Myocardial tolerance to ischemia-reperfusion injury, training intensity and cessation.

Training has been shown to induce cardioprotection. The mechanisms involved remain still poorly understood. Aims of the study were to examine the relevance of training intensity on myocardial protection against ischemia/reperfusion (I/R) injury, and to which extent the beneficial effects persist after training cessation in rats. Sprague-Dawley rats trained at either low (60% [Formula: see text]) or high (80% [Formula: see text]) intensity for 10 weeks. An additional group of highly trained rats was detrained for 4 weeks. Untrained rats served as controls. At the end of treatment, rats of all groups were split into two subgroups. In the former, rats underwent left anterior descending artery (LAD) ligature for 30 min, followed by 90-min reperfusion, with subsequent measurement of the infarct size. In the latter, biopsies were taken to measure heat-shock proteins (HSP) 70/72, vascular endothelial growth factor (VEGF) protein levels, and superoxide dismutase (SOD) activity. Training reduced infarct size proportionally to training intensity. With detraining, infarct size increased compared to highly trained rats, maintaining some cardioprotection with respect to controls. Cardioprotection was proportional to training intensity and related to HSP70/72 upregulation and Mn-SOD activity. The relationship with Mn-SOD was lost with detraining. VEGF protein expression was not affected by either training or detraining. Stress proteins and antioxidant defenses might be involved in the beneficial effects of long-term training as a function of training intensity, while HSP70 may be one of the factors accounting for the partial persistence of myocardial protection against I/R injury in detrained rats.

Modulation of paraoxonase 1 and 3 expression after moderate exercise training in the rat.

Paraoxonases (PONs) are a small family of antioxidant enzymes whose antiatherogenic activity is well known. The aim of the present study was the evaluation of the effects of moderate aerobic training on their expression using a rat model. In order to discriminate between PON1 and PON3 enzymatic activity, we took advantage of some differences in their substrate preferences. PON1 and PON3 enzymatic activities and their protein levels were analyzed in plasma and in liver microsomes, and their mRNA levels in the liver. Exercise training did not affect PON1 expression or enzymatic activity but increased PON3 mRNA, protein levels, and enzymatic activity. Training also induced variations in plasma membrane composition, including an increase in polyunsaturated and a decrease in mono- and di-unsaturated fatty acids. On the other hand, acute exercise inhibited PON activities while increasing PON3 protein content in liver microsomes and reversing the relative composition in mono-, di-, and poly-unsaturated fatty acids, suggesting that physical stress, by altering membrane composition, may impair PON release from liver membranes. In conclusion, we documented, for the first time, the presence of PON3 in rat serum and, notably, found that the upregulation of PON3, rather than PON1, appears to be associated with physical training.

Gene expression profile of rat left ventricles reveals persisting changes following chronic mild exercise protocol: implications for cardioprotection.

BACKGROUND: Epidemiological studies showed that physical exercise, specifically moderate lifelong training, is protective against cardiovascular morbidity and mortality. Most experimental work has focused into the effects and molecular mechanisms underlying intense, rather than mild exercise, by exploring the acute effect of training. Our study aims at investigating the cardioprotective effect of mild chronic exercise training and the gene expression profile changes at 48 hrs after the exercise cessation. Rats were trained at mild intensity on a treadmill: 25 m/min, 10%incline, 1 h/day, 3 days/week, 10 weeks; about 60% of the maximum aerobic power. By Affymetrix technology, we investigated the gene expression profile induced by exercise training in the left ventricle (LV) of trained (n = 10) and control (n = 10) rats. Cardioprotection was investigated by ischemia/reperfusion experiments (n = 10 trained vs. n = 10 control rats). RESULTS: Mild exercise did not induce cardiac hypertrophy and was cardioprotective as demonstrated by the decreased infarct size (p = 0.02) after ischemia/reperfusion experiments in trained with respect to control rats. Ten genes and 2 gene sets (two pathways) resulted altered in LV of exercised animals with respect to controls. We validated by real-time PCR the increased expression of four genes: similar to C11orf17 protein (RGD1306959), caveolin 3, enolase 3, and hypoxia inducible factor 1 alpha. Moreover, caveolin 3 protein levels were higher in exercised than control rats by immunohistochemistry and Western Blot analysis. Interestingly, the predicted gene similar to C11orf17 protein (RGD1306959) was significantly increased by exercise. This gene has a high homology with the human C11orf17 (alias: protein kinase-A interacting protein 1 or breast cancer associated gene 3). This is the first evidence that this gene is involved in the response to the exercise training. CONCLUSION: Our data indicated that few, but significant, genes characterize the gene expression profile of the rat LV, when examined 48 hrs since the last training section and that mild exercise training determines cardioprotection without the induction of hypertrophy.

Persistence of regenerative myogenesis in spite of down-regulation of activity-dependent genes in long-term denervated rat muscle.

Contrary to general expectation, in humans, we have recently shown that after complete conus cauda lesion, the lower motoneuron denervated myofibers may survive several years. In adult rats, the sciatectomized muscle progresses in 4-6 months from severe atrophy to a dystrophic stage and undergoes a dramatic weight loss; during this process, myofiber death/regeneration processes maintain a decreasing population of very small, but vital myofibers. At the same time, in vitro electrophysiologic recordings show that denervated fibers can maintain membrane excitability longer than they can retain contractile properties. A certain level of myofiber regeneration seems to have a role in the process, with the early re-expression of embryonic subunits of integrins and acetylcholine receptor subunits. In the present work, using the reliable real-time quantitative PCR, we confirm the long-lasting occurrence of myoblast proliferation-dependent events and their focal nature. In fact, we show here that in sciatectomized muscle, the expression of 12 selected genes was differentially regulated after 3 and 9 month denervation. At both time points, indexes of muscle activity/inactivity and tissue remodeling (proteolysis, energy usage and angiogenic factors) were down-regulated, while indexes of regenerative myogenesis (Myogenin, MyoD, MRF4 and MHCemb) were up-regulated. Immunohistochemistry with anti-MHCemb and anti-NCAM monoclonal antibodies show that such regeneration events were focally distributed. We conclude that myofiber regeneration is a non-compensatory mechanism, which prolongs the chance of reinnervation during long-lasting denervation. It may also contribute to muscle recovery in paraplegic patients, even when rehabilitation strategies based on functional electric stimulation start late after spinal cord injury (SCI).

Oxidative stress in the denervated muscle.

Following experimental hind limb denervation in rats, this study demonstrates that oxidative stress occurs and advances an hypothesis about its origin. In fact: (i) ROS are formed; (ii) membrane lipids are oxidized; (iii) oxidized ion channels and pumps may lead to increased [Ca(2+)](i); all the above mentioned events increase with denervation time. In the denervated muscle, (iv) mRNA abundance of cytoprotective and anti-oxidant proteins (Hsp70, Hsp27, Sod1, Catalase, Gpx1, Gpx4, Gstm1), as well as (v) SOD1 enzymatic activity and HSP70i protein increase; (vi) an unbalance in mitochondrial OXPHOS enzymes occurs, presumably leading to excess mitochondrial ROS production; (vii) increased cPLA2alpha expression (mRNA) and activation (increased [Ca(2+)](i)) may lead to increased hydroperoxides release. Since anti-oxidant defences appear inadequate to counterbalance increased ROS production with increased denervation time, an anti-oxidant therapeutic strategy seems to be advisable in the many medical conditions where the nerve-muscle connection is impaired.

Partial persistence of exercise-induced myocardial angiogenesis following 4-week detraining in the rat.

Enhanced angiogenesis, or capillary growth, has a prominent role among the various beneficial effects of exercise training on the myocardium. The aim of the present study is to assess if training-induced increases in capillarity and vascularization persist after 4 weeks of detraining. Adult male rats were trained to run on a treadmill for 10 weeks at approximately 60% VO2max, which did not induce cardiac hypertrophy, but increased (P < 0.05) the soleus/body weight ratio, left ventricle capillarity and von Willebrand-positive cell density (n = 6). In another group of animals (n = 6) subjected to training followed by 4-week detraining, the soleus/body weight ratio returned to normal, with only partial reversal of left ventricle capillarity and von Willebrand-positive cell density. Markers of angiogenesis (VEGF, KDR/VEGF-R2 and HIF-1alpha mRNA, studied by real-time RT-PCR) were upregulated at the end of training, and returned to baseline value after detraining. Electron microscopy highlighted some morphological features in trained hearts (endothelial cell sprouting and bridges and pericyte detachment), suggestive of endothelial cell proliferation and capillary growth that were absent in untrained and detrained hearts. We conclude that the training-induced increase in cardiac capillarity and vascularization are retained for some time upon cessation of the training program even in the absence of angiogenic stimuli.