Dominique Fortin

Depressed mitochondrial transcription factors and oxidative capacity in rat failing cardiac and skeletal muscles

Congestive heart failure (CHF) induces alterations in energy metabolism and mitochondrial function that span cardiac as well as skeletal muscles. Whether these defects originate from altered mitochondrial DNA copy number and/or mitochondrial gene transcription is not known at present, nor are the factors that control mitochondrial capacity in different muscle types completely understood. We used an experimental model of CHF induced by aortic banding in the rat and investigated mitochondrial respiration and enzyme activity of biochemical mitochondrial markers in cardiac, slow and fast skeletal muscles. We quantified mitochondrial DNA (mtDNA), expression of nuclear (COX IV) and mitochondrial (COX I) encoded cytochrome c oxidase subunits as well as nuclear factors involved in mitochondrial biogenesis and in the necessary coordinated interplay between nuclear and mitochondrial genomes in health and CHF. CHF induced a decrease in oxidative capacity and mitochondrial enzyme activities with a parallel decrease in the mRNA level of COX I and IV, but no change in mtDNA content. The expression of the peroxisome proliferator activated receptor gamma co‐activator 1α (PGC‐1α) gene was downregulated in CHF, as well as nuclear respiratory factor 2 and mitochondrial transcription factor A, which act downstream from PGC‐1α. Most interestingly, only the level of PGC‐1α expression was strongly correlated with muscle oxidative capacity in cardiac and skeletal muscles, both in healthy and CHF rats. Mitochondrial gene transcription is reduced in CHF, and PGC‐1α appears as a potential modulator of muscle oxidative capacity under these experimental conditions.

Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle

We examined the transcriptional signal‐ ing cascade involved in the changes of mitochondrial biogenesis and mitochondrial function of skeletal mus‐ cle and of the exercise capacity of humans in response to long‐term physical activity and chronic heart failure (CHF). Biopsy samples of vastus lateralis muscle were obtained from 18 healthy subjects with different fitness levels (assessed by maximal oxygen uptake, VO2 peak). We compared 9 sedentary subjects with 10 CHF pa‐ tients undergoing transplantation. Muscle oxidative ca‐ pacity was measured in permeabilized fibers (Vmax). Transcript levels of target genes were quantified by RT‐PCR. In healthy subjects, VO2 peak was linearly related to Vmax (P<0.01) and to the gene expression of mitochondrial proteins and of the coactivator PGC‐1α and its downstream transcription factors. A coordinate increase in PGC‐1ɑ and mRNA levels of proteins involved in degradation, fusion, and fission of mitochondria was observed associated with calcineurin activation. Despite decreased VO2 peak, in CHF pa‐ tients skeletal muscles showed preserved Vmax in ac‐ cordance with preserved markers and transcription factors of mitochondrial biogenesis and dynamics, with no calcineurin activation. The results provide strong support for a central role for PGC‐1ɑ and calcineurin activation in mitochondrial biogenesis in healthy and diseased human skeletal muscles.—Garnier, A., Fortin, D., Zoll, J., NGuessan, B., Mettauer, B., Lampert, E., Veksler, V., Ventura‐Clapier, R. Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle. FASEB J. 19, 43‐52 (2005)

Physical activity changes the regulation of mitochondrial respiration in human skeletal muscle

This study explores the importance of creatine kinase (CK) in the regulation of muscle mitochondrial respiration in human subjects depending on their level of physical activity. Volunteers were classified as sedentary, active or athletic according to the total activity index as determined by the Baecke questionnaire in combination with maximal oxygen uptake values (peak V̇O2, expressed in ml min−1 kg−1). All volunteers underwent a cyclo‐ergometric incremental exercise test to estimate their peak V̇O2 and V̇O2 at the ventilatory threshold (VT). Muscle biopsy samples were taken from the vastus lateralis and mitochondrial respiration was evaluated in an oxygraph cell on saponin permeabilised muscle fibres in the absence (V̇0) or in the presence (V̇max) of saturating [ADP]. While V̇0 was similar, V̇max differed among groups (sedentary, 3.7 ± 0.3, active, 5.9 ± 0.9 and athletic, 7.9 ± 0.5 μmol O2 min−1 (g dry weight)−1). V̇max was correlated with peak V̇O2 (P < 0.01, r= 0.63) and with V̇T (P < 0.01, r= 0.57). There was a significantly greater degree of coupling between oxidation and phosphorylation (V̇max/V̇0) in the athletic individuals. The mitochondrial Km for ADP was significantly higher in athletic subjects (P < 0.01). Mitochondrial CK (mi‐CK) activation by addition of creatine induced a marked decrease in Km in athletic individuals only, indicative of an efficient coupling of mi‐CK to ADP rephosphorylation in the athletic subjects only. It is suggested that increasing aerobic performance requires an enhancement of both muscle oxidative capacity and mechanisms of respiratory control, attesting to the importance of temporal co‐ordination of energy fluxes by CK for higher efficacy.

Functional Adiponectin Resistance at the Level of the Skeletal Muscle in Mild to Moderate Chronic Heart Failure

Background—Adiponectin is an antiinflammatory, insulin-sensitizing, and antiatherogenic adipocytokine that plays a fundamental role in energy homeostasis. In patients with chronic heart failure (CHF), high circulating adiponectin levels are associated with inverse outcome. Recently, adiponectin expression has been identified in human skeletal muscle fibers. We investigated the expression of adiponectin, the adiponectin receptors, and genes involved in the downstream lipid and glucose metabolism in the skeletal muscle of patients with CHF. Methods and Results—Muscle biopsies (vastus lateralis muscle) were obtained from 13 patients with CHF and 10 healthy subjects. mRNA transcript levels of adiponectin, adiponectin receptors (AdipoR1 and AdipoR2), and downstream adiponectin-related enzymes were quantified by real-time reverse transcriptase polymerase chain reaction. Adiponectin expression in the skeletal muscle of patients with CHF was 5-fold higher than in healthy subjects (P<0.001), whereas AdipoR1 was downregulated (P=0.005). In addition, the expression of the main genes involved in downstream pathway (peroxisome proliferator-activated receptor-&agr; [PPAR-&agr;] and both AMP-activated protein kinase-&agr;1 and -&agr;2 subunits) as well as their target genes in lipid (acyl-coenzyme A dehydrogenase C-14 to C-12 straight chain) and glucose metabolism (hexokinase-2) were significantly reduced in CHF. The strong positive correlation found between AdipoR1 and PPAR-&agr;/AMP-activated protein kinase gene expression was confirmed in PPAR-&agr; null mice, suggesting a cause-and-effect relationship. Immunohistochemical staining confirmed the presence of adiponectin in the skeletal muscle. Conclusions—Despite increased adiponectin expression in the skeletal muscle, patients with CHF are characterized by downregulation of AdipoR1 that is most probably linked to deactivation of the PPAR-&agr;/AMP-activated protein kinase pathway. These facts suggest functional adiponectin resistance at the level of the skeletal muscle in CHF.

Down-regulation of OPA1 alters mouse mitochondrial morphology, PTP function, and cardiac adaptation to pressure overload

AIMSnThe optic atrophy 1 (OPA1) protein is an essential protein involved in the fusion of the mitochondrial inner membrane. Despite its high level of expression, the role of OPA1 in the heart is largely unknown. We investigated the role of this protein in Opa1(+/-) mice, having a 50% reduction in OPA1 protein expression in cardiac tissue.nnnMETHODS AND RESULTSnIn mutant mice, cardiac function assessed by echocardiography was not significantly different from that of the Opa1(+/+). Electron and fluorescence microscopy revealed altered morphology of the Opa1(+/-) mice mitochondrial network; unexpectedly, mitochondria were larger with the presence of clusters of fused mitochondria and altered cristae. In permeabilized mutant ventricular fibres, mitochondrial functional properties were maintained, but direct energy channelling between mitochondria and myofilaments was weakened. Importantly, the mitochondrial permeability transition pore (PTP) opening in isolated permeabilized cardiomyocytes and in isolated mitochondria was significantly less sensitive to mitochondrial calcium accumulation. Finally, 6 weeks after transversal aortic constriction, Opa1(+/-) hearts demonstrated hypertrophy almost two-fold higher (P< 0.01) than in wild-type mice with altered ejection fraction (decrease in 43 vs. 22% in Opa1(+/+) mice, P< 0.05).nnnCONCLUSIONSnThese results suggest that, in adult cardiomyocytes, OPA1 plays an important role in mitochondrial morphology and PTP functioning. These properties may be critical for cardiac function under conditions of chronic pressure overload.

Activation of AMP kinase α1 subunit induces aortic vasorelaxation in mice

Vasodilatation is a vital mechanism of systemic blood flow regulation that occurs during periods of increased energy demand. The AMP‐dependent protein kinase (AMPK) is a serine/threonine kinase that is activated by conditions that increase the AMP‐to‐ATP ratio, such as exercise and metabolic stress. We hypothesized that AMPK could trigger vasodilatation and participate in blood flow regulation. Rings of thoracic aorta were isolated from C57Bl6 mice and mice deficient in the AMPK catalytic α1 (AMPKα1−/−) or α2 (AMPKα2−/−) subunit and their littermate controls, and mounted in an organ bath. Aortas were preconstricted with phenylephrine (1 μm) and activation of AMPK was induced by addition of increasing concentrations of 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR). AICAR (0.1–3 mm) dose‐dependently induced relaxation of precontracted C57BL6, AMPKα1+/+ and α2+/+ aorta (P < 0.001, n= 5–7 per group). This AICAR induced vasorelaxation was not inhibited by the addition of adenosine receptor antagonists. Moreover, when aortic rings were freed of endothelium by gentle rubbing, AICAR still induced aortic ring relaxation, suggesting a direct effect of AICAR on smooth muscle cells. When aortic rings were pretreated with l‐NMMA (30 μm) to inhibit nitric oxide synthase activity, AICAR still induced relaxation. Western blot analysis of C57Bl6 mice denuded aorta showed that AMPK was phosphorylated after incubation with AICAR and that AMPKα1 was the main catalytic subunit expressed. Finally, AICAR‐induced relaxation of aortic rings was completely abolished in AMPKα1−/− but not AMPKα2−/− mice. Taken together, the results show that activation of AMPKα1 but not AMPKα2 is able to induce aortic relaxation in mice, in an endothelium‐ and eNOS‐independent manner.

Resveratrol Improves Survival, Hemodynamics and Energetics in a Rat Model of Hypertension Leading to Heart Failure

Heart failure (HF) is characterized by contractile dysfunction associated with altered energy metabolism. This study was aimed at determining whether resveratrol, a polyphenol known to activate energy metabolism, could be beneficial as a metabolic therapy of HF. Survival, ventricular and vascular function as well as cardiac and skeletal muscle energy metabolism were assessed in a hypertensive model of HF, the Dahl salt-sensitive rat fed with a high-salt diet (HS-NT). Resveratrol (18 mg/kg/day; HS-RSV) was given for 8 weeks after hypertension and cardiac hypertrophy were established (which occurred 3 weeks after salt addition). Resveratrol treatment improved survival (64% in HS-RSV versus 15% in HS-NT, p<0.001), and prevented the 25% reduction in body weight in HS-NT (P<0.001). Moreover, RSV counteracted the development of cardiac dysfunction (fractional shortening −34% in HS-NT) as evaluated by echocardiography, which occurred without regression of hypertension or hypertrophy. Moreover, aortic endothelial dysfunction present in HS-NT was prevented in resveratrol-treated rats. Resveratrol treatment tended to preserve mitochondrial mass and biogenesis and completely protected mitochondrial fatty acid oxidation and PPARα (peroxisome proliferator-activated receptor α) expression. We conclude that resveratrol treatment exerts beneficial protective effects on survival, endothelium–dependent smooth muscle relaxation and cardiac contractile and mitochondrial function, suggesting that resveratrol or metabolic activators could be a relevant therapy in hypertension-induced HF.

Preserved response of Mitochondrial function to short-term endurance training in skeletal muscle of heart transplant recipients

OBJECTIVESnWe sought to determine whether intrinsic mitochondrial function and regulation were altered in heart transplant recipients (HTRs) and to investigate the response of mitochondrial function to six-week endurance training in these patients.nnnBACKGROUNDnDespite the normalization of central oxygen transport during exercise, HTRs are still characterized by limited exercise capacity, which is thought to result from skeletal muscle metabolic abnormalities.nnnMETHODSnTwenty HTRS agreed to have vastus lateralis biopsies and exercise testing: before and after training for 12 of them and before and after the same control period for eight subjects unwilling to train. Mitochondrial respiration was evaluated on saponin-permeabilized muscle fibers in the absence or presence (maximum respiration rate [V(max)]) of saturating adenosine diphosphate.nnnRESULTSnMitochondrial function was preserved at the level of sedentary subjects in untrained HTRs, although they showed 28 +/- 5% functional aerobic impairment (FAI). After training, V(max), citrate synthase, cytochrome c oxidase, and mitochondrial creatine kinase (CK) activities were significantly increased by 48%, 40%, 67%, and 53%, respectively (p < 0.05), whereas FAI decreased to 12 +/- 5% (p < 0.01). The control of mitochondrial respiration by creatine and mitochondrial CK was also improved (p < 0.01), suggesting that phosphocreatine synthesis and transfer by the mitochondrial CK become coupled to oxidative phosphorylation, as shown in trained, healthy subjects.nnnCONCLUSIONSnIn HTRs, the mitochondrial properties of skeletal muscle were preserved and responded well to training, reaching values of physically active, healthy subjects. This suggests that, in HTRs, immunosuppressive drugs do not alter the intrinsic muscle oxidative capacities and that the patients physical handicap results from nonmitochondrial mechanisms.

Control by circulating factors of mitochondrial function and transcription cascade in heart failure: a role for endothelin-1 and angiotensin II.

Background—Evidence is emerging to support the concept that the failing heart is “energy depleted” and that defects in energy metabolism are important determinants in the development and the progression of the disease. We have shown previously that depressed mitochondrial function in cardiac and skeletal muscles in chronic heart failure is linked to decreased expression of the gene encoding transcriptional proliferator-activated receptor-γ coactivator-1α, the inducible regulator of mitochondrial biogenesis and its transcription cascade, leading to altered expression of mitochondrial proteins. However, oxidative capacity of the myocardium of patients treated for chronic heart failure and pathophysiological mechanisms of mitochondrial dysfunction are still largely unknown. Methods and Results—In patients with chronic heart failure treated with angiotensin-converting enzyme inhibition, cardiac oxidative capacity, measured in saponin-permeabilized fibers, was 25% lower, and proliferator-activated receptor-γ coactivator-1α protein content was 34% lower compared with nonfailing controls. In a rat model of myocardial infarction, angiotensin-converting enzyme inhibition therapy was only partially able to protect cardiac mitochondrial function and transcription cascade. Expression of proliferator-activated receptor-γ coactivator-1α and its transcription cascade were evaluated after a 48-hour exposure of cultured adult rat ventricular myocytes to endothelin-1, angiotensin II, aldosterone, phenylephrine, or isoprenaline. Endothelin-1 (−30%) and, to a lesser degree, angiotensin II (−20%) decreased proliferator-activated receptor-γ coactivator-1α mRNA content, whereas other hormones had no effect (phenylephrine) or even increased it (aldosterone, isoprenaline). Conclusions—Taken together, these results show that, despite angiotensin-converting enzyme inhibition treatment, oxidative capacity is reduced in human and experimental heart failure and that endothelin-1 and angiotensin II could be involved in the downregulation of the mitochondrial transcription cascade.

Differential effects of thyroid hormones on energy metabolism of rat slow- and fast-twitch muscles.

Thyroid hormone (TH) is an important regulator of mitochondrial content and activity. As mitochondrial content and properties differ depending on muscle‐type, we compared mitochondrial regulation and biogenesis by T3 in slow‐twitch oxidative (soleus) and fast‐twitch mixed muscle (plantaris). Male Wistar rats were treated for 21 to 27 days with T3 (200 μg/kg/day). Oxidative capacity, regulation of mitochondrial respiration by substrates and phosphate acceptors, and transcription factors were studied. In soleus, T3 treatment increased maximal oxygen consumption (Vmax) and the activities of citrate synthase (CS) and cytochrome oxidase (COX) by 100%, 45%, and 71%, respectively (Pu2009<u20090.001), whereas in plantaris only Vmax increased, by 39% (Pu2009<u20090.01). ADP‐independent respiration rate was increased in soleus muscle by 216% suggesting mitochondrial uncoupling. Mitochondrial substrate utilization in soleus was also influenced by T3, as were mitochondrial enzymes. Lactate dehydrogenase (LDH) activity was elevated in soleus and plantaris by 63% and 11%, respectively (Pu2009<u20090.01), and soleus creatine kinase was increased by 48% (Pu2009<u20090.001). T3 increased the mRNA content of the transcriptional co‐activator of mitochondrial genes, PGC‐1α, and the I and IV COX subunits in soleus. The muscle specific response to thyroid hormones could be explained by a lower content of TH receptors in plantaris than soleus. Moreover, TRα mRNA level decreased further after T3 treatment. These results demonstrate that TH has a major effect on mitochondrial content, regulation and coupling in slow oxidative muscle, but to a lesser extent in fast muscle, due to the high expression of TH receptors and PGC‐1α transcription factor.