasts to differentiate into myotubes.

Proteomic investigation of changes in human vastus lateralis muscle in response to interval-exercise training.

No previous study has used proteomics to investigate the effects of exercise training on human skeletal muscle. Five recreationally active men completed a 6‐wk training programme involving three sessions per week, utilising six 1‐min bouts at maximum oxygen uptake (V̇ O2max) interspersed with 4 min at 50% V̇ O2max. Vastus lateralis was biopsied at standardised times before and after the training intervention. Protein expression profiling was performed using differential analysis of 2‐DE gels; complemented with quantitative analysis (iTRAQ) of tryptic peptides from 1‐DE gel lane‐segments using LC‐MALDI MS/MS. Interval training increased average V̇ O2max (7%; p<0.001) and was associated with greater expression of mitochondrial components, including succinate dehydrogenase, trifunctional protein‐α and ATP synthase α‐ and β‐chains. 2‐DE resolved 256 spots, and paired t‐tests identified 20 significant differences in expression (false discovery rate <10%). Each differentially expressed gene product was present as multiple isoelectric species. Therefore, the differences in spot expression represent changes in post‐transcriptional or post‐translational processing. In particular, modulation of muscle creatine kinase and troponin T were prominent. Pro‐Q Diamond staining revealed these changes in expression were associated with phosphorylated protein species, which provides novel information regarding muscle adaptation to interval training.

Lifelong training preserves some redox-regulated adaptive responses after an acute exercise stimulus in aged human skeletal muscle

Several redox-regulated responses to an acute exercise bout fail in aged animal skeletal muscle, including the ability to upregulate the expression of antioxidant defense enzymes and heat shock proteins (HSPs). These findings are generally derived from studies on sedentary rodent models and thus may be related to reduced physical activity and/or intraspecies differences as opposed to aging per se. This study, therefore, aimed to determine the influence of age and training status on the expression of HSPs, antioxidant enzymes, and NO synthase isoenzymes in quiescent and exercised human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis before and 3 days after an acute high-intensity-interval exercise bout in young trained, young untrained, old trained, and old untrained subjects. Levels of HSP72, PRX5, and eNOS were significantly higher in quiescent muscle of older compared with younger subjects, irrespective of training status. 3-NT levels were elevated in muscles of the old untrained but not the old trained state, suggesting that lifelong training may reduce age-related macromolecule damage. SOD1, CAT, and HSP27 levels were not significantly different between groups. HSP27 content was upregulated in all groups studied postexercise. HSP72 content was upregulated to a greater extent in muscle of trained compared with untrained subjects postexercise, irrespective of age. In contrast to every other group, old untrained subjects failed to upregulate CAT postexercise. Aging was associated with a failure to upregulate SOD2 and a downregulation of PRX5 in muscle postexercise, irrespective of training status. In conclusion, lifelong training is unable to fully prevent the progression toward a more stressed muscular state as evidenced by increased HSP72, PRX5, and eNOS protein levels in quiescent muscle. Moreover, lifelong training preserves some (e.g., CAT) but not all (e.g., SOD2, HSP72, PRX5) of the adaptive redox-regulated responses after an acute exercise bout. Collectively, these data support many but not all of the findings from previous animal studies and suggest parallel aging effects in humans and mice at rest and after exercise that are not modulated by training status in human skeletal muscle.

Characterization of adrenoceptor involvement in skeletal and cardiac myotoxicity induced by sympathomimetic agents: Toward a new bioassay for β-blockers

Excessive levels of catecholamines have long been known to be cardiotoxic, but less well known are their toxic effects on skeletal muscle. By using an antimyosin monoclonal antibody and quantitative methods to measure the extent of myocyte necrosis, and by employing modulators of adrenoceptors (ARs), including clenbuterol, bupranolol, propranolol, bisoprolol, atenolol, ICI-118551, phenoxybenzamine, prazosin, and yohimbine, the involvement of ARs in isoproterenol-induced myotoxicity was characterized. In the myocardium, the toxic effects were predominantly mediated via the &bgr;1-ARs. In the soleus muscle, it was almost solely via the &bgr;2-ARs. Myotoxicity was also observed in the myocardium when challenged with the &bgr;2-AR agonist clenbuterol. This was found to be mediated via sympathetic presynaptic &bgr;2-ARs, leading to enhanced release of norepinephrine. This effect was abolished by prior treatment with reserpine. The skeletal muscle was found to be more sensitive to the myotoxic effects than cardiac muscle at lower doses of &bgr;-AR agonists. These experiments introduce a new way of assaying &bgr;-AR antagonists by classifying them according to their ability to prevent catecholamine-induced myotoxicity. Further research along these lines may deepen understanding of which &bgr;-blockers work best in heart failure therapy.

Preferential utilization of perilipin 2‐associated intramuscular triglycerides during 1 h of moderate‐intensity endurance‐type exercise

The lipid droplet (LD)‐associated protein perilipin 2 (PLIN2) appears to colocalize with LDs in human skeletal muscle fibres, although the function of PLIN2 in the regulation of intramuscular triglyceride (IMTG) metabolism is currently unknown. Here we investigated the hypothesis that the presence of PLIN2 in skeletal muscle LDs is related to IMTG utilisation during exercise. We therefore measured exercise‐induced changes in IMTG and PLIN2 distribution and changes in their colocalization. Muscle biopsies from the vastus lateralis were obtained from seven lean, untrained men (22 ± 2 years old, body mass index 24.2 ± 0.9 kg m−2 and peak oxygen uptake 3.35 ± 0.13 l min−1) before and after 1 h of moderate‐intensity cycling at ∼65% peak oxygen uptake. Cryosections were stained for perilipin 2, IMTG and myosin heavy chain type I and viewed using wide‐field and confocal fluorescence microscopy. Exercise induced a 50 ± 7% decrease in IMTG content in type I fibres only (P < 0.05), but no change in PLIN2 content. Colocalization analysis showed that the fraction of PLIN2 associated with IMTG was 0.67 ± 0.03 before exercise, which was reduced to 0.51 ± 0.01 postexercise (P < 0.05). Further analysis revealed that the number of PLIN2‐associated LDs was reduced by 31 ± 10% after exercise (P < 0.05), whereas the number of PLIN2‐null LDs was unchanged. No such changes were seen in type II fibres. In conclusion, this study shows that PLIN2 content in skeletal muscle is unchanged in response to a single bout of endurance exercise. Furthermore, the PLIN2 and IMTG association is reduced postexercise, apparently due to preferential utilization of PLIN2‐associated LDs. These results confirm the hypothesis that the PLIN2 association with IMTG is related to the utilization of IMTG as a fuel during exercise.

Adaptation of the rat cardiac proteome in response to intensity-controlled endurance exercise

Endurance training improves cardiac function and protects against heart disease. The rodent intensity‐controlled running model replicates endurance exercise in humans and can be used to investigate molecular adaptations in the heart. Rats (n = 6, 280 ± 3 g) performed exercise tests to measure their peak oxygen uptake (

Proteomic responses of skeletal and cardiac muscle to exercise

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