Physiological characterization of 2-week regenerated soleus muscle fibres

The functional properties of 14-day regenerated rat soleus muscle was investigated in chemically skinned fibres. Regeneration was induced by myotoxic injury with bupivacaine. Myofibrillar and sarcoplasmic reticulum properties of single fibres were correlated to the expression of specific protein isoforms. The maximal specific tension of regenerating fibres was not different from that of controls, despite the smaller CSA (about 45%) of regenerating fibres. The 14-day regenerating soleus fibres expressed type 1 or 1+2A myosin heavy chain isoforms in addition to residual embryonic and/or neonatal isoforms. The regenerating fibres showed a significant right shift of pCa-tension relationships and a higher pCa threshold. SDS-PAGE analysis showed regenerated fibres containing the slow troponin C isoform as well as fibres with both the slow and fast isoform. The presence of the fast isoform was correlated to the higher pCa threshold of fibres. The caffeine threshold concentration of sarcoplasmic reticulum Ca2+ release was significantly higher in regenerated than in control fibres. Consistent with the lower sensitivity to caffeine of RyR-3, regenerated muscles expressed a significant higher level of this RyR isoform. The amount of Ca2+ released at maximally-activating caffeine concentration (20 mM) was higher than in controls. Moreover, sarcoplasmic reticulum Ca2+ capacity, measured by a light-scattering method, was higher in regenerated fibres than in controls. In contrast, Western blot analysis in the whole muscle did not show appreciable differences in SERCA isoform expression between regenerated and control muscles, suggesting that the large sarcoplasmic reticulum Ca2+ capacity of regenerating fibres is likely due to a larger volume. Consistently, the rate of Ca2+ uptake, was not different in 14-day regenerating fibres with respect to control. Finally, the regenerated muscle shows a larger amount of the cardiac isoform of DHPR with respect to the control

Isoform switching in myofibrillar and excitation-contraction coupling proteins contributes to diminished contractile function in regenerating rat soleus muscle.

Postnatal development of skeletal muscle occurs through the progressive transformation of diverse biochemical, metabolic, morphological, and functional characteristics from the embryonic to the adult phenotype. Since muscle regeneration recapitulates postnatal development of muscle fiber, it offers an appropriate experimental model to investigate the existing relationships between diverse muscle functions and the expression of key protein isoforms, particularly at the single-fiber level. This study was carried out in regenerating soleus muscle 14 days after injury. At this intermediate stage, the regenerating muscle exhibited a recovery of mass greater than its force generation capacity. The lower specific tension of regenerating muscle suggested intrinsic defective excitation-contraction coupling and/or contractility processes. The presence of developmental isoforms of both the voltage-gated Ca(2+) channel (alpha(1)C) and of ryanodine receptor 3, paralleled by an abnormal caffeine contracture development, confirms the immature excitation-contraction coupling of the regenerating muscle. The defective Ca(2+) handling could also be confirmed by the lower sarcoplasmic reticulum caffeine sensitivity of regenerating single fibers. Also, regenerating single fibers revealed a lower maximal specific tension, which was associated with the residual presence of embryonic myosin heavy chains. Moreover, the fibers showed a reduced Ca(2+) sensitivity of myofibrillar proteins, particularly those simultaneously expressing the slow and fast isoforms of troponin C. The present results indicate that the expression of developmental proteins determines the incomplete functional recovery of regenerating soleus

Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling

Age-associated mitochondrial dysfunction and oxidative damage are primary causes for multiple health problems including sarcopenia and cardiovascular disease (CVD). Though the role of Nrf2, a transcription factor that regulates cytoprotective gene expression, in myopathy remains poorly defined, it has shown beneficial properties in both sarcopenia and CVD. Sulforaphane (SFN), a natural compound Nrf2-related activator of cytoprotective genes, provides protection in several disease states including CVD and is in various stages of clinical trials, from cancer prevention to reducing insulin resistance. This study aimed to determine whether SFN may prevent age-related loss of function in the heart and skeletal muscle. Cohorts of 2-month-old and 21- to 22-month-old mice were administered regular rodent diet or diet supplemented with SFN for 12 weeks. At the completion of the study, skeletal muscle and heart function, mitochondrial function, and Nrf2 activity were measured. Our studies revealed a significant drop in Nrf2 activity and mitochondrial functions, together with a loss of skeletal muscle and cardiac function in the old control mice compared to the younger age group. In the old mice, SFN restored Nrf2 activity, mitochondrial function, cardiac function, exercise capacity, glucose tolerance, and activation/differentiation of skeletal muscle satellite cells. Our results suggest that the age-associated decline in Nrf2 signaling activity and the associated mitochondrial dysfunction might be implicated in the development of age-related disease processes. Therefore, the restoration of Nrf2 activity and endogenous cytoprotective mechanisms by SFN may be a safe and effective strategy to protect against muscle and heart dysfunction due to aging.Foundation for the National Institutes of Health, Grant/Award Number: AG032643, GM109005; American Heart Association, Grant/Award Number: 14GRNT1889008

Expression and characterization of Edg-1 receptors in rat cardiomyocytes - Calcium deregulation in response to sphingosine 1-phosphate

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus