Inês O. Gonçalves

Acute exercise protects against calcium-induced cardiac mitochondrial permeability transition pore opening in doxorubicin-treated rats

The use of DOX (doxorubicin), an antibiotic used in oncological treatments, is limited by a dose-related cardiotoxicity against which acute exercise is protective. However, the mitochondrial-related mechanisms of this protection remain unknown. Therefore the present study aimed to determine the effects of an acute endurance exercise bout performed 24 h before DOX treatment on heart and liver mitochondrial function. A total of 20 adult male Wistar rats were divided into groups as follows: non-exercised with saline (NE + SAL), non-exercised DOX-treated (NE + DOX), exercised with saline (EX + SAL) and exercised DOX-treated (EX + DOX). The animals performed a 60 min exercise bout on a treadmill or remained sedentary 24 h before receiving either a DOX bolus (20 mg/kg of body weight) or saline. Heart and liver mitochondrial function [oxygen consumption, membrane potential (DeltaPsi) and cyclosporin-A-sensitive calcium-induced MPTP (mitochondrial permeability transition pore) opening] were evaluated. The activities of the respiratory complex, Mn-SOD (superoxide dismutase), caspases 3 and 9, as well as the levels of ANT (adenine nucleotide translocase), VDAC (voltage-dependent anion channel), CypD (cyclophilin D), Bax and Bcl-2, were measured. Acute exercise prevented the decreased cardiac mitochondrial function (state 3, phosphorylative lagphase; maximal DeltaPsi generated both with complex I- and II-linked substrates and calcium-induced MPTP opening) induced by DOX treatment. Exercise also prevented the DOX-induced decreased activity of cardiac mitochondrial chain complexes I and V, and increased caspase 3 and 9 activities. DOX administration and exercise caused increased cardiac mitochondrial SOD activity. Exercise ameliorated liver mitochondrial complex activities. No alterations were observed in the measured MPTP and apoptosis-related proteins in heart and liver mitochondria. The results demonstrate that acute exercise protects against cardiac mitochondrial dysfunction, preserving mitochondrial phosphorylation capacity and attenuating DOX-induced decreased tolerance to MPTP opening.

Modulation of cardiac mitochondrial permeability transition and apoptotic signaling by endurance training and intermittent hypobaric hypoxia

BACKGROUND Modulation of the mitochondrial permeability transition pore (MPTP) and inhibition of the apoptotic signaling are critically associated with the cardioprotective phenotypes afforded by both intermittent hypobaric-hypoxia (IHH) and endurance-training (ET). We recently proposed that IHH and ET improve cardiac function and basic mitochondrial capacity, although without showing addictive effects. Here we investigate whether a combination of IHH and ET alters cardiac mitochondrial vulnerability to MPTP and related apoptotic signaling. METHODS Male Wistar rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE, 1h/day/5 week treadmill-running), hypoxic-sedentary (HS, 6000 m, 5h/day/5 weeks) and hypoxic-exercised (HE) to study susceptibility to calcium-induced cardiac MPTP opening. Mitochondrial cyclophilin D (CypD), adenine nucleotide translocator (ANT), Bax and Bcl-2 protein contents were semi-quantified by Western blotting. Cardiac caspase 3-, 8- and 9-like activities were measured. Mitochondrial aconitase and superoxide dismutase (MnSOD) activity and malondialdehyde (MDA) and sulphydryl group (-SH) content were determined. RESULTS Susceptibility to MPTP decreased in NE and HS vs. NS and even further in HE. The ANT content increased in HE vs. NS. Bcl-2/Bax ratio increased in NE and HS compared to NS. Decreased activities in tissue caspase 3-like (HE vs. NS) and caspase 9-like (HS and HE vs. NS) were observed. Mitochondrial aconitase increased in NE and HS vs. NS. No alterations between groups were observed for caspase 8-like activity, MnSOD, CypD, MDA and -SH. CONCLUSIONS Data confirm that IHH and ET modulate cardiac mitochondria to a protective phenotype characterized by decreased MPTP induction and apoptotic signaling, although without visible addictive effects as initially hypothesized.

Exercise as a therapeutic tool to prevent mitochondrial degeneration in nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease, encompassing hepatic steatosis, nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, is a significant health problem associated with modern lifestyle, based on caloric overconsumption and physical inactivity. Although the mechanisms associated with progression from the ‘benign’ steatosis to NASH are still elusive, mitochondrial dysfunction seems to play an important role in this degenerative process. Degeneration of mitochondrial function during NASH has been associated with impaired β‐oxidation, oxidative phosphorylation and increased reactive oxygen species production, contributing to hepatocyte death and inflammatory response. Despite the fact that several therapeutic approaches can be used in the context of NASH, including insulin‐sensitizing agents, anti‐obesity drugs, lipid‐lowering drugs or mitochondrial‐targeted drugs, dietary and physical activity are still the most effective strategies. In fact, active lifestyles decrease insulin resistance and body weight and result in decreased histological signs of liver injury. In fatty liver, physical activity prevents the disease progression through mitochondrial adaptations, namely by increasing cytochrome c content, enzyme activities and fatty acid oxidation, which are lost after some days of physical inactivity. However, less is known about the effect of physical activity on NASH‐associated mitochondrial dysfunction. After a brief characterization of NASH and its association with liver mitochondrial (dys)function, the present review addresses the impact of physical (in)activity on NASH and, particularly, the possible contribution of active lifestyles to the modulation of liver mitochondrial dysfunction.

Modulation of hepatic redox status and mitochondrial metabolism by exercise: therapeutic strategy for liver diseases.

Liver steatosis (non-alcoholic fatty liver disease, NAFLD) is deemed as the hepatic face of the metabolic syndrome, with both physical inactivity and hypercaloric/unbalanced diet, together with increasing age playing a role as predisposing factors. Consequently, one of the most effective strategies used to counteract this scenario is physical exercise. Given the importance of redox signaling in cellular remodeling, in which mitochondria are closely implicated along with important roles on substrate oxidation, here we briefly review the effects of both acute and chronic forms of physical exercise on the modulation of hepatic redox state, highlighting the relevance of mitochondrial metabolism and function in the induction of liver phenotypes that antagonize metabolic alterations associated with liver metabolic diseases.

Physical exercise prevents and mitigates non-alcoholic steatohepatitis-induced liver mitochondrial structural and bioenergetics impairments.

Exercise is considered a non-pharmacological tool against several lifestyle disorders in which mitochondrial dysfunction is involved. The present study aimed to analyze the preventive (voluntary physical activity-VPA) and therapeutic (endurance training-ET) role of exercise against non-alcoholic steatohepatitis (NASH)-induced liver mitochondrial dysfunction. Sixty male Sprague-Dawley rats were divided into standard-diet sedentary (SS, n=20), standard-diet VPA (SVPA, n=10), high-fat diet sedentary (HS, n=20) and high-fat diet VPA (HVPA, n=10). After 9weeks of diet-treatment, half of SS and HS animals were engaged in an ET program (SET and HET) for 8weeks, 5days/week and 60min/day. Liver mitochondrial oxygen consumption and transmembrane-electric potential (ΔΨ) were evaluated in the presence of glutamate-malate (G/M), palmitoyl-malate (P/M) and succinate (S/R). Mitochondrial enzymes activity, lipid and protein oxidation, oxidative phosphorylation (OXPHOS) subunits, cytochrome c, adenine nucleotide translocator (ANT) and uncoupling protein-2 (UCP2) content were assessed. HS groups show the histological features of NASH in parallel with decreased ΔΨ and respiratory control (RCR) and ADP/O ratios (G/M and P/M). A state 3 decrease (G/M and S/R), FCCP-induced uncoupling respiration (S/R) and ANT content were also observed. Both exercise types counteracted oxygen consumption (RCR, ADP/O and FCCP-uncoupling state) impairments and improved ΔΨ (lag-phase). In conclusion, exercise prevented or reverted (VPA and ET, respectively) the bioenergetic impairment induced by NASH, but only ET positively remodeled NASH-induced liver structural damage and abnormal mitochondria. It is possible that alterations in inner membrane integrity and fatty acid oxidation may be related to the observed phenotypes induced by exercise.

Endurance training and chronic intermittent hypoxia modulate in vitro salicylate-induced hepatic mitochondrial dysfunction.

Mitochondrial function is modulated by multiple approaches including physical activity, which can afford cross-tolerance against a variety of insults. We therefore aimed to analyze the effects of endurance-training (ET) and chronic-intermittent hypobaric-hypoxia (IHH) on liver mitochondrial bioenergetics and whether these effects translate into benefits against in vitro salicylate mitochondrial toxicity. Twenty-eight young-adult male rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE), hypoxic-sedentary (HS) and hypoxic-exercised (HE). ET consisted of 1h/days of treadmill running and IHH of simulated atmospheric pressure of 49.3 kPa 5h/days during 5weeks. Liver mitochondrial oxygen consumption, transmembrane-electric potential (ΔΨ) and permeability transition pore induction (MPTP) were evaluated in the presence and absence of salicylate. Aconitase, MnSOD, caspase-3 and 8 activities, SH, MDA, SIRT3, Cyp D, HSP70, and OXPHOS subunit contents were assessed. ET and IHH decreased basal mitochondrial state-3 and state-4 respiration, although no alterations were observed in ΔΨ endpoints evaluated in control mitochondria. In the presence of salicylate, ET and IHH decreased state-4 and lag-phase of ADP-phosphorylation. Moreover, ADP-lag phase in hypoxic was further lower than in normoxic groups. Neither ET nor IHH altered the susceptibility to calcium-induced MPTP. IHH lowered MnSOD and increased aconitase activities. ET and IHH decreased caspase 8 activity whereas no effect was observed on caspase 3. The levels of SIRT3 increased with ET and IHH and Cyp D decreased with IHH. Data suggest that ET and IHH do not alter general basal liver mitochondrial function, but may attenuate some adverse effects of salicylate.

Exercise mitigates mitochondrial permeability transition pore and quality control mechanisms alterations in nonalcoholic steatohepatitis

Mitochondrial quality control and apoptosis have been described as key components in the pathogenesis of nonalcoholic steatohepatitis (NASH); exercise is recognized as a nonpharmacological strategy to counteract NASH-associated consequences. We aimed to analyze the effect of voluntary physical activity (VPA) and endurance training (ET) against NASH-induced mitochondrial permeability transition pore (mPTP) opening and mitochondrial and cellular quality control deleterious alterations. Forty-eight male Sprague-Dawley rats were divided into standard-diet sedentary (SS, n = 16), standard-diet VPA (n = 8), high-fat diet sedentary (HS, n = 16), and high-fat diet VPA (n = 8). After 9 weeks of diet treatment, half of the SS and HS groups were engaged in an ET program for 8 weeks, 5 days/week, 1 h/day. Liver mPTP susceptibility through osmotic swelling, mPTP-related proteins (cyclophilin D, Sirtuin3, Cofilin-1), markers of mitochondrial biogenesis ((mitochondrial transcription factor A (Tfam) and peroxisome proliferator-activated receptor gamma co-activator protein (PGC-1α)), dynamics (Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2), Dynamin related protein 1, and Optic atrophy 1)), auto/mitophagy (Beclin-1, microtubule-associated protein 1 light chain 3, p62, PINK1, and Parkin), and apoptotic signaling (Bax, Bcl-2) and caspases-like activities were assessed. HS animals showed an increased susceptibility to mPTP, compromised expression of Tfam, Mfn1, PINK1, and Parkin and an increase in Bax content (HS vs. SS). ET and VPA improved biogenesis-related proteins (PGC-1α) and autophagy signaling (Beclin-1 and Beclin-1/Bcl-2 ratio) and decreased apoptotic signaling (caspases 8 activity, Bax content, and Bax/Bcl-2 ratio). However, only ET decreased mPTP susceptibility and positively modulated Bcl-2, Tfam, Mfn1, Mfn2, PINK1, and Parkin content. In conclusion, exercise reduces the increased susceptibility to mPTP induced by NASH and promotes the increase of auto/mitophagy and mitochondrial fusion towards a protective phenotype.

Exercise alters liver mitochondria phospholipidomic profile and mitochondrial activity in non-alcoholic steatohepatitis

Mitochondrial membrane lipid composition is a critical factor in non-alcoholic steatohepatitis (NASH). Exercise is the most prescribed therapeutic strategy against NASH and a potential modulator of lipid membrane. Thus, we aimed to analyze whether physical exercise exerted preventive (voluntary physical activity - VPA) and therapeutic (endurance training - ET) effect on NASH-induced mitochondrial membrane changes. Sprague-Dawley rats (n=36) were divided into standard-diet sedentary (SS, n=12), standard-diet VPA (SVPA, n=6), high-fat diet sedentary (HS, n=12) and high-fat diet VPA (HVPA, n=6). After 9 weeks of diet-specific feeding, half of SS and HS group were engaged in an ET program for 8 weeks/5 day/week/1h/day (SET, HET). Liver mitochondria were isolated for oxygen consumption and transmembrane-electric potential (ΔΨ) assays. Mitochondrial phospholipid classes and fatty acids were quantified through thin layer chromatography and gas chromatography, respectively, while cardiolipin (CL), phosphatidylcholine (PC) phosphatidylethanolamine (PE) and phosphatidylinositol (PI) molecular profile was determined by electrospray mass spectrometry. In parallel with histological signs of NASH, high-fat diet decreased PI, CL and PC/PE ratio, whereas PE and phosphatidic acid content increased in sedentary animals (HS vs. SS). Moreover, a decrease in linolelaidic, monounsaturated fatty acids content and an increase in saturated fatty acids (SFAS) were observed. Along with phospholipidomic alterations, HS animals showed a decrease in respiratory control ratio (RCR), ΔΨ and FCCP-induced uncoupling respiration (HS vs. SS). Both phospholipidomic (PC/PE, SFAS) and mitochondrial respiratory alterations were counteracted by exercise interventions. Exercise used as preventive (VPA) or therapeutic (ET) strategies preserved liver mitochondrial phospholipidomic profile and maintained mitochondrial function in a model of NASH.

Synergistic impact of endurance training and intermittent hypobaric hypoxia on cardiac function and mitochondrial energetic and signaling

BACKGROUND Intermittent hypobaric-hypoxia (IHH) and endurance-training (ET) are cardioprotective strategies against stress-stimuli. Mitochondrial modulation appears to be an important step of the process. This study aimed to analyze whether a combination of these approaches provides additive or synergistic effects improving heart-mitochondrial and cardiac-function. METHODS Two-sets of rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE, 1 h/day/5 weeks treadmill-running), hypoxic-sedentary (HS, 6000 m, 5h/day/5 weeks) and hypoxic-exercised (HE) to study overall cardiac and mitochondrial function. In vitro cardiac mitochondrial oxygen consumption and transmembrane potential were evaluated. OXPHOS subunits and ANT protein content were semi-quantified by Western blotting. HIF-1α, VEGF, VEGF-R1 VEGF-R2, BNP, SERCA2a and PLB expressions were measured by qRT-PCR and cardiac function was characterized by echocardiography and hemodynamic parameters. RESULTS Respiratory control ratio (RCR) increased in NE, HS and HE vs. NS. Susceptibility to anoxia/reoxygenation-induced dysfunction decreased in NE, HS and HE vs. NS. HS decreased mitochondrial complex-I and -II subunits; however HE completely reverted the decreased content in complex-II subunits. ANT increased in HE. HE presented normalized ventricular-arterial coupling (Ea) and BNP myocardial levels and significantly improved myocardial performance as evaluated by increased cardiac output and normalization of the Tei index vs. HS CONCLUSION Data demonstrates that IHH and ET confer cardiac mitochondria with a more resistant phenotype although without visible addictive effects at least under basal conditions. It is suggested that the combination of both strategies, although not additive, results into improved cardiac function.

Physical exercise antagonizes clinical and anatomical features characterizing Lieber-DeCarli diet-induced obesity and related metabolic disorders

BACKGROUND & AIMS Lieber-DeCarli diet has been used to induce obesity and non-alcoholic steatohepatitis (NASH). As scarce anatomical and clinical-related information on this diet model exists and being exercise an advised strategy to counteract metabolic diseases, we aimed to analyze the preventive (voluntary physical activity - VPA) and therapeutic (endurance training - ET) effect of exercise on clinical/anatomical features of rats fed with Lieber-DeCarli diet. METHODS In the beginning of the protocol, Sprague-Dawley rats were divided into standard-diet sedentary (SS, n = 20), standard-diet VPA (SVPA, n = 10), high-fat diet sedentary (HS, n = 20) and high-fat diet VPA (HVPA, n = 10) groups. After 9-weeks, half (n = 10) of SS and HS groups were engaged in an ET program (8 wks/5 d/wk/60 min/day). At this time, a blood sample was collected for biochemical analysis. At the end of protocol (17-weeks) anatomic measures were assessed. Heart, liver, femur and visceral fat were weighted and blood was collected again. Liver section was used for histopathological examination. RESULTS At 17-weeks, high-fat diet increased visceral adiposity (HS vs. SS), which was counteracted by both exercises. However, ET was the only intervention able to diminished obesity-related measures and the histological features of NASH. Moreover, blood analysis at 9 weeks showed that high-fat diet increased ALT, AST, cholesterol and HDL while VLDL and TG levels were decreased (HS vs. SS). Notably, although these parameters were counteracted after 9-weeks of VPA, they were transitory and not observed after 17-weeks. CONCLUSIONS ET used as a therapeutic tool mitigated the clinical/anatomical-related features induced by Liber-DeCarli diet, thus possibly contributing to control obesity and metabolic disorders.