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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.)

Because the question "is AMP-activated protein kinase (AMPK) alpha(2)-isoform a friend or a foe in the protection of the myocardium against ischemia-reperfusion injury?" is still in debate, we studied the functional consequence of its deletion on the contractility, the energetics, and the respiration of the isolated perfused heart and characterized the response to low-flow ischemia and reperfusion with glucose and pyruvate as substrates. alpha(2)-AMPK deletion did not affect basal contractility, respiration, and high-energy phosphate contents but induced a twofold reduction in glycogen content and a threefold reduction in glucose uptake. Low-flow ischemia increased AMPK phosphorylation and stimulated glucose uptake and phosphorylation in both alpha(2)-knockout (alpha(2)-KO) and wild-type (WT) groups. The high sensitivity of alpha(2)-KO to the development of ischemic contracture was attributed to the constitutive impairment in glucose transport and glycogen content and not to a perturbation of the energy transfer by creatine kinase (CK). The functional coupling of MM-CK to myofibrillar ATPase and the CK fluxes were indeed similar in alpha(2)-KO and WT. Low-flow ischemia impaired CK flux by 50% in both strains, showing that alpha(2)-AMPK does not control CK activity. Despite the higher sensitivity to contracture, the postischemic contractility recovered to similar levels in both alpha(2)-KO and WT in the absence of fatty acids. In their presence, alpha(2)-AMPK deletion also accelerated the contracture but delayed postischemic contractile recovery. In conclusion, alpha(2)-AMPK is required for a normal glucose uptake and glycogen content, which protects the heart from the development of the ischemic contracture, but not for contractile recovery in the absence of fatty acids.

It is generally considered that mitochondria regulate cardiac cell contractility by providing ATP for cellular ATPases and by participating in Ca2+ homeostasis. However, other possible mechanisms by which mitochondria can influence contractility have been largely overlooked. Here, we demonstrate that inhibition of the mitochondrial electron transport chain strongly increases Ca2+-dependent and independent isometric force development in rat ventricular fibers with selectively permeabilized sarcolemma. This effect is unrelated to the ATP-generating activity of mitochondria or Ca2+ homeostasis. Furthermore, various conditions that increase K+ accumulation in the mitochondrial matrix (activation of ATP- or Ca2+-dependent K+ channels as well as inhibition of the K+ efflux pathway via the K+/H+ exchanger) induce a similar mechanical response. Modulators of mitochondrial function that augment isometric force also cause swelling of mitochondria in the vicinity of myofibrils in situ, as shown by confocal microscopy. Osmotic compression of intracellular structures abolishes the effect of mitochondria-induced force modulation, suggesting a mechanical basis for the interaction between the organelles. These findings suggest a novel mechanism for cellular regulation of myofibrillar function, whereby increases in mitochondrial volume can impose mechanical constraints inside the cell, leading to an increase in force developed by myofibrils.

To understand the role of creatine kinase (CK) in cardiac excitation-contraction coupling, CK-deficient mice (CK-/-) were studied in vitro and in vivo. In skinned fibers, the kinetics of caffeine-induced release of Ca2+ was markedly slowed in CK-/- mice with a partial restoration when glycolytic substrates were added. These abnormalities were almost compensated for at the cellular level: the responses of Ca2+ transient and cell shortening to an increased pacing rate from 1 Hz to 4 Hz were normal with a normal post-rest potentiation of shortening. However, the post-rest potentiation of the Ca2+ transient was absent and the cellular contractile response to isoprenaline was decreased in CK-/- mice. In vivo, echocardiographically determined cardiac function was normal at rest but the response to isoprenaline was blunted in CK-/- mice. Previously described compensatory pathways (glycolytic pathway and closer sarcoplasmic reticulum-mitochondria interactions) allow a quasi-normal SR function in isolated cells and a normal basal in vivo ventricular function, but are not sufficient to cope with a large and rapid increase in energy demand produced by beta-adrenergic stimulation. This shows the specific role of CK in excitation-contraction coupling in cardiac muscle that cannot be compensated for by other pathways.

The subcellular mechanisms responsible for myocardial depression during sepsis remain unclear. Recent data suggest a role for impaired energy generation and utilization, resulting in altered contractile function. Here, we studied the energetic and mechanical properties of skinned fibers isolated from rabbit ventricle in a nonlethal but hypotensive model of endotoxemia. Thirty-six hours after lipopolysaccharide (LPS) injection (in the presence of altered myocardial contractility), mitochondrial respiration, coupling between oxidation and phosphorylation, and creatine kinase function were similar in preparations from endotoxemic (LPS) and control animals. The maximal Ca2+-activated force was similar in LPS and control preparations. However, the Ca2+ concentration corresponding to half-maximal force (pCa50, where pCa = -log10[Ca2+]) was 5.55 +/- 0.01 (n = 11) in LPS fibers versus 5.61 +/- 0.01 (n = 10) in control fibers (p < 0.01). Both protein kinase A (PKA) and alkaline phosphatase treatment led to the disappearance in the difference between control and LPS pCa50 values. Incubation of control fibers with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) did not change the Ca2+ sensitivity after subsequent skinning, whereas isoproterenol decreased pCa50 from 5.62 +/- 0.01 to 5.55 +/- 0.01 (p < 0.01). These data suggest that during sepsis, cardiac mitochondrial and creatine kinase systems remain unaltered, whereas protein phosphorylation decreases myofibrillar Ca2+ sensitivity and may contribute to the depression of cardiac contractility.

Objective: To elucidate the role of bound creatine kinase in adenine nucleotide compartmentation in myofibrils, the effects of this enzyme's substrates and products on rigor tension were studied in using isolated skinned rat cardiomyocytes rather than fibers, to avoid restrictions due to concentration gradients within the multicellular preparations., Methods: A new experimental set-up was built to allow continuous and stable measurements of force developed by cells. Triton X-100-treated cardiomyocytes were glued between a glass holder and the needle of a galvanometer. A feedback system allowed the precise measurement of force by recording the coil current necessary to prevent movement of the needle., Results: At very low [Ca2+] (pCa 7), as MgATP level decreased, rigor tension appeared. In the absence of phosphocreatine (PCr), this tension started to rise at MgATP concentrations several times higher than in the presence of 12 mM PCr. In the absence of PCr, the pMgATP/tension curves of single cells usually had a complicated relationship which could not be analyzed by a simple Hill equation. In the absence of PCr, 250 microM MgADP strongly potentiated rigor tension development in the 1 mM-3 microM range of [MgATP]; at 100 microM MgATP, in the presence of MgADP, the tension was 4.6 times higher than in the absence of MgADP. Addition of 12 mM PCr immediately eliminated rigor. Finally, in the presence of 100 microM MgATP and 250 microM MgADP, a decrease in PCr resulted in rigor; the half-maximal contracture being recorded at 1 mM PCr., Conclusions: These results indicate a myofibrillar compartmentation of adenine nucleotides influenced by bound creatine kinase, since at equal MgATP concentrations in extramyofibrillar milieu the response of myofibrils strongly depends on the presence of PCr. Local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar creatine kinase to rephosphorylate ADP produced by myosin ATPase could be an important mechanism of diastolic tension rise in ischaemic conditions.

The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+ dT/dt) and relaxation (-dT/dt) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas + dT/dt and -dT/dt (normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+ concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+ adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca(2+)-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the alpha-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.

Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.

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.

Effects of alkalosis (pH 7.4) or acidosis (pH 6.8) on the intrinsic mechanical properties of control and pressure-overloaded rat hearts were studied in Triton X-100-treated left ventricular fiber bundles. In control bundles, Ca sensitivity [pCa required for one-half maximal response (pCa50)] was 5.520 +/- 0.012 at pH 7.1. Alkalosis increased it by 0.357 +/- 0.018 pCa unit, whereas acidosis decreased it by 0.365 +/- 0.014 pCa unit with no change in Hill coefficient. Maximal tension was decreased by acidic pH and increased by alkaline pH. Stiffness was measured by the response to quick length changes. Acidosis decreased maximal stiffness but increased the stiffness-to-force ratio, whereas alkalosis increased maximal stiffness but had no effect on stiffness-to-force ratio, suggesting that acidosis decreased the force generated per cross bridge. Alkalosis increased the time constant of tension recovery following a quick stretch from 10.6 +/- 0.66 to 17.45 +/- 1.83 ms, suggesting a decreased cross-bridge cycling rate. Pressure overload induced by thoracic aortic stenosis for 4-6 wk led to a 200% cardiac hypertrophy associated with a shift from fast to slow ventricular myosin. pCa50 of hypertrophied bundles was not different from control (5.541 +/- 0.012). Ca sensitivity was increased by 0.383 +/- 0.008 in alkaline medium and decreased by 0.325 +/- 0.009 in acidic medium. Stiffness-to-force ratio was decreased in acidic pH, and the time constant of tension recovery was increased from 31.0 +/- 0.4 to 34.9 +/- 0.25 ms by alkalosis. In hypertrophied bundles, maximal tension was decreased by acidic pH but not changed by alkalosis. These results show that in the small pH range of our study 1) pH changes have symmetrical effects on Ca sensitivity in both control and hypertrophied bundles, 2) a decrease or an increase in H+ concentration does not have symmetrical effects on the mechanics of the cross bridges, and 3) changes in the phenotype of contractile proteins induced by aortic stenosis do not influence Ca sensitivity, only moderately influence the response to pH changes, and mainly affect the cross-bridge cycling rate.

Myocardial ischemia is characterized by a decrease in phosphocreatine (PCr) and Mg(2+)-ATP contents as well as an accumulation of myosin ATPase reaction products (inorganic phosphate [P(i)], protons, and Mg(2+)-ADP). The possibility that these metabolites play a role in rigor tension development was checked in rat ventricular Triton X-100-skinned fibers. Rigor tension was induced by stepwise decreasing [Mg(2+)-ATP] in the presence or in the absence of 12 mmol/L PCr. To mimic the diastolic ionic environment of the myofibrils, [free Ca2+] was set at 100 nmol/L (pCa 7); [free Mg2+], at 1 mmol/L; and ionic strength, at 160 mmol/L. In control conditions (pH 7.1, with no added P(i) or Mg(2+)-ADP), the pMg(2+)-ATP for half-maximal rigor tension (pMg(2+)-ATP50) was 5.07 +/- 0.03 in the presence of PCr. After withdrawal of PCr, the pMg2+)-ATP50 value was shifted toward higher Mg(2+)-ATP values (3.57 +/- 0.03). Addition of 20 mmol/L P(i) shifted the pMg(2+)-ATP50 to 3.71 +/- 0.04 (P < .05) in the absence of PCr and in the opposite direction to 4.98 +/- 0.02 (P < .01) in the presence of PCr. Acidic pH (6.6) strongly increased pMg(2+)-ATP50 in both the absence (3.90 +/- 0.03, P < .001) and presence (5.44 +/- 0.02, P < .001) of PCr. Conversely, Mg(2+)-ADP (250 mumol/L) decreased pMg(2+)-ATP50 to 3.26 +/- 0.06 (P < .001) in the absence of PCr; at pMg(2+)-ATP 4, no rigor tension was observed until PCr concentration was decreased to < 2 mmol/L. At acidic pH, maximal rigor tension was lower by 29% compared with control conditions, whereas in the presence of Mg(2+)-ADP, maximal rigor tension developed to 143% of the control value; P(i) had no effect. The tension-to-stiffness (measured by the quick length-change technique) ratio was lower in rigor (no PCr and pMg(2+)-ATP 6) than during Ca2+ activation in the presence of both PCr and ATP. Compared with control rigor conditions, this parameter was unchanged by Mg(2+)-ADP and decreased by acidic pH, suggesting a proton-induced decrease in the amount of force per crossbridge. In addition to their known effects on active tension, Mg(2+)-ADP and protons affect rigor tension and influence ischemic contracture development. It is concluded that ischemic contracture and increased myocardial stiffness may be mediated by a decreased PCr and local Mg(2+)-ADP accumulation. This emphasizes the importance of myofibrillar creatine kinase activity in preventing ischemic contracture.

This article is a review on the organization and function of myofibrillar creatine kinase in striated muscle. The first part describes myofibrillar creatine kinase as an integral structural part of the complex organization of myofibrils in striated muscle. The second part considers the intrinsic biochemical and mechanical properties of myofibrils and the functional coupling between myofibrillar CK and myosin ATPase. Skinned fiber studies have been developed to evidence this functional coupling and the consequences for cardiac contraction. The data show that creatine kinase in myofibrils is effective enough to sustain normal tension and relaxation, normal Ca sensitivity and kinetic characteristics. Moreover, the results suggest that myofibrillar creatine kinase is essential in maintaining adequate ATP/ADP ratio in the vicinity of myosin ATPase active site to prevent dysfunctioning of this enzyme. Implications for the physiology and physiopathology of cardiac muscle are discussed.

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.

Chronic growth hormone (GH) hypersecretion in rats leads to increased isometric force without affecting the unloaded shortening velocity of isolated cardiac papillary muscles, despite a marked isomyosin shift toward V3. To determine if alterations occurred at the level of the contractile proteins in rats bearing a GH-secreting tumor (GH rats), we examined the mechanical properties of skinned fibers to eliminate the early steps of the excitation-contraction coupling mechanism. We found that maximal active tension and stiffness at saturating calcium concentrations (pCa 4.5) were markedly higher in GH rats than in control rats (tension, 52.9 +/- 5.2 versus 38.1 +/- 4.6 mN.mm-2, p < 0.05; stiffness, 1,105 +/- 120 versus 685 +/- 88 mN.mm-2.microns-1, p < 0.01), whereas values at low calcium concentrations (pCa 9) were unchanged. In addition, the calcium sensitivity of the contractile proteins was slightly but significantly higher in GH rats than in control rats (delta pCa 0.04, p < 0.001). The crossbridge cycling rate, reflected by the response to quick length changes, was lower in GH rats than in control rats (62.0 +/- 2.6 versus 77.4 +/- 6.6 sec-1, p < 0.05), in good agreement with a decrease in the proportion of alpha-myosin heavy chains in the corresponding papillary muscles (45.5 +/- 2.0% versus 94.6 +/- 2.4%, p < 0.001). The changes in myosin heavy chain protein phenotype were paralleled by similar changes of the corresponding mRNAs, indicating that the latter occurred mainly at a pretranslational level. These results demonstrate that during chronic GH hypersecretion in rats, alterations at the myofibrillar level contribute to the increase in myocardial contractility observed in intact muscle.

To investigate the changes in the properties of cardiac contractile proteins due to neurohormonal stimulation, different agonists were applied to single cells isolated from rat ventricle. Cells were then rapidly skinned by Triton X-100, and force was recorded after gluing the cells to a strain gauge. The skinned cells had mechanical properties very similar to those described for thin trabeculas. Tension-pCa relations were highly reproducible from one cell to another, with sarcomere length fixed at 2.1 microns. The application of alpha 1-adrenergic and muscarinic agonists, which increase the turnover of phosphatidylinositol, for 5 minutes before skinning the cells increased the sensitivity of the myofilaments to calcium, as indicated by a leftward shift of the tension-pCa relation, whereas beta-adrenergic stimulation induced a rightward shift. The increase in calcium sensitivity was also evoked by protein kinase C activators such as 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate but not by protein kinase C itself or by purinergic agonists, although the latter also increased the turnover of phosphatidylinositol. Incubation of the skinned cells with phosphatase reversed the alterations in calcium sensitivity induced by previous agonist stimulation of the intact cells. In conclusion, this study demonstrates a potentially influential mechanism for the physiological regulation of cardiac muscle contractility.

Volatile anesthetics were demonstrated to decrease calcium sensitivity and maximal developed force of detergent-treated rat cardiac skinned fibers. To further investigate the possible mechanisms involved in the decrease of force production, stiffness measurements were performed at defined levels of activation with the use of quick length changes of 0.3 to 4% of initial muscle length in the absence and in the presence of 2 MAC of halothane, enflurane, or isoflurane. The results of various series of experiments suggest that these anesthetics have multiple sites of action on cardiac myofibrillar proteins: 1) they decreased active stiffness indicating a decreased number of attached force-generating cross-bridges; 2) they increased the stiffness/force ratio suggesting that the individual force developed by each cross-bridge was decreased during anesthetic exposure; and 3) they increased the time constant of force recovery, which is consistent with the decreased rate of ATP hydrolysis described by others. These changes in cross-bridges kinetics and efficiency may result from conformational changes in all the protein systems involved in force production, and especially actin-myosin attachment and detachment. However, the changes observed were small despite a relatively high concentration of anesthetics; therefore, they will probably participate only to a moderate extent in the overall negative inotropic effect of these agents.

Immature hearts of various animal species and humans have been demonstrated to be more sensitive than adult hearts to the myocardial depressant effects of volatile anesthetics. To further investigate the mechanisms involved, the calcium sensitivity and maximal activated tension of detergent-treated left ventricular fibres of fetuses (30 days), newborn (1-day-old), immature (3-, 8-, and 17-day-old), and adult rabbits were determined by stepwise exposure to increasing Ca2+ concentrations. Responses were measured prior to and after exposure to equianesthetic concentrations of halothane (1%) or isoflurane (1.5%) applied in a random order. In control conditions maximal developed tension was the lowest in fetuses (11.1 +/- 0.6 mN.mm-2), intermediate in newborn and immature rabbits, and highest in adults (25.6 +/- 2.9 mN.mm-2). There were also age-related changes in calcium sensitivity; pCa (= -log10[Ca2+]) for half-activation (pCa50) was significantly less in 1-, 3-, and 8-day-old rabbits (5.444 +/- 0.036, 5.425 +/- 0.017, and 5.385 +/- 0.019, respectively) than in adults (5.517 +/- 0.010), whereas it was not different in fetuses (5.521 +/- 0.017). During anesthetic exposure both calcium sensitivity and maximal developed tension decreased significantly in all age groups of animals, with both anesthetics having a similar effect in animals of identical age. However, calcium sensitivity decreased significantly more in newborn animals (0.192 and 0.196 pCa unit for halothane and isoflurane, respectively) compared with adults (0.122 and 0.137 pCa units, respectively). By contrast, fetuses were less sensitive to the myocardial depressant effects of anesthetics than were newborn animals.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

In the absence of creatine phosphate, MgATP produced relaxation of rigor tension in chemically-skinned right papillary muscles of the rat, the half maximal effect being obtained at 1.8 mM MgATP. In the presence of 12 mM creatine phosphate and 250 microM ADP, a decrease in MgATP concentration even to 10(-9) M never induced rigor tension. At a very low MgATP concentration (10(-6) M), the half maximal relaxing effect was obtained with 2 mM creatine phosphate, a value close to the Km of isolated MM-creatine kinase for this substrate, or with 14 microM MgADP, a value 5 times lower than the reported Km. An exogenous MgATP regenerating system (phosphoenol pyruvate + pyruvate kinase) was not able to fully relax the fibres. When MM-creatine kinase was inhibited by fluorodinitrobenzene, the dependency of rigor tension on MgATP became the same as it was without creatine phosphate. After washing out the fluorodinitrobenzene the addition of exogenous MM-creatine kinase for half an hour fully relaxed rigor tension; moreover, this effect persisted even after prolonged washout. These results show that endogenous MM-creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a localized high MgATP/MgADP ratio; they also suggest the existence of rapidly exchangeable binding sites for MM-creatine kinase in cardiac myofibrils.

It has been realized for a century that Ca2+ is important in the initiation and the control of mechanical activity. The present paper does not cover the field of excitation-contraction coupling in heart but mainly reports some modern and controversial aspects about the regulation of the internal Ca concentration by the sarcolemma while the role of internal stores is only discussed. The first part deals with flux experiments; the second with the slow inward current mainly carried by Ca ions. An analysis of the mechanical activity reveals that only a part of tension is triggered by this slow current; besides, a second component of tension is demonstrated and an exchange mechanism of Na and Ca ions is described in detail. This countertransport, during depolarization, facilitates an influx of Ca ions (coupled to an efflux of Na ions). During hyperpolarization, or even at the resting membrane potential, it promotes an efflux of Ca ions. Thus, the same mechanism may account in part for the development or for the relaxation of tension according to the membrane potential.

The effects of inorganic phosphate (Pi) on mechanical properties of Triton X100 treated ventricular fibres have been studied in different substrate conditions. In the presence of both MgATP and phosphocreatine, increasing concentrations of Pi progressively decreased maximal active force, up to 50-60% at 20 mM Pi. The reduction in stiffness was slightly less. These effects appeared nearly independent of the diameter of the preparations. 20 mM Pi decreased Ca sensitivity of the myofilaments and increased the Hill coefficient of the tension/pCa relationship; furthermore, the time constant of tension recovery was decreased from 12.9 to 8.9 ms suggesting that the cycling rate of cross-bridges was increased in the presence of Pi. When MgATP was regenerated by the myofilament bound creatine kinase in the presence of phosphocreatine, Pi was less efficient in decreasing the maximal tension and it weakened the relaxing effect of MgATP upon rigor tension. These effects are related to the inhibition of creatine kinase by Pi. The effects of Pi on maximal force and kinetics of contraction were antagonized by the effects of a decrease in phosphocreatine. The results are discussed in terms of the antagonistic role of Pi increase and phosphocreatine decrease upon contractile properties of myofilaments during hypoxia in heart muscle.

Frog heart relaxation was analysed under voltage clamp conditions as the tension decay observed after membrane potential had been stepped back to its resting value. It appeared mostly exponential with a time constant of about 190 msec. Relaxation rate decreased with the external Na concentration. It fell to 1/10 in a Na-free solution. Relaxation rate decreased also following an increase in the intracellular Na content by application of veratrine. When switching from a high to a low Na-containing solution the relaxation rate reached rapidly a value transiently higher than the steady-state's (and vice versa). In Na-free solution, adrenaline accelerated tension decay, an effect not noticeable on frog heart in Ringer solution. In frog myocardium, part of the intracellular Ca concentration decrease which occurs during each cardiac cycle directly results from a calcium extrusion carried out by the Na-Ca exchange. Such a Ca decrease, besides internal Ca storage, should play a primary role in the relaxation of contractile tissues whose cells have a large surface/volume ratio.

The hypodynamic state of frog heart has been investigated from the energetic point of view. The amplitude of tension was enhanced when the oxidative metabolism was stimulated by oxygen and pyruvate. However, a time-dependent tension decrease still occurred with both oxygen and pyruvate. Hypodynamia, although slowed down under these conditions, was accompanied by a decrease in CP rather than in ATP. It is shown that the decrease in CP was due to a leak of creatine out of the cell, consequent to the non-equilibration of the oncotic pressure in the perfusion fluid. Creatine (20 mM) or any plasma expander, prevented the fall in both CP and tension. These results are in agreement with the proposal that the CP/CK/Cr system is the major metabolic intermediate between production and use of energy for contraction and that, besides Ca ions, it participates in the regulation of tension. It is suggested that hypodynamia mostly originates in a decreased Ca utilization due to shortage of energy.

The influence of phosphocreatine in the presence or absence of MgATP and MgADP was studied in Triton X-100-treated thin papillary muscles and ventricular strips of the rat heart. The pCa/tension relationships, the pMgATP/tension relationships, and the tension responses to quick length changes were analyzed. The results show three major consequences of the reduction of the phosphocreatine concentration in the presence of millimolar concentrations of the MgATP. (a) The resting tension and the maximal Ca2+-activated tension were increased, and the pCa/tension relationship was shifted toward higher pCa values and its steepness was decreased; these effects were enhanced by the inclusion of MgADP. (b) The time constant of tension recoveries after quick stretches applied during maximal activation was increased, while the extent of these recoveries was decreased. (c) The study of pMgATP/tension relationships in low Ca concentrations showed that the decrease in phosphocreatine induced a shift toward higher MgATP values with no changes in maximal rigor tension or the slope coefficient; these effects were increased by the increase in MgADP and were independent of the preparation diameter. Thus, modifications of the apparent Ca sensitivity and resting and maximal tension when phosphocreatine is decreased seem to be due to an increasing participation of rigor-like or slowly cycling cross-bridges spending more time in the attached state. These results suggest that endogenous creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a local high MgATP/MgADP ratio.

After prolonged ischemia followed by reperfusion of the isolated rat heart, irreversible heart failure is associated with creatine kinase leakage from the cells. The possible implications of MM creatine kinase leakage from myofibrillar compartments on the contractile properties of ventricular muscle have been studied in control versus ischemic hearts. Total creatine kinase activity decreased in ischemic cells while creatine kinase and ATPase activities were not modified in isolated myofibrils. The efficiency of creatine kinase and phosphocreatine in the relaxation of rigor tension in skinned ventricular preparations was not changed after ischemia. Furthermore, neither the pCa/tension relationship nor the rate of tension development following length changes were modified by ischemia. These results show that the contractile properties of myofilaments as well as the functional coupling between myosin ATPase and creatine kinase are preserved in ischemic hearts suffering irreversible contractile failure.

Due to glycolysis, anaerobic ATP supports normal electrical activity and allows only a reduced but sustained mechanical activity; however, frog myocardium metabolism is mainly aerobic dependent. Krebs cycle provides ATP for both mechanical and electrical acitivites even when glycolysis is inhibited. The close correlation between amplitude of contraction and metabolic state suggests that ATP is a limiting factor of mechanical activity even in normal conditions. Therefore it is necessary to use oxygen and to add pyruvate which partly compensate for the decrease of mechanical activity observed during the occurrence of hypodynamic state. Moreover equilibrating the oncotic pressure prevents hypodynamia and considerably improves the experimental conditions.

The state of myofibril creatinkinase and contractile properties of chemically skinned myocardial fibers of rat after 70-90 min ischemia and 15-30 min reperfusion was studied. In spite of sharp fall in the total creatinkinase activity in the tissue, the enzyme activity in myofibrils does not change greatly. Ischemia does not change the functional abilities of myofibril creatinkinase as well as characteristics of Ca-activated contraction of skinned fibers. The results show that irreversible loss of myocardial contractile activity after prolonged ischemia and reperfusion is not connected with violation of myofilament characteristics or deterioration of functional association between myofibril creatinkinase and ATPase.