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 autophagylysosome 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.\ud Methods/Principal Findings: Wistar rats underwent MI or Sham surgeries, and after 12 weeks were submitted toechocardiography, exercise tolerance and histology evaluations. Cathepsin L activity and expression of autophagy-related\ud 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\ud specifically in plantaris muscle of MI rats.\ud 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.Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP #2010/14567-4).FAPESP (#2010/50048-1)Conselho Nacional de Pesquisa e Desenvolvimento (CNPq #302201/2011-4

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. Student's 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 Dunn's test or a one-way analysis of variance followed by Tukey's test (

Effects of a physical exercise program previous to myocardial infarction on the left ventricle morphology and contractile function of rats

O estudo teve como objetivo verificar os efeitos de um programa de corrida em esteira realizado previamente ao infarto do miocárdio (IM) sobre a morfologia e função contrátil do miocárdio de ratos. Ratos Wistar com idade de 30 dias foram divididos em 3 grupos: infartado sedentário (INF SED, n=13), infartado exercício (INF EX, n=26) e infarto simulado (SHAM, n=12). Antes da cirurgia os animais do grupo INF EX foram submetidos a um programa progressivo de corrida em esteira (5 dias/semana, 60min/dia, 18 m/min einclinação de 10o) por 8 semanas. Após este período os animais dos grupos INFEX e INF SED foram submetidos a uma cirurgia para indução do IM (oclusão da artéria) e nos animais do grupo SHAM a artéria não foi ocluída. Quinze dias após a indução do IM, os cardiomiócitos localizados na borda (2mm) da área infartada de metade dos animais de cada grupo foram isolados por dispersão enzimática. Os cardiomiócitos foram estimulados a 0,5 e 3 Hz, em temperatura ambiente (~25ºC), para mensuração da função contrátil em diferentes concentrações externas de cálcio (0.6, 1 e 5 mM). O restante dos animais foi usado para obtenção dos registros hemodinâmicos. Após este procedimento os corações foram removidos, fixados e corados com Picrosirius red ou Tricrômico de Masson para análise histológica. Os resultados mostram que os animais exercitados tiveram melhor capacidade de corrida, comparados aos sedentários (P<0,05). O programa de corrida aplicado não reduziu significativamente a mortalidade dos animais infartados. O peso do coração, peso relativo do coração, peso do pulmão, peso relativo do pulmão e peso líquido pulmão foram maiores nos animais infartos em comparação com o SHAM, mas sem diferença entre os grupos INF SED e INF EX (P<0,05). Entretanto, a extensão do infarto e o conteúdo de colágeno no miocárdio sobrevivente foram reduzidos pelo exercício físico, apesar da densidade capilar não ter sido alterada (P<0,05). O comprimento, a largura e o volume celular foram maiores no grupo INF EX (P<0,05), quando comparado aos demais grupos. Os animais exercitados previamente apresentam maior amplitude de contração e velocidades máximas de contração e relaxamento, quando comparados ao grupo INF SED (P<0,05). Além disso, o exercício físico prévio aumentou a dP/dt+ e reduziu a dP/dt- (P<0,05). Conclui-se que o programa de corrida em esteira realizado previamente ao IM atenuou os efeitos deletérios do IM sobre a morfologia e a função contrátil do miocárdio. As principais adaptações do exercício: a) redução do tamanho do IM; b) atenuação das alterações provocadas pelo IM na amplitude de contração e aumento da velocidade do ciclo contração-relaxamento de cardiomiócitos isolados; c) aumento do comprimento, largura e volume dos cardiomiócitos; d) redução da deposição de colágeno no miocárdio sobrevivente; e) atenuação das alterações provocadas pelo IM nos parâmetros hemodinâmicos.The aim of this study was to verify the effects of a treadmill running program previous to myocardial infarction (MI) on the myocardium morphology and contractile function of rats. One-month old male Wistar rats were randomly divided into three groups: sedentary infarcted (INF SED, n = 13), exercised infarcted (INF EX, n = 26) and simulated infarction (SHAM, n = 12). Before surgery animals from INF EX were subjected to a program of treadmill running (5 days / week, 1 hour/day, 18 m / min and inclination of 10 °) for 8 weeks. Then animals from INF EX and INF SED had the left descending coronary artery occluded with a suture thread. The same procedure was performed with SHAM animals, but without artery occlusion. Fifteen days after MI induction myocytes located at the border zone (2mm) of the infarcted area were isolated from some animals of each group and stimulated at 0.5 and 3 Hz at room temperature (~ 25 ° C) to measure cell contractile function at different extracellular calcium (0.6, 1 e 5 mM). The remaining animals were used to obtain hemodynamic records. After this procedure, the hearts were removed, fixed and stained with picrosirius red and Masson Trichrome for histological analysis. The results show that the exercised animals had a better running capacity than the sedentary ones (P <0.05). Treadmill running reduced the mortality of infarcted animals, but without statistical difference (P <0.05). Body weight, heart weight, relative weight of heart, lung weight, relative lung weight and lung fluid were not affected by the exercise program (P <0.05). However, the infarct size and collagen content in the surviving myocardium were reduced in previously exercised animals, although the capillary density was not changed (P <0.05). The length, width and cell volume were greater in INF EX group (P <0.05), compared to INF-SED. Exercised animals showed higher contraction amplitude and maximal velocity of contraction and relaxation when compared to sedentary control group (P <0.05). Furthermore, treadmill running increased the dP / dt + and reduced the xiii dP/dt- (P <0.05). It was concluded that the program of treadmill running attenuated the deleterious effects caused by MI on the morphology and contractile function of the myocardium. The main adaptations of exercise program were: a) attenuation of the changes in the amplitude of cell contraction and improvement of the contraction- relaxation cycle; b) increases in the length, width and volume of cardiomyocytes, c) reduction of collagen deposition in the surviving myocardium; and e) attenuation of the changes in the hemodynamic parameters

Characterization of endoplasmic reticulum stress-induced cardiac dysfunction: role of aerobic exercise training

As doenças cardiovasculares são a principal causa de morte no mundo, sendo a cardiomiopatia isquêmica a mais prevalente. Independente da sua etiologia, a via final comum da maioria das doenças cardiovasculares é a insuficiência cardíaca. Nos últimos anos, tem sido reportado que o acúmulo de proteínas mal enoveladas no retículo endoplasmático (estresse do RE) pode contribuir para redução da função cardíaca e instalação da insuficiência cardíaca. Apesar do mecanismo responsável pela disfunção contrátil induzida pelo estresse do RE ainda não ser conhecido, evidências sugerem que a inibição da via de sinalização PI3K/AKT pela proteína JNK pode estar envolvida nessa resposta. Na primeira parte desta tese, verificamos que a indução do estresse do RE em cardiomiócitos isolados ativou a JNK, mas não inibiu a via de sinalização PI3K/AKT. A inativação de JNK reverteu a disfunção contrátil e a redução da amplitude do transiente de Ca+2 de cardiomiócitos causados pelo estresse do RE. Pelo fato da via sinalização PI3K/AKT não estar envolvida na disfunção contrátil causada pelo estresse do RE, analisamos outro alvo de JNK, a proteína BNIP3, proteína pró-apoptótica e envolvida no controle de qualidade mitocondrial promovendo mitofagia quando ativada. O estresse do RE aumentou a expressão de BNIP3, a qual foi atenuada pela inibição de JNK. A depleção de BNIP3 impediu a disfunção contrátil dos cardiomiócitos e a redução da amplitude do transiente de Ca+2 induzidos pelo estresse do RE. Na segunda parte da tese, o objetivo foi avaliar se os efeitos observados em cardiomiócitos submetidos a estresse do RE poderiam ser observados em modelo experimental de doença cardiovascular. Nesse sentido, observamos que a disfunção cardíaca provocada pelo infarto do miocárdio em ratos foi acompanhada pelo quadro de estresse do RE e pela ativação da via de sinalização JNK/BNIP3. Entretanto, o treinamento físico aeróbico (TFA), uma das principais terapias não farmacológicas mais eficazes das doenças cardiovasculares, foi capaz de atenuar o estresse RE, a ativação da via de sinalização JNK/BNIP3 e a disfunção cardíaca de ratos infartados. Na terceira parte da tese, verificamos que o TFA aumentou a expressão proteica de DERLIN-1, uma proteína que atua retro-translocando proteínas mal enoveladas para o citosol, no miocárdio de ratos saudáveis. O aumento dos níveis proteicos de DERLIN-1 observado em ratos infartados foi atenuado pelo TFA. Apesar do aumento da proteína DERLIN-1, observamos que nos animais infartados as proteínas mal enoveladas acumulavam na forma de oligômeros e que o TFA atenuou essa resposta. Em conjunto, os resultados da presente tese sugerem que a ativação da via de sinalização JNK/BNIP3 pelo estresse do RE causa disfunção contrátil de cardiomiócitos e que o TFA é capaz de atenuar essa resposta no coração de ratos infartados, melhorando o controle de qualidade de proteína no músculo cardíacoCardiovascular diseases are currently the main cause of death worldwide, with the ischemic cardiomyopathy as the most prevalent ethiology. This is of particular interest, since ischemic cardiomyopathy advances to heart failure, a common endpoint of the most cardiovascular disease. In the last years, it has been showed that accumulation of unfolded protein in the endoplasmic reticulum (ER stress) may cause cardiac dysfunction and heart failure development. Despite the mechanisms behind this cardiac deterioration is still unknown, evidences suggest that ER stress-induced cardiomyocytes contractile dysfunction results from PI3K/AKT signaling pathway inhibition, which would be caused by JNK activation. In the first part of this thesis, we found that the ER stress activated JNK, but different from our hypothesis it was not accompanied by an inactivation of PI3K/AKT signaling pathway. The inhibition of JNK mitigated the reduction in cardiomyocytes shortening and amplitude of Ca+2 transient caused by ER stress. Once the PI3K/AKT signaling pathway was not involved in the ER stress-induced cardiomyocytes contractile dysfunction, we have analyzed protein expression of BNIP3, another JNK target involved in apoptosis and mitochondria quality control. We observed that the elevation in BNIP3 proteins levels after ER stress induction was prevented by inhibition of JNK. BNIP3 depletion attenuated the reduction in cardiomyocytes contractility and amplitude of Ca+2 transient induced by ER stress. In the second part of the thesis, we found that myocardial infarction-induced cardiac dysfunction in rats was accompanied by ER stress and activation of JNK/BNIP3 signaling pathway. However, the AET mitigated ER stress, activation of JNK/BNIP3 signaling pathway and cardiac dysfunction in infarcted rats. In third part of the thesis, we have identified that AET increased the protein expression of DERLIN-1 an ER membrane protein that retro-translocates unfolded proteins to cytosol in the myocardial of healthy rats. We observed that the increased DERLIN-1 protein levels in infarcted rats were mitigated by AET. Despite increased DERLIN-1 protein expression, we found high levels of oligomers in the myocardium of infarcted rat, which was reduced by AET. It suggests that unfolded protein degradation was reduced in infarcted hearts. Taken together, these results suggest that ER stress causes cardiomyocytes contractile dysfunction through JNK/BNIP3 signaling pathway activation and that AET mitigates the myocardial infarction-induced ER stress and activation of JNK/BNIP3 signaling pathway by restoring of ER-associated protein quality control in the cardiac muscl

Lactate up-regulates the expression of lactate oxidation complex-related genes in left ventricular cardiac tissue of rats.

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.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.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

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 programon 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

Activities of antioxidant enzymes and gene expression.

<p>Activities of (<b>A</b>) catalase and <b>(B)</b> SOD. mRNA levels of <b>(C)</b> NRF-1, <b>(D)</b> NRF-2, <b>(E)</b> SOD1, <b>(F)</b> SOD2 and <b>(G)</b> SOD3. <b>(H)</b> Nuclear NRF-2 expression in hearts perfused with KH or KH + lactate (20 mM) solutions during 120 min. Values are mean ± SE of 6–9 hearts; *indicates p<0.05 vs. control group.</p

pH values of perfusate during 120 min of perfusion with KH or KHL.

<p>KH = Krebs-Henseleit and KHL Krebs-Henseleit + Lactate (20 mM); Values are mean ± SE of 5–7 hearts.</p><p>pH values of perfusate during 120 min of perfusion with KH or KHL.</p

Hemodynamic parameters measured in perfused hearts.

<p><b>(A)</b> Left ventricular developed pressure, <b>(B)</b> Heart rate, <b>(C)</b> +dP/dt_<b>max</b>, <b>(D)</b>-dP/dt_<b>max</b> and <b>(E)</b> Perfusion pressure after 120 min of perfusion with KH or KH + lactate (20mM) solutions. Values are mean ± SE of 10–15 hearts; *indicates p<0.05 vs. control group.</p

Activities of enzymes involved in lactate turnorver.

<p>Activities of <b>(A)</b> HK, <b>(B)</b> LDH, <b>(C)</b> PDH, and <b>(D)</b> CS in hearts perfused with KH or KH + lactate (20 mM) solutions during 120 min. Values are mean ± SE of 6 hearts.</p