Ana C. Moreira

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.

Endurance training reverts heart mitochondrial dysfunction, permeability transition and apoptotic signaling in long-term severe hyperglycemia

The present study analyzed the effects of endurance training against cardiac mitochondrial dysfunction, particularly on the susceptibility to mitochondrial permeability transition pore (MPTP) induction in streptozotocin (STZ)-induced hyperglycemia. Twenty-four young male Wistar rats were randomly assigned into sedentary citrate (SED+CIT), sedentary type I diabetes (SED+STZ; 50mg/kg), T+CIT (14-week treadmill running, 60min/day) and T+STZ (injected 4weeks before training). After 18weeks, isolated heart mitochondria were used for in vitro oxygen consumption and transmembrane potential (∆Ψ) assessment. Cyclosporin-A (CyclA)-sensitive osmotic swelling and Ca(2+) fluxes were measured to study MPTP susceptibility. Voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), cyclophilin D (CypD), transcription factor A (Tfam), Bax, Bcl-2 contents, caspase-3 and -9 activities were determined. In the sedentary group, long-term severe hyperglycemia decreased state 3, CCCP-induced uncoupling and increased oligomycin-inhibited respiration, state 4 and lag phase with glutamate-malate. A decreased state 3 and state 4 with succinate were observed. Moreover, hyperglycemia decreased Ca(2+) uptake and increased CyclA-sensitive Ca(2+) release and Ca(2+)-induced mitochondrial swelling. The oxygen consumption and ∆Ψ parameters impaired by long-term severe hyperglycemia were reverted by endurance training (SED+STZ vs. T+STZ). Training increased mitochondrial Ca(2+) uptake and decreased Ca(2+) release in hyperglycemic groups. Additionally, endurance training reverted the hyperglycemia-induced CypD elevation, attenuating decrease of ANT, VDAC and Tfam. Moreover, training prevented the STZ-induced elevation in Bax, Bax-to-Bcl-2 ratio, caspase-3 and -9 and the increased Bcl-2. Endurance training reestablished heart mitochondrial respiratory dysfunction caused by long-term severe hyperglycemia and reduced the increased susceptibility to MPTP induction probably by modulation of MPTP regulatory proteins.

Isoproterenol Cytotoxicity is Dependent on the Differentiation State of the Cardiomyoblast H9c2 Cell Line

H9c2 cells are used as a surrogate for cardiac cells in several toxicological studies, which are usually performed with cells in their undifferentiated state, raising questions on the applicability of the results to adult cardiomyocytes. Since H9c2 myoblasts have the capacity to differentiate into skeletal and cardiac muscle cells under different conditions, the hypothesis of the present work was that cells in different differentiation states differ in their susceptibility to toxicants. In order to test the hypothesis, the effects of the cardiotoxicant isoproterenol (ISO) were investigated. The present work demonstrates that differentiated H9c2 cells are more susceptible to ISO toxicity. Cellular content of beta1-adrenergic receptors (AR), beta3-AR, and calcineurin is decreased as cells differentiate, as opposed to the content on the mitochondrial voltage-dependent anion channel (VDAC) and phosphorylated p38-MAPK, which increase. After ISO treatment, the pro-apoptotic protein Bax increases in all experimental groups, although only undifferentiated myoblasts up-regulate the anti-apoptotic Bcl-2. Calcineurin is decreased in differentiated H9c2 cells, which suggests an important role against ISO-induced cell death. The results indicate that the differentiation state of H9c2 myoblasts influence ISO toxicity, which may involve calcineurin, p38-MAPK, and Bax/Bcl-2 alterations. The data also provide new insights into cardiovascular toxicology during early development.

Phytoestrogens as alternative hormone replacement therapy in menopause: What is real, what is unknown.

Menopause is characterized by an altered hormonal status and by a decrease in life quality due to the appearance of uncomfortable symptoms. Nowadays, with increasing life span, women spend one-third of their lifetime under menopause. Understanding menopause-associated pathophysiology and developing new strategies to improve the treatment of menopausal-associated symptoms is an important topic in the clinic. This review describes physiological and hormone alterations observed during menopause and therapeutic strategies used during this period. We critically address the benefits and doubts associated with estrogen/progesterone-based hormone replacement therapy (HRT) and discuss the use of phytoestrogens (PEs) as a possible alternative. These relevant plant-derived compounds have structural similarities to estradiol, interacting with cell proteins and organelles, presenting several advantages and disadvantages versus traditional HRT in the context of menopause. However, a better assessment of PEs safety/efficacy would warrant a possible widespread clinical use.

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.

Differentiation-Dependent Doxorubicin Toxicity on H9c2 Cardiomyoblasts

A characteristic component of the anti-neoplastic doxorubicin (DOX)-induced cardiac toxicity is the delayed and persistent toxicity, with cancer childhood survivors developing cardiac failure later in life. The mechanisms behind this persistent toxicity are unknown, although one of the consequences of early childhood treatment with DOX is a specific removal of cardiac progenitor cells. DOX treatment may be more toxic to undifferentiated muscle cells, contributing to impaired cardiac development and toxicity persistence. H9c2 myoblasts, a rat embryonic cell line, which has the ability to differentiate into a skeletal or cardiac muscle phenotype, can be instrumental in understanding DOX cytotoxicity in different differentiation stages. H9c2 cell differentiation results in decreased cell proliferation and increased expression of a differentiated muscle marker. Differentiated H9c2 cells accumulated more DOX and were more susceptible to DOX-induced cytotoxicity. Differentiated cells had increased levels of mitochondrial superoxide dismutase and Bcl-xL, an anti-apoptotic protein. Of critical importance for the mechanisms of DOX toxicity, p53 appeared to be equally activated regardless of the differentiation state. We suggest that although more differentiated H9c2 muscle cells appear to have more basal mechanisms that would predict higher protection, DOX toxicity is higher in the differentiated population. The results are instrumental in the understanding of stress responses of this specific cell line in different differentiation stages to the cardiotoxicity caused by anthracyclines.

Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts

The cardiotoxicity induced by the anti-cancer doxorubicin involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the apoptosis-inducing factor from mitochondria is involved in doxorubicin toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent doxorubicin toxicity in this cell line. Doxorubicin treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, doxorubicin treatment of H9c2 cardiomyoblasts resulted in the appearance of 50kbp DNA fragments, a hallmark of apoptosis-inducing factor nuclear effects. Apoptosis-inducing factor knockdown using a small-interfering RNA approach in H9c2 cells resulted in a reduction of doxorubicin toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase cleavage. Among the proteases that could be responsible for apoptosis-inducing factor cleavage, doxorubicin decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing doxorubicin toxicity. Altogether, the results support that apoptosis-inducing factor release is involved in doxorubicin-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.

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.

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.

Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria

BACKGROUND Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are used in many African countries as food spices or in traditional medicine to treat several maladies. In this work, we (a) investigate whether the crude spice extracts present selective cytotoxicity for breast cancer cell lines and (b) investigate whether the same extracts affect the bioenergetics and calcium susceptibility of isolated liver mitochondrial fractions. RESULTS All extracts were cytotoxic to the cell lines studied, with the exception of MW, which was less toxic for a normal cell line. Interestingly, some of the extracts did not depolarize mitochondria in intact breast cancer MCF-7 cells, although this effect was observed in a normal breast cancer cell line (MCF-12A). All extracts increased hepatic mitochondrial state 2/4 respiration and decreased the respiratory control ratio and the transmembrane electric potential. Also, the extracts induced the mitochondrial permeability transition (MPT). CONCLUSIONS Mitochondrial toxicity may be part of the mechanism by which the spices tested cause inhibition of proliferation and death in the cell lines tested. This study also warrants caution in the excessive use of these spices for human consumption.