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PGC-1α, a key regulator of energy metabolism, seems to be a relevant therapeutic target to rectify the energy deficit observed in heart failure (HF). Since our previous work has shown positive effects of cobalamin (Cb) on PGC-1α cascade, we investigate the protective role of Cb in pressure overload-induced myocardial dysfunction. Mice were fed with normal diet (ND) or with Cb and folate supplemented diet (SD) 3weeks before and 4weeks after transverse aortic constriction (TAC). At the end, left ventricle hypertrophy and drop of ejection fraction were significantly lower in SD mice than in ND mice. Alterations in mitochondrial oxidative capacity, fatty acid oxidation and mitochondrial biogenesis transcription cascade were markedly improved by SD. In SD-TAC mice, lower expression level of the acetyltransferase GCN5 and upregulation of the methyltransferase PRMT1 were associated with a lower protein acetylation and a higher protein methylation levels. This was accompanied by a sustained expression of genes involved in mitochondrial biogenesis transcription cascade (Tfam, Nrf2, Cox1 and Cox4) after TAC in SD mice, suggesting a preserved activation of PGC-1α; this could be at least partly due to corrected acetylation/methylation status of this co-activator. The beneficial effect of the treatment would not be due to an effect of Cb and folate on oxidative stress or on homocysteinemia, which were unchanged by SD. These results showed that Cb and folate could protect the failing heart by preserving energy status through maintenance of mitochondrial biogenesis. It reinforces the concept of a metabolic therapy of HF., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recently, we have demonstrated that heart failure in rats is associated with a myopathy altering energy metabolism in different muscles, but the origin of this myopathy is still unknown. Here, we studied the possible involvement of increased angiotensin II (Ang II) by treatment with perindopril, an inhibitor of angiotensin-converting enzyme (ACE). The beneficial effects of ACE inhibition could result either from vasodilatation-induced cardiac unloading or from inhibition of the direct angiotensin action on the muscle cells. The model of aortic banding with persisting left ventricular (LV) overload where the cardiac unloading does not occur allows to distinguish between the two effects of ACE inhibition. Four months after aortic clipping (just before the treatment), echocardiographic study showed an impairment of the systolic function (decrease of the LV shortening by 30% and ejection fraction by 21%). Ten-week treatment with perindopril dramatically decreased Ang II plasma level but did not reduce LV hypertrophy though a significant decrease in right ventricular (RV) hypertrophy occurred. Perindopril did not improve alterations in activities of energy metabolism enzymes (creatine kinase, citrate synthase, cytochrome c oxidase, lactate dehydrogenase) either in ventricular or in skeletal (gastrocnemius) muscle. Similarly, ACE inhibition did not improve the main parameters of mitochondrial respiration in permeabilized muscle fibers. These data suggest that the generalized metabolic myopathy induced by the hemodynamic abnormalities conditioned by the continuous LV overload (aorta clipping) does not result from the increase in Ang II level per se. Correction of hemodynamic parameters and LV unloading seem to be the prerequisite for the improvement of muscle energy metabolism abnormalities.

Several works have shown the importance of the creatine kinase (CK) system for cardiac energetics and Ca2+ homeostasis. Nevertheless, CK-deficient mice have cardiac function close to normal, at least under conditions of low or moderate workload. To characterize possible adaptive changes of the sarcoplasmic reticulum (SR) and potential role of glycolytic support in cardiac contractility we used the skinned fibre technique to study properties of the SR and myofibrils, in control and muscle-type homodimer (MM-/mitochondrial-CK)-deficient mice. In control fibres, SR Ca2+ loading with ATP and phosphocreatine (solution PL) was significantly better than loading with ATP alone (solution AL), as determined by analysis of caffeine-induced tension transients. Loading in the presence of ATP and glycolytic intermediates (solution GL) was not significantly different from solution PL. These data indicate that Ca2+ uptake by the SR in situ depends on a local ATP:ADP ratio that is controlled by both CK and glycolytic enzymes. In CK-deficient mice, Ca2+ loading was impaired in solution PL due to the absence of CK. In solution GL, loading was significantly increased, such that calculated Ca2+ release parameters were normalized to those in control fibres in solution PL. In CK-deficient mice, fibre kinetic parameters of tension recovery were impaired after quick stretch in solution PL and were not improved in solution GL. These results show that in CK-deficient mice, at least under basal conditions, glycolysis can replace the CK system in fueling the SR Ca2+ ATPase, but not the myosin ATPase, and may in part explain the limited phenotypic alterations seen in the hearts of these mice.

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.

Ferret right ventricular hypertrophy is characterized by a decreased and prolonged isometric contraction, associated with altered intracellular calcium (Ca2+) regulation. However myofibrillar composition, cross-bridge function and/or energy transfer may also be involved in these contractile disturbances. Therefore, mechanical properties of myofibrils have been studied with Triton X-100-skinned fibres and troponin (Tn) T and I composition has been examined. Mitochondrial function and functional activity of creatine kinase (CK) isoforms have been studied in saponin-skinned fibres of control (C) and hypertrophied (H) ferret right ventricle, to check for a possible mismatch between energy production and utilization. Our results show that neither TnT nor TnI isoform expression, nor myofibrillar Ca2+ responsiveness (similar apparent Ca2+ sensitivity and Hill coefficient) were affected by pressure-overload. Similarly, maximal tension and stiffness, as well as cross-bridge cycling rate (v)--assessed by quick length changes--were not significantly altered. Importantly, passive stiffness was dramatically increased (163 +/- 30 mN/mm2/microns for C v 500 +/- 121 mN/mm2/microns for H; P < 0.02). Moreover, there was a significant correlation between passive stiffness and cross-bridge cycling rate, indicating that a factor involved in the passive stiffness may affect cross-bridge kinetics. Oxidative capacity (normalized to ventricular dry weight), reflecting mitochondrial ATP production and mitochondrial CK efficacy, as well as myofibrillar CK efficacy (assessed by the shift of MgATP-rigor tension curves before and after phosphocreatine addition), were similar in both groups. These results demonstrate that ferret right ventricular pressure-overload was accompanied by a development of myofibrils and a parallel increase of energy production capacity, transfer and utilization. Decreased compliance, probably linked to an increase in the collagen fraction and/or alterations of the cytoskeletal architecture of the overloaded ventricle, could contribute to the slower time course and decreased amplitude of the isometric twitch.

Saponin-skinned rat cardiac fibres were used to study the influence of ischaemic factors (high [Pi] and decreased pH) on functional activity of mitochondrial creatine kinase (mi-CK) in situ by evaluation of the stimulation of respiration by creatine. High (20 mM) [Pi] known to solubilize mi-CK, decreased this stimulation significantly, though mi-CK was still present in the mitochondrial compartment. Acidosis (pH 6.6) increased the stimulation of respiration by creatine at low [Pi], and prevented the decrease in mi-CK functional activity at high [Pi]. Thus, these two ischaemic factors act in opposite directions. The data obtained suggest that under physiological or pathological conditions, inorganic phosphate and protons, by changing the functional coupling between creatine kinase and translocase, may influence the distribution of energy fluxes through adenylate and creatine kinase systems in cardiac tissue. Presence of creatine kinase in mitochondrial compartment is not alone sufficient for functional efficacy of the enzyme.

Energy dissipation and the bearing of tension during ionic contracture in myocardium may not result from one and the same process. To test this, comparative indirect and direct microcalorimetry determinations were made in non-perfused tissue-cell preparations under optimal oxygenation conditions (right papillary muscles, high-pO2 superfusion, 30 degrees C) before, during and after exposure to low-Na, high-K solutions. Over a 15-min contracture plus 45-min recovery cycle, both heat production rate (E), and the indirectly determined heat production rate (EO2) which is oxygen uptake multiplied by the overall energetic equivalent of O2 for nutrient oxidations, were constantly larger than basal rates. The two 60-min time integrals of this increase in metabolic rate were equal [30.3 +/- 3.7 and 31.0 +/- 3.9 (SE) J/g muscle wet weight (n = 9) for E and EO2 respectively]. During contracture however, E exceeded EO2 by 24% (4.7 +/- 1.7 J/g), and during the recovery period EO2 exceeded E by 21% (5.4 +/- 2.6 J/g). Whereas oxidative recovery of the energy lost by the preparation during 15-min contractures was complete, after longer contractures recovery did not occur or was incomplete. In keeping with the now prevalent idea that ion--namely Ca--transport activities are maintained foremost among cellular ATP-dependent processes and consume significant amounts of energy, the present finding that in a 15-min ionic contracture myocardium incurs not only some, but the maximum oxygen debt still compatible with complete oxidative recovery suggests that contracture tension is maintained at low energy cost, essentially by slow-cycling or "rigor" bridges as in hypoxic contractures, whereas heat is mainly related to intracellular calcium homeostasis.

Mechanical performance, energy-rich phosphates and cyclic nucleotides of frog heart were measured during energetic deficiency and subsequent addition of adrenaline. When oxidative metabolism was inhibited by 3 mM cyanide, tension decrease was accompanied by a decrease in creatine phosphate while ATP and cyclic nucleotides did not vary significantly. After subsequent addition of adrenaline mechanical activity remained less than control value; creatine phosphate (CP) concentration was further decreased while cAMP was increased in the same proportion as when adrenaline alone was added. In the presence of cyanide, the weak inotropic effect of adrenaline is not due to an alteration of cyclic nucleotides but is rather correlated to a further decrease in energy-rich phosphates, mainly in creatine phosphate. These results suggest that creatine phosphate may control contractile activity and may be a limiting factor for inotropic interventions at least during energetic inhibition.

Functional states of cardiac contractile apparatus and mitochondria were studied in hereditary cardiomyopathic hamsters (CHF 146) and control golden hamsters using cardiac fibers skinned by two different techniques. The Triton X-100 skinned fibers obtained from diseased animals of 175 to 200 days old, or from control animals, demonstrated the same resting and maximal Ca-activated tensions, the same stiffness, the same rate of tension recovery after quick stretch; the fibers from cardiomyopathic animals differed only by a slightly increased calcium sensitivity. Functional activity of myofibrillar creatine kinase in cardiomyopathy was decreased as indicated by a smaller shift in the pMgATP/rigor tension curve to lower [MgATP] in the presence of phosphocreatine and by a slower rate of the tension recovery after quick stretch in the presence of phosphocreatine and ADP (without ATP). The saponin-skinned fibers allow evaluation of the respiration properties of the total tissue mitochondria. Data obtained in the preparations isolated from diseased animals of two ages (75 to 100 and 175 to 200 days) showed that the ratio of maximal ADP-stimulated respiration rate to the respiration rate in the absence of ADP (an analog of respiration control index) was unchanged in myopathy as compared with age-matched controls. However stimulation of respiration after an addition of creatine at submaximal ADP concentration was observed to be respectively 1.45 times and 3.5 times less in the preparations from younger and older myopathic animals as compared with their respective controls, thus indicating the impairment of functional coupling between mitochondrial creatine kinase reaction and oxidative phosphorylation. These results suggest that hereditary cardiomyopathy is associated with alterations in myocardial creatine kinase system, while myofilaments and mitochondria preserve their basic functional properties.

The effects of clinical concentrations of halothane (1 and 2% v/v) on detergent treated cardiac fibers were studied in two different models of cardiomyopathic animals, the Syrian hamster UM-X7.1, and the streptozotocin-induced diabetic rat. The changes of contractile properties in cardiac muscle observed on cardiomyopathic animals, although of moderate importance, were different in these two models. The cardiomyopathic hamsters exhibited macroscopic structural changes in cardiac muscle responsible for a significant decrease in maximal activated tension, but myocardial calcium sensitivity was unchanged. On the other hand, in diabetic rats, maximal activated tension was unchanged, while a slight but significant increase in myocardial calcium sensitivity was observed. Addition of halothane produced a similar dose-dependent decrease in myocardial calcium sensitivity, in both the controls and the two groups of cardiomyopathic animals. Halothane exposure was also associated with a dose-dependent decrease in maximal calcium activated tension in all groups, an effect that was more pronounced in cardiomyopathic hamsters than in their control at the lowest anesthetic concentration. These results indicate that the negative inotropic effects of halothane are additive to the myocardial depression observed in these cardiomyopathies.