Luiz Henrique Marchesi Bozi

High- versus moderate-intensity aerobic exercise training effects on skeletal muscle of infarcted rats

Poor skeletal muscle performance was shown to strongly predict mortality and long-term prognosis in a variety of diseases, including heart failure (HF). Despite the known benefits of aerobic exercise training (AET) in improving the skeletal muscle phenotype in HF, the optimal exercise intensity to elicit maximal outcomes is still under debate. Therefore, the aim of the present study was to compare the effects of high-intensity AET with those of a moderate-intensity protocol on skeletal muscle of infarcted rats. Wistar rats underwent myocardial infarction (MI) or sham surgery. MI groups were submitted either to an untrained (MI-UNT); moderate-intensity (MI-CMT, 60% Vo(2)(max)); or matched volume, high-intensity AET (MI-HIT, intervals at 85% Vo(2)(max)) protocol. High-intensity AET (HIT) was superior to moderate-intensity AET (CMT) in improving aerobic capacity, assessed by treadmill running tests. Cardiac contractile function, measured by echocardiography, was equally improved by both AET protocols. CMT and HIT prevented the MI-induced decay of skeletal muscle citrate synthase and hexokinase maximal activities, and increased glycogen content, without significant differences between protocols. Similar improvements in skeletal muscle redox balance and deactivation of the ubiquitin-proteasome system were also observed after CMT and HIT. Such intracellular findings were accompanied by prevented skeletal muscle atrophy in both MI-CMT and MI-HIT groups, whereas no major differences were observed between protocols. Taken together, our data suggest that despite superior effects of HIT in improving functional capacity, skeletal muscle adaptations were remarkably similar among protocols, leading to the conclusion that skeletal myopathy in infarcted rats was equally prevented by either moderate-intensity or high-intensity AET.

Combined Effect of AMPK/PPAR Agonists and Exercise Training in mdx Mice Functional Performance

The present investigation was undertaken to test whether exercise training (ET) associated with AMPK/PPAR agonists (EM) would improve skeletal muscle function in mdx mice. These drugs have the potential to improve oxidative metabolism. This is of particular interest because oxidative muscle fibers are less affected in the course of the disease than glycolitic counterparts. Therefore, a cohort of 34 male congenic C57Bl/10J mdx mice included in this study was randomly assigned into four groups: vehicle solution (V), EM [AICAR (AMPK agonist, 50 mg/Kg-1.day-1, ip) and GW 1516 (PPARδ agonist, 2.5 mg/Kg-1.day-1, gavage)], ET (voluntary running on activity wheel) and EM+ET. Functional performance (grip meter and rotarod), aerobic capacity (running test), muscle histopathology, serum creatine kinase (CK), levels of ubiquitined proteins, oxidative metabolism protein expression (AMPK, PPAR, myoglobin and SCD) and intracellular calcium handling (DHPR, SERCA and NCX) protein expression were analyzed. Treatments started when the animals were two months old and were maintained for one month. A significant functional improvement (p<0.05) was observed in animals submitted to the combination of ET and EM. CK levels were decreased and the expression of proteins related to oxidative metabolism was increased in this group. There were no differences among the groups in the intracellular calcium handling protein expression. To our knowledge, this is the first study that tested the association of ET with EM in an experimental model of muscular dystrophy. Our results suggest that the association of ET and EM should be further tested as a potential therapeutic approach in muscular dystrophies.

Autophagy Signaling in Skeletal Muscle of Infarcted Rats

Background Heart failure (HF)-induced skeletal muscle atrophy is often associated to exercise intolerance and poor prognosis. Better understanding of the molecular mechanisms underlying HF-induced muscle atrophy may contribute to the development of pharmacological strategies to prevent or treat such condition. It has been shown that autophagy-lysosome system is an important mechanism for maintenance of muscle mass. However, its role in HF-induced myopathy has not been addressed yet. Therefore, the aim of the present study was to evaluate autophagy signaling in myocardial infarction (MI)-induced muscle atrophy in rats. Methods/Principal Findings Wistar rats underwent MI or Sham surgeries, and after 12 weeks were submitted to echocardiography, exercise tolerance and histology evaluations. Cathepsin L activity and expression of autophagy-related genes and proteins were assessed in soleus and plantaris muscles by fluorimetric assay, qRT-PCR and immunoblotting, respectively. MI rats displayed exercise intolerance, left ventricular dysfunction and dilation, thereby suggesting the presence of HF. The key findings of the present study were: a) upregulation of autophagy-related genes (GABARAPL1, ATG7, BNIP3, CTSL1 and LAMP2) was observed only in plantaris while muscle atrophy was observed in both soleus and plantaris muscles, and b) Cathepsin L activity, Bnip3 and Fis1 protein levels, and levels of lipid hydroperoxides were increased specifically in plantaris muscle of MI rats. Conclusions Altogether our results provide evidence for autophagy signaling regulation in HF-induced plantaris atrophy but not soleus atrophy. Therefore, autophagy-lysosome system is differentially regulated in atrophic muscles comprising different fiber-types and metabolic characteristics.

Resistance training regulates cardiac function through modulation of miRNA-214.

Aims: To determine the effects of resistance training (RT) on the expression of microRNA (miRNA)-214 and its target in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), and on the morphological and mechanical properties of isolated left ventricular myocytes. Main methods: Male Wistar rats were divided into two groups (n = 7/group): Control (CO) or trained (TR). The exercise-training protocol consisted of: 4 × 12 bouts, 5×/week during 8 weeks, with 80% of one repetition maximum. Key findings: RT increased the left ventricular myocyte width by 15% and volume by 12%, compared with control animals (p < 0.05). The time to half relaxation and time to peak were 8.4% and 4.4% lower, respectively, in cells from TR group as compared to CO group (p < 0.05). RT decreased miRNA-214 level by 18.5% while its target SERCA2a expression were 18.5% higher (p < 0.05). Significance: Our findings showed that RT increases single left ventricular myocyte dimensions and also leads to faster cell contraction and relaxation. These mechanical adaptations may be related to the augmented expression of SERCA2a which, in turn, may be associated with the epigenetic modification of decreased miRNA-214 expression.

Exercise training prior to myocardial infarction attenuates cardiac deterioration and cardiomyocyte dysfunction in rats

OBJECTIVES: The present study was performed to investigate 1) whether aerobic exercise training prior to myocardial infarction would prevent cardiac dysfunction and structural deterioration and 2) whether the potential cardiac benefits of aerobic exercise training would be associated with preserved morphological and contractile properties of cardiomyocytes in post-infarct remodeled myocardium. METHODS: Male Wistar rats underwent an aerobic exercise training protocol for eight weeks. The rats were then assigned to sham surgery (SHAM), sedentary lifestyle and myocardial infarction or exercise training and myocardial infarction groups and were evaluated 15 days after the surgery. Left ventricular tissue was analyzed histologically, and the contractile function of isolated myocytes was measured. Students t-test was used to analyze infarct size and ventricular wall thickness, and the other parameters were analyzed by the Kruskal-Wallis test followed by Dunns test or a one-way analysis of variance followed by Tukeys test (p<0.05). RESULTS: Myocardial infarctions in exercise-trained animals resulted in a smaller myocardial infarction extension, a thicker infarcted wall and less collagen accumulation as compared to myocardial infarctions in sedentary animals. Myocardial infarction-induced left ventricular dilation and cardiac dysfunction, as evaluated by +dP/dt and -dP/dt, were both prevented by previous aerobic exercise training. Moreover, aerobic exercise training preserved cardiac myocyte shortening, improved the maximum shortening and relengthening velocities in infarcted hearts and enhanced responsiveness to calcium. CONCLUSION: Previous aerobic exercise training attenuated the cardiac dysfunction and structural deterioration promoted by myocardial infarction, and such benefits were associated with preserved cardiomyocyte morphological and contractile properties.

Aerobic exercise training rescues cardiac protein quality control and blunts endoplasmic reticulum stress in heart failure rats

Cardiac endoplasmic reticulum (ER) stress through accumulation of misfolded proteins plays a pivotal role in cardiovascular diseases. In an attempt to reestablish ER homoeostasis, the unfolded protein response (UPR) is activated. However, if ER stress persists, sustained UPR activation leads to apoptosis. There is no available therapy for ER stress relief. Considering that aerobic exercise training (AET) attenuates oxidative stress, mitochondrial dysfunction and calcium imbalance, it may be a potential strategy to reestablish cardiac ER homoeostasis. We test the hypothesis that AET would attenuate impaired cardiac ER stress after myocardial infarction (MI). Wistar rats underwent to either MI or sham surgeries. Four weeks later, rats underwent to 8 weeks of moderate‐intensity AET. Myocardial infarction rats displayed cardiac dysfunction and lung oedema, suggesting heart failure. Cardiac dysfunction in MI rats was paralleled by increased protein levels of UPR markers (GRP78, DERLIN‐1 and CHOP), accumulation of misfolded and polyubiquitinated proteins, and reduced chymotrypsin‐like proteasome activity. These results suggest an impaired cardiac protein quality control. Aerobic exercise training improved exercise capacity and cardiac function of MI animals. Interestingly, AET blunted MI‐induced ER stress by reducing protein levels of UPR markers, and accumulation of both misfolded and polyubiquinated proteins, which was associated with restored proteasome activity. Taken together, our study provide evidence for AET attenuation of ER stress through the reestablishment of cardiac protein quality control, which contributes to better cardiac function in post‐MI heart failure rats. These results reinforce the importance of AET as primary non‐pharmacological therapy to cardiovascular disease.

Exercise reestablishes autophagic flux and mitochondrial quality control in heart failure

ABSTRACT We previously reported that facilitating the clearance of damaged mitochondria through macroautophagy/autophagy protects against acute myocardial infarction. Here we characterize the impact of exercise, a safe strategy against cardiovascular disease, on cardiac autophagy and its contribution to mitochondrial quality control, bioenergetics and oxidative damage in a post-myocardial infarction-induced heart failure animal model. We found that failing hearts displayed reduced autophagic flux depicted by accumulation of autophagy-related markers and loss of responsiveness to chloroquine treatment at 4 and 12 wk after myocardial infarction. These changes were accompanied by accumulation of fragmented mitochondria with reduced O2 consumption, elevated H2O2 release and increased Ca2+-induced mitochondrial permeability transition pore opening. Of interest, disruption of autophagic flux was sufficient to decrease cardiac mitochondrial function in sham-treated animals and increase cardiomyocyte toxicity upon mitochondrial stress. Importantly, 8 wk of exercise training, starting 4 wk after myocardial infarction at a time when autophagy and mitochondrial oxidative capacity were already impaired, improved cardiac autophagic flux. These changes were followed by reduced mitochondrial number:size ratio, increased mitochondrial bioenergetics and better cardiac function. Moreover, exercise training increased cardiac mitochondrial number, size and oxidative capacity without affecting autophagic flux in sham-treated animals. Further supporting an autophagy mechanism for exercise-induced improvements of mitochondrial bioenergetics in heart failure, acute in vivo inhibition of autophagic flux was sufficient to mitigate the increased mitochondrial oxidative capacity triggered by exercise in failing hearts. Collectively, our findings uncover the potential contribution of exercise in restoring cardiac autophagy flux in heart failure, which is associated with better mitochondrial quality control, bioenergetics and cardiac function.

Modulation of inflammatory and oxidative status by exercise attenuates cardiac morphofunctional remodeling in experimental Chagas cardiomyopathy.

AIMS The rational basis that explains the benefits of exercise therapy on Chagas cardiomyopathy (ChC) is poorly understood. This study investigated the impact of an exercise program on exercise performance, heart parasitism, immunoinflammatory response, fibrogenesis, oxidative damage, and cardiomyocytes contractility in experimental ChC. MAIN METHODS Wistar rats were subjected to a 9-week treadmill running training and challenged with Trypanosoma cruzi. Control animals remained sedentary. Physical and metabolic performance, cardiac morphology, cytokines, chemokines, nitric oxide, oxidative tissue damage, cardiomyocyte morphology and contractility were analyzed. KEY FINDINGS Exercise training was efficient to improve physical performance and anaerobic threshold in trained animals. By increasing cardiac and serum levels of cytokines (TNF-α, IFN-γ, and IL-6), chemokines (MCP-1 and CX3CL1), the myocardial activity catalase and superoxide dismutase, and reducing lipid and protein oxidation in cardiac tissue, exercise training seem to be a beneficial strategy to mitigate the progression and severity of Chagas-associated cardiomyopathy. SIGNIFICANCE The protective adaptations to the host triggered by exercise training contributed to reduce cardiac parasitism, inflammation, fibrosis and cardiomyocytes atrophy. Although exercise training does not affect nitric oxide levels in cardiac tissue from infected animals, this strategy enhanced the efficiency of endogenous antioxidant mechanisms, restricting oxidative tissue damage with positive repercussions to cardiomyocytes biomechanics in rats.

Mitochondrial Quality Control in Cardiac Diseases

Disruption of mitochondrial homeostasis is a hallmark of cardiac diseases. Therefore, maintenance of mitochondrial integrity through different surveillance mechanisms is critical for cardiomyocyte survival. In this review, we discuss the most recent findings on the central role of mitochondrial quality control processes including regulation of mitochondrial redox balance, aldehyde metabolism, proteostasis, dynamics, and clearance in cardiac diseases, highlighting their potential as therapeutic targets.

Lactate Up-Regulates the Expression of Lactate Oxidation Complex-Related Genes in Left Ventricular Cardiac Tissue of Rats

Background Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex. Methods/Principal Findings Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O2 ●-/H2O2) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O2 ●-/H2O2 levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O2 ●-/H2O2. Conclusions Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O2 ●-/H2O2 driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle.