Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

Abstract-We have previously shown that exercise training activates nucleus tractus solitarii (NTS) oxytocinergic projections, resulting in blunted exercise tachycardia. The objective of this study was to determine the effects of hypertension and training on oxytocin (OT) and OT receptor expression in the hypothalamic paraventricular nucleus (PVN) and projection areas (dorsal brain stem [DBS]). Male, normotensive, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were trained (55% maximal exercise capacity, 3 months) or kept sedentary, and pressure was measured weekly. DBS sections were processed for immunohistochemistry (polyclonal guinea pig anti-OT) or in situ hybridization for OT and OT receptor ( 35 S-oligonucleotide probes). Other groups of rats had brains removed and frozen to isolate the DBS and PVN; samples were processed for OT and OT receptor cDNA reverse transcription-polymerase chain reaction amplification with ␤-actin as the housekeeping gene. Training was equally effective in improving running distance in both groups, with pressure reduction only in SHR (Ϫ10%, PϽ0.05). In trained WKY, baseline bradycardia (PϽ0.05) occurred simultaneously with increased NTS OT immunostaining and mRNA expression (ϩ3.5-fold), without any change in OT receptor mRNA expression. PVN OT mRNA and DBS OT receptor mRNA expressions were significantly lower in SHR versus WKY (Ϫ39% and Ϫ56%, respectively). Training did not alter DBS OT receptor density in the SHR group but increased OT mRNA in both PVN and DBS areas (ϩ78% and ϩ45%, respectively). Our results show a marked hypertension-induced reduction in OT receptor mRNA expression, not altered by training. In contrast, training increased OT mRNA expression in sedentary and hypertensive rats, which may facilitate traininginduced cardiac performance. Key Words: hypothalamus Ⅲ exercise Ⅲ rats, spontaneously hypertensive Ⅲ neurotransmitter Ⅲ genetics Ⅲ autonomic nervous system Ⅲ immunohistochemistry H ypertension is a highly prevalent disease and a common risk factor for different cardiovascular diseases, with a major impact on morbidity and mortality. 1 Hypertensive individuals present with a series of functional and anatomic deficits, such as increased vascular resistance, vessel rarefaction, increased heart energy expenditure, increased stroke work, impaired baroreceptor reflex control, hormonal imbalance with overactivation of the renin-angiotensin system, and increased insulin resistance. Most of these effects contribute to increased sympathetic activity and depressed cardiovascular control. 1 On the other hand, exercise training has been associated with a variety of beneficial cardiovascular adjustments in hypertensive individuals, such as eutrophic remodeling of arterioles causing wall-lumen ratio normalization, 2,3 capillary angiogenesis and venule neoformation in exercised muscles resulting in increased vascular capacity and O 2 extraction, 3-7 and remodeling of the heart with simultaneous stroke volume increase and heart rate decrease. 8,9 Although there are ...

Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca(2+) spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca(2+) sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca(2+) responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca(2+) transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca(2+) spark activity, which was suppressed by ROS-reducing agents and by inhibitors of NAD(P)H oxidase. These Ca(2+) signals resulted in mitochondrial Ca(2+) accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca(2+) signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.

Ca2+ sparks, localized elevations in cytosolic [Ca 2+ ], are rarely detected in intact adult mammalian skeletal muscle under physiological conditions. However, they have been observed in permeabilized cells and in intact fibres subjected to stresses, such as osmotic shock and strenuous exercise. Our previous studies indicated that an excess in cellular reactive oxygen species (ROS) generation over the ROS scavenging capabilities could be one of the up-stream causes of Ca 2+ spark appearance in permeabilized muscle fibres. Here we tested whether the cytosolic ROS balance is compromised in intact skeletal muscle fibres that underwent osmotic shock and whether this misbalance contributes to unmasking Ca 2+ sparks. Spontaneous Ca 2+ sparks and the rate of ROS generation were assessed with single photon confocal microscopy and fluorescent indicators fluo-4, CM-H 2 DCFDA and MitoSOX Red. Osmotic shock produced spontaneous Ca 2+ sparks and a concomitant significant increase in ROS production. Preincubation of muscle cells with ROS scavengers (e.g. MnTBAP, Mn-cpx 3, TIRON) nearly eliminated Ca 2+ sparks. In addition, inhibitors of NAD(P)H oxidase (DPI and apocynin) significantly reduced ROS production and suppressed the appearance of Ca 2+ sparks. Taken together, the data suggest that ROS contribute to the abnormal Ca 2+ spark activity in mammalian skeletal muscle subjected to osmotic stress and also indicate that NAD(P)H oxidase is a possible source of ROS. We propose that ROS-dependent Ca 2+ sparks are an important component of adaptive/maladaptive muscle responses under various pathological conditions such as eccentric stretch, osmotic changes during ischaemia and reperfusion, and some muscle diseases.

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Background Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy (HCM), but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in-vivo. The goal of this study was to determine whether TNNC1 can be categorized as an HCM susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results The TNNC1-A8V proband diagnosed with severe obstructive HCM at 34-years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left-ventricular dimensions, left-atrial enlargement and hyperdynamic left-ventricular systolic function. Genetically-engineered knock-in mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure-volume studies. Three-month-old, KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, while KI-TnC-A8V+/− mice displayed cardiac restriction at 14-months of age. KI hearts exhibited atrial enlargement, papillary-muscle hypertrophy and fibrosis. Liquid chromatography-mass spectroscopy was used to determine incorporation of mutant cTnC (~21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+-sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+ > KI-TnC-A8V+/− > WT, while KI-TnC-A8V+/+ relaxed more slowly upon flash-photolysis of diazo-2. Conclusions The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament, and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in-vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.

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