Gender issues in cardiovascular diseases. Focus on energy metabolism.

It is increasingly recognized that sex and gender differences (S&G) influence cardiovascular diseases (CVD), greatly impacting disease management. In terms of definition, sex refers to biological aspects, gender effects being mainly related to socio-cultural factors. Both sex and gender are interpenetrated in humans and difficult to separate. This is more clearly feasible in animal models where sex effects largely predominate. As alterations in energy metabolism are essential features of cardiovascular diseases, sexual dimorphism of energy metabolism and more specifically mitochondria occupies a place of choice. This review presents the basis of sex and gender differences in the cardiovascular pathophysiology, and how it mainly affects woman diseases, effectiveness of therapies and clinical outcome. These differences rely on complex molecular mechanisms that are still poorly understood because of the under-representation of females/women in experimental and clinical studies. Finally, the differing psychological and biological phases of woman's life are largely underestimated. This review presents an overview of the field with focus on differences in cardiac energy metabolism, which are illustrated with specific examples.

Skeletal Muscle Response to Endurance Training in IL-6-/- Mice.

We examined effects of moderate-intensity endurance training on muscle COX/CS activities and V'O2max in control WT and IL-6(-/-) mice. Animals were exercised for 10 weeks on treadmill for 1 h, 5 days a week at velocity of 6 m·min(-1) which was increased by 0.5 m·min(-1) every 2 weeks up to 8 m·min(-1) . Training triggered an increase of enzyme activities in soleus muscle of WT mice (COX: 480.3±8.9 U·g(-1) in sedentary group vs. 773.3±62.6 U·g(-1) in trained group, P<0.05 and CS: 374.0±6.0 U·g(-1) in sedentary group vs. 534.2±20.5 U·g(-1) in trained group, P<0.01, respectively) whereas no changes were observed in soleus of IL6(-/-) mice. Moreover, in mixed gastrocnemius muscle of trained IL-6(-/-) mice enzyme activities tended to be lower (COX: 410.7±48.4 U·g(-1) for sedentary vs. 277.0±36.5 U·g(-1) for trained group and CS: 343.8±24.6 U·g(-1) for sedentary vs. 251.7±27.1 U·g(-1) for trained group). No changes in V'O2max were observed in WT and IL-6(-/-) mice after training. Concluding, moderate-velocity endurance training-induced increase in COX and CS activities in muscles of WT mice only which suggests that IL-6 regulates training-induced skeletal muscle responses to exercise.

Altered skeletal muscle mitochondrial biogenesis but improved endurance capacity in trained OPA1-deficient mice.

The role of OPA1, a GTPase dynamin protein mainly involved in the fusion of inner mitochondrial membranes, has been studied in many cell types, but only a few studies have been conducted on adult differentiated tissues such as cardiac or skeletal muscle cells. Yet OPA1 is highly expressed in these cells, and could play different roles, especially in response to an environmental stress like exercise. Endurance exercise increases energy demand in skeletal muscle and repeated activity induces mitochondrial biogenesis and activation of fusion-fission cycles for the synthesis of new mitochondria. But currently no study has clearly shown a link between mitochondrial dynamics and biogenesis. Using a mouse model of haploinsufficiency for the Opa1 gene (Opa1(+/-)), we therefore studied the impact of OPA1 deficiency on the adaptation ability of fast skeletal muscles to endurance exercise training. Our results show that, surprisingly, Opa1(+/-) mice were able to perform the same physical activity as control mice. However, the adaptation strategies of both strains after training differed: while in control mice mitochondrial biogenesis was increased as expected, in Opa1(+/-) mice this process was blunted. Instead, training in Opa1(+/-) mice led to an increase in endurance capacity, and a specific adaptive response involving a metabolic remodelling towards enhanced fatty acid utilization. In conclusion, OPA1 appears necessary for the normal adaptive response and mitochondrial biogenesis of skeletal muscle to training. This work opens new perspectives on the role of mitochondrial dynamics in skeletal muscle cells and during adaptation to stress.

Stimulus specific changes of energy metabolism in hypertrophied heart.

Cardiac energy metabolism is a determinant of the response to hypertrophic stimuli. To investigate how it responds to physiological or pathological stimuli, we compared the energetic status in models of hypertrophy induced by physiological stimuli (pregnancy or treadmill running) and by pathological stimulus (spontaneously hypertensive rats, SHR) in 15 week-old female rats, leading to a 10% cardiac hypertrophy. Late stage of compensated hypertrophy was also studied in 25 week-old SHR (35% of hypertrophy). Markers of cardiac remodelling did not follow a unique pattern of expression: in trained rats, only ANF was increased; in gravid rats, calcineurin activation and BNP expression were reduced while beta-MHC expression was enhanced; all markers were clearly up-regulated in 25 week-old SHR. Respiration of permeabilized fibers revealed a 17% increase in oxidative capacity in trained rats only. Mitochondrial enzyme activities, expression of the master regulator PGC-1alpha and mitochondrial transcription factor A, and content of mitochondrial DNA were not consistently changed, suggesting that compensated hypertrophy does not involve alterations of mitochondrial biogenesis. Mitochondrial fatty acid utilization tended to increase in trained rats and decreased by 14% in 15 week-old SHR. Expression of markers of lipid oxidation, PPARalpha and its down-stream targets MCAD and CPTI, was up-regulated after training and tended to decrease in gravid and 15 week-old SHR rats. Taken together these results show that there is no univocal pattern of cardiac adaptation in response to physiological or pathological hypertrophic stimuli, suggesting that other factors could play a role in determining adaptation of energy metabolism to increased workload.

Mitochondrial biogenesis in fast skeletal muscle of CK deficient mice.

Creatine kinase (CK) is a phosphotransfer kinase that catalyzes the reversible transfer of a phosphate moiety between ADP and creatine and that is highly expressed in skeletal muscle. In fast glycolytic skeletal muscle, deletion of the cytosolic M isoform of CK in mice (M-CK-/-) leads to a massive increase in the oxidative capacity and of mitochondrial volume. This study was aimed at investigating the transcriptional pathways leading to mitochondrial biogenesis in response to CK deficiency. Wild type and M-CK-/- mice of eleven months of age were used for this study. Gastrocnemius muscles of M-CK-/- mice exhibited a dramatic increase in citrate synthase (+120%) and cytochrome oxidase (COX, +250%) activity, and in mitochondrial DNA (+60%), showing a clear activation of mitochondrial biogenesis. Similarly, mRNA expression of the COXI (mitochondria-encoded) and COXIV (nuclear-encoded) subunits were increased by +103 and +94% respectively. This was accompanied by an increase in the expression of the nuclear respiratory factor (NRF2alpha) and the mitochondrial transcription factor (mtTFA). Expression of the co-activator PGC-1alpha, a master gene in mitochondrial biogenesis was not significantly increased while that of PGC-1beta and PRC, two members of the same family, was moderately increased (+45% and +55% respectively). While the expression of the modulatory calcineurin-interacting protein 1 (MCIP1) was dramatically decreased (-68%) suggesting inactivation of the calcineurin pathway, the metabolic sensor AMPK was activated (+86%) in M-CK-/- mice. These results evidence that mitochondrial biogenesis in response to a metabolic challenge exhibits a unique pattern of regulation, involving activation of the AMPK pathway.

Heart failure: a model of cardiac and skeletal muscle energetic failure.

Chronic heart failure (CHF), the new epidemic in cardiology, is characterized by energetic failure of both cardiac and skeletal muscles. The failing heart wastes energy due to anatomical changes that include cavity enlargement, altered geometry, tachycardia, mitral insufficiency and abnormal loading, while skeletal muscle undergoes atrophy. Cardiac and skeletal muscles also have altered high-energy phosphate production and handling in CHF. Nevertheless, there are differences in the phenotype of myocardial and skeletal muscle myopathy in CHF: cardiomyocytes have a lower mitochondrial oxidative capacity, abnormal substrate utilisation and intracellular signalling but a maintained oxidative profile; in skeletal muscle, by contrast, mitochondrial failure is less clear, and there is altered microvascular reactivity, fibre type shifts and abnormalities in the enzymatic systems involved in energy distribution. Underlying these phenotypic abnormalities are changes in gene regulation in both cardiac and skeletal muscle cells. Here, we review the latest advances in cardiac and skeletal muscle energetic research and argue that energetic failure could be taken as a unifying mechanism leading to contractile failure, ultimately resulting in skeletal muscle energetic failure, exertional fatigue and death.

Adaptive changes in cardiac myosin heavy chain and creatine kinase isozymic profiles in rats native of altitude.

AIM: The developmental changes in the myosin heavy chain (MHC) profile, creatine kinase (CK) and lactate dehydrogenase (LDH) activities and isozyme expression occurring in heart were examined in rats born and living at altitude (La Paz, Bolivia, 3700 m, H(LP)) for 16 generations. We hypothesized that H(LP) rats respond differently to hypoxia than rats born and living at sea level, and secondarily exposed to altitude during 3 weeks (H(3W)). METHODS: The cardiac expression of MHC, CK and LDH was studied in left (LV) and right ventricle (RV) of H(LP) animals 1, 2, 3, 4 and 18 weeks after birth, and compared with control normoxic (C groups) and H(3W) animals. RESULTS: Rats secondarily exposed to hypoxia showed a lower alpha-MHC content than C or H(LP) rats in both LV and RV, 3 weeks after birth (P < 0.05), consistent with a delay in the maturation of the heart contractile phenotype. A global increase in the total CK activity was observed in the LV of H(3W) animals in comparison with C rats (P < 0.05), while no change was reported in H(LP) animals. In both ventricles, M-LDH activity was higher in H(3W) than in H(LP) and C rats (P < 0.05). The relative amount of alpha-MHC decreased by 20% in RV of 18-week-old H(LP) and H(3W) rats in comparison with C animals, consistent with the hypoxia-induced ventricular enlargement (P < 0.01). An increased activity of the foetal B-CK subunit was observed in both LV and RV of H(3W) rats in comparison with H(LP) and C animals (P < 0.05). CONCLUSION: This study demonstrates that rats native and living at altitude for several generations present some features relevant to genetic selection to altitude.

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 microg/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 (P < 0.001), whereas in plantaris only Vmax increased, by 39% (P < 0.01). 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 (P < 0.01), and soleus creatine kinase was increased by 48% (P < 0.001). T3 increased the mRNA content of the transcriptional co-activator of mitochondrial genes, PGC-1alpha, 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, TRalpha 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-1alpha transcription factor.

Mitochondrial tissue specificity of substrates utilization in rat cardiac and skeletal muscles.

As energetic metabolism is crucial for muscles, they develop different adaptations to respond to fluctuating demand among muscle types. Whereas quantitative characteristics are known, no study described simultaneously quantitative and qualitative differences among muscle types in terms of substrates utilization patterns. This study thus defined the pattern of substrates preferential utilization by mitochondria from glycolytic gastrocnemius (GAS) and oxidative soleus (SOL) skeletal muscles and from heart left ventrical (LV) in rats. We measured in situ, ADP (2 mM)-stimulated, mitochondrial respiration rates from skinned fibers in presence of increasing concentrations of pyruvate (Pyr) + malate (Mal), palmitoyl-carnitine (Palm-C) + Mal, glutamate (Glut) + Mal, glycerol-3-phosphate (G3-P), lactate (Lact) + Mal. Because the fibers oxygen uptake (Vs) followed Michaelis-Menten kinetics in function of substrates level we determined the Vs and Km, representing maximal oxidative capacity and the mitochondrial sensibility for each substrate, respectively. Vs were in the order GAS < SOL < LV for Pyr, Glu, and Palm-C substrates, whereas in the order SOL = LV < GAS with G3-P. Moreover, the relative capacity to oxidize Palm-C is extremely higher in LV than in SOL. Vs was not stimulated by the Lact substrate. The Km was equal for Pyr among muscles, but much lower for G3-P in GAS and lower for Palm-C in LV. These results demonstrate qualitative mitochondrial tissue specificity for metabolic pathways. Mitochondria of glycolytic muscle fibers are well adapted to play a central role for maintaining a satisfactory cytosolic redox state in these fibers, whereas mitochondria of LV developed important capacities to use fatty acids.

Structural and functional adaptations of striated muscles to CK deficiency.

In adult mammalian muscle cells, energy consuming processes are mainly localized to the sarcolemma, sarcoplasmic reticulum (SR) and myofibrillar compartments, while energy production occurs within mitochondria or glycolytic complexes. Due to the restricted diffusion of adenine nucleotides near the active sites of ATPases involved in contractile activity and calcium homeostasis, there are multiple local systems that can locally rephosphorylate ADP and provide ATP. The creatine kinase (CK) system, with specific isoenzymes localized within each compartment, efficiently controls local adenylate pools and links energy production and utilization. However, mice lacking one or both of the MM-CK and mi-CK isoforms (CK-/-) are viable and develop almost normal cardiac and skeletal muscle function under the conditions of moderate workload, suggesting adaptations or other mechanisms that may ensure efficient energy transfer. While fixed CK is essentially important, other systems could also be involved as well, such as bound glycolytic enzymes or adenylate kinase. We have shown that, additionally, a direct functional interplay exists between mitochondria and sarcoplasmic reticulum, or between mitochondria and myofilaments in muscle cells, that catalyzes direct energy and signal transfer between organelles. In cardiac cells of CK-/- mice, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was very effective to rescue SR and myofilament functions. In fast skeletal muscles, increased oxidative capacity also indicates compensatory mechanisms. In mutant mice, mitochondrial capacity increases and a direct energy channeling occurs between mitochondria on one hand and ATP consuming sites on the other. However, these systems appear to be insufficient to fully compensate for the lack of CK at high workload. It can be concluded that local rephosphorylation of ADP is a crucial regulatory point in highly differentiated and organized muscle cells to ensure contractile diversity and efficiency and that the CK system is important to control energy fluxes and energy homeostasis.

Does ACE inhibition enhance endurance performance and muscle energy metabolism in rats?

The renin-angiotensin-aldosterone system plays an important role in the hydroelectrolytic balance, blood pressure regulation, and cell growth. In some studies, the insertion (I) allele of the angiotensin-converting enzyme (ACE) gene, associated with a lower ACE activity, has been found in excess frequency in elite endurance athletes, suggesting that decreased ACE activity could be involved in endurance performance (Myerson S, Hemingway H, Budget R, Martin J, Humphries S, and Montgomery H. J Appl Physiol 87: 1313-1316, 1999). To test this hypothesis, we evaluated whether ACE inhibition could be associated with improved endurance performance and muscle oxidative capacity in rats. Eight male Wistar rats were treated for 10-12 wk with an ACE inhibitor, perindopril (2 mg.kg-1.day-1), and compared with eight control rats. Endurance time was measured on a treadmill, and oxidative capacity and regulation of mitochondrial respiration by substrates were evaluated in saponin-permeabilized fibers of slow soleus and fast gastrocnemius muscles. Endurance time did not differ between groups (57 +/- 5 min for perindopril vs. 55 +/- 6 min for control). Absolute and relative (to body weight) left ventricular weight was 20% (P < 0.01) and 12% (P < 0.01) lower, respectively, in the treated group. No difference in oxidative capacity, mitochondrial enzyme activities, or mitochondrial regulation by ADP was observed in soleus or gastrocnemius. Mitochondrial respiration with glycerol 3-phosphate was 17% higher in gastrocnemius (P < 0.03) and with octanoylcarnitine 14% greater in soleus (P < 0.01) of treated rats. These results demonstrate that ACE inhibition was not associated with improved endurance time and maximal oxidative capacity of skeletal muscles. This suggests that ACE activity has no implication in endurance capacity and only minor effects on mitochondrial function in sedentary animals.

Recovery of contractile and metabolic phenotypes in regenerating slow muscle after notexin-induced or crush injury.

The recovery of metabolic pathways after muscle damage has been poorly studied. We investigated the myosin heavy chain (MHC) isoform transitions and the recovery of citrate synthase (CS) activity, isoform distribution of lactate dehydrogenase (LDH) and creatine kinase (CK) in slow muscles after two types of injury. Muscle degeneration was induced in left soleus muscles of male Wistar rats by either notexin injection or crushing and the regenerative process was examined from 2 to 56 days after injury. Myosin transition occurred earlier after notexin than after crush injury. Fast-type IIx and more particularly type IIa MHC isoform disappeared by day 28 after notexin inoculation, while they were still detected long after in crushed muscles. A full recovery of both the CS activity and the specific activity of the H-LDH subunit was observed from day 42 in notexin-treated muscles, while values measured in crushed muscles remained significantly lower than in non-injured muscles (P < 0.05). The activity of the mitochondrial isoform of CK (mi-CK) was markedly affected by the type of injury (P < 0.001), and failed to reach normal levels after crush injury (P < 0.05). The results of this study show that the relatively rapid MHC transitions during regeneration contrasts with the slow recovery in the oxidative capacity. The recovery of the oxidative capacity remained incomplete after crush injury, a model of injury known to lead to disruption of the basal lamina and severe interruption of the vascular and nerve supply.

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 1 alpha (PGC-1 alpha) gene was downregulated in CHF, as well as nuclear respiratory factor 2 and mitochondrial transcription factor A, which act downstream from PGC-1 alpha. Most interestingly, only the level of PGC-1 alpha 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-1 alpha appears as a potential modulator of muscle oxidative capacity under these experimental conditions.

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 V(O2), expressed in ml min(-1) kg(-1)). All volunteers underwent a cyclo-ergometric incremental exercise test to estimate their peak V(O2) and V(O2) 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 (V(0)) or in the presence (V(max)) of saturating [ADP]. While V(0) was similar, V(max) differed among groups (sedentary, 3.7 +/- 0.3, active, 5.9 +/- 0.9 and athletic, 7.9 +/- 0.5 micromol O2 min(-1) (g dry weight)(-1)). V(max) was correlated with peak V(O2) (P < 0.01, r = 0.63) and with V(T) (P < 0.01, r = 0.57). There was a significantly greater degree of coupling between oxidation and phosphorylation (V(max)/V(0)) in the athletic individuals. The mitochondrial K(m) 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 K(m) 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.

Influence of overload on phenotypic remodeling in regenerated skeletal muscle.

We studied the effects of 10 wk of functional overload on the expression of myosin heavy chain (MHC), sarcoplasmic reticulum Ca(2+)-ATPase isoforms (SERCA), and the activity of several metabolic enzymes in sham and regenerated plantaris muscles. Overload was accomplished by bilateral surgical ablation of its synergists 4 wk after right plantaris muscles regenerated after myotoxic infiltration. The overload-induced muscle enlargement was slightly less in regenerated than in sham muscles [28% (P < 0.005) and 43% (P < 0.001), respectively]. Overload led to an increase in type I MHC expression (P < 0.01) to a similar extent in sham and regenerated plantaris, while the expected shift from type IIb to type IIa MHC was less marked in regenerated than in sham plantaris. The overload-induced decrease in the expression of the fast SERCA isoform and in the activity of the M subunit of lactate dehydrogenase occurred to a similar extent in sham and regenerated plantaris [66% (P < 0.01) and 27% (P < 0.005), respectively]. In conclusion, the lesser responses of muscle mass and fast MHC composition of regenerated plantaris to mechanical overload suggest an alteration of the transcriptional, translational, and/or posttranslational control of gene expression in regenerated muscle.

Oxidative capacity of skeletal muscle in heart failure patients versus sedentary or active control subjects.

OBJECTIVES: We investigated the in situ properties of muscle mitochondria using the skinned fiber technique in patients with chronic heart failure (CHF) and sedentary (SED) and more active (ACT) controls to determine: 1) whether respiration of muscle tissue in the SED and ACT groups correlates with peak oxygen consumption (pVO(2)), 2) whether it is altered in CHF, and 3) whether this results from deconditioning or CHF-specific myopathy. BACKGROUND: Skeletal muscle oxidative capacity is thought to partly determine the exercise capacity in humans and its decrease to participate in exercise limitation in CHF. METHODS: M. Vastus lateralis biopsies were obtained from 11 SED group members, 10 ACT group members and 15 patients with CHF at the time of transplantation, saponine-skinned and placed in an oxygraphic chamber to measure basal and maximal adenosine diphosphate (ADP)-stimulated (V(max)) respiration rates and to assess mitochondrial regulation by ADP. All patients received angiotensin-converting enzyme (ACE) inhibitors. RESULTS: The pVO(2) differed in the order CHF < SED < ACT. Compared with SED, muscle alterations in CHF appeared as decreased citrate synthase, creatine kinase and lactate dehydrogenase, whereas the myosin heavy chain profile remained unchanged. However, muscle oxidative capacity (V(max), CHF: 3.53 +/- 0.38; SED: 3.17 +/- 0.48; ACT: 7.47 +/- 0.73, micromol O(2).min(-1).g(-1)dw, p < 0.001 vs. CHF and SED) and regulation were identical in patients in the CHF and SED groups, differing in the ACT group only. In patients with CHF, the correlation between pVO(2) and muscle oxidative capacity observed in controls was displaced toward lower pVO(2) values. CONCLUSIONS: In these patients, the disease-specific muscle metabolic impairments derive mostly from extramitochondrial mechanisms that disrupt the normal symmorphosis relations. The possible roles of ACE inhibitors and level of activity are discussed.

Response of mitochondrial function to hypothyroidism in normal and regenerated rat skeletal muscle.

Although thyroid hormones induce a well known decrease in muscle oxidative capacity, nothing is known concerning their effects on mitochondrial function and regulation in situ. Similarly, the influence of regeneration process is not completely understood. We investigated the effects of hypothyroidism on mitochondrial function in fast gastrocnemius (GS) and slow soleus (SOL) muscles either intact or having undergone a cycle of degeneration/regeneration (Rg SOL) following a local injection of myotoxin. Thyroid hormone deficiency was induced by thyroidectomy and propylthiouracyl via drinking water. Respiration was measured in muscle fibres permeabilised by saponin in order to assess the oxidative capacity of the muscles and the regulation of mitochondria in situ. Oxidative capacities were 8.9 in SOL, 8.5 in Rg SOL and 5.9 micromol O2/min/g dry weight in GS and decreased by 52, 42 and 39% respectively (P < 0.001) in hypothyroid rats. Moreover, the Km of mitochondrial respiration for the phosphate acceptor ADP exhibited a two-fold decrease in Rg SOL and intact SOL by hypothyroidism (P < 0.01), while mitochondrial creatine kinase activity and sensitivity of mitochondrial respiration to creatine were not altered. The results of this study demonstrate that hypothyroidism markedly altered the sensitivity of mitochondrial respiration to ADP but not to creatine in SOL muscles, suggesting that mitochondrial regulation could be partially controlled by thyroid hormones. On the other hand, mitochondrial function completely recovered following regeneration/degeneration, suggesting that thyroid hormones are not involved in the regeneration process per se.

Effect of cyclosporin A and its vehicle on cardiac and skeletal muscle mitochondria: relationship to efficacy of the respiratory chain.

Although cyclosporin (CsA) is considered to be the best immunosuppressive molecule in transplantation, it has been suspected to alter mitochondrial respiration of various tissues. We evaluated the acute effect of CsA and its vehicle on maximal oxidative capacity (V(max)) of cardiac, soleus and gastrocnemius muscles of rats by an oxygraphic method in saponin skinned muscle fibres. The effects of Sandimmun (a formulation of CsA), vehicle of Sandimmun (cremophor and ethanol (EtOH)), CsA in EtOH and EtOH alone were tested. Increasing concentrations (5 - 20 - 50 - 100 microM) of CsA (or vehicles) were used. Sandimmun profoundly altered the V(max) of all muscles. For example, at 20 microM, inhibition reached 18+/-3, 23+/-5, 45+/-5%, for heart, soleus and gastrocnemius respectively. There were only minor effects of CsA diluted in EtOH and EtOH alone on V(max) of cardiac muscle. Because the effects of vehicle on V(max) were similar or higher than those of Sandimmun, the inhibition of oxidative capacity could be entirely attributed to the vehicle for all muscles. Next, we investigated the potential sites of action of the vehicle on the different complexes of the mitochondrial respiratory chain by using specific substrates and inhibitors. The vehicle affected mitochondrial respiration mainly at the level of complex I ( approximately -85% in skeletal muscles, and -32% in heart), but also at complex IV ( approximately -26% for all muscles). The mechanism of action of the vehicle on the mitochondrial membrane and the implications for the clinical use of immunosuppressive drugs are discussed.

Energetic crosstalk between organelles: architectural integration of energy production and utilization.

Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads.