Phosphotransfer dynamics in skeletal muscle from creatine kinase gene-deleted mice

To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant ATPase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.     

Creatine kinase-deficient hearts exhibit increased susceptibility to ischemia-reperfusion injury and impaired calcium homeostasis

The creatine kinase (CK) system is involved in the rapid transport of high-energy phosphates from the mitochondria to the sites of maximal energy requirements such as myofibrils and sarcolemmal ion pumps. Hearts of mice with a combined knockout of cytosolic M-CK and mitochondrial CK (M/Mito-CK(-/-)) show unchanged basal left ventricular (LV) performance but reduced myocardial high-energy phosphate concentrations. Moreover, skeletal muscle from M/Mito-CK(-/-) mice demonstrates altered Ca2+ homeostasis. Our hypothesis was that in CK-deficient hearts, a cardiac phenotype can be unmasked during acute stress conditions and that susceptibility to ischemia-reperfusion injury is increased because of altered Ca2+ homeostasis. We simultaneously studied LV performance and myocardial Ca2+ metabolism in isolated, perfused hearts of M/Mito-CK(-/-) (n = 6) and wild-type (WT, n = 8) mice during baseline, 20 min of no-flow ischemia, and recovery. Whereas LV performance was not different during baseline conditions, LV contracture during ischemia developed significantly earlier (408 +/- 72 vs. 678 +/- 54 s) and to a greater extent (50 +/- 2 vs. 36 +/- 3 mmHg) in M/Mito-CK(-/-) mice. During reperfusion, recovery of diastolic function was impaired (LV end-diastolic pressure: 22 +/- 3 vs. 10 +/- 2 mmHg), whereas recovery of systolic performance was delayed, in M/Mito-CK(-/-) mice. In parallel, Ca2+ transients were similar during baseline conditions; however, M/Mito-CK(-/-) mice showed a greater increase in diastolic Ca2+ concentration ([Ca2+]) during ischemia (237 +/- 54% vs. 167 +/- 25% of basal [Ca2+]) compared with WT mice. In conclusion, CK-deficient hearts show an increased susceptibility of LV performance and Ca2+ homeostasis to ischemic injury, associated with a blunted postischemic recovery. This demonstrates a key function of an intact CK system for maintenance of Ca2+ homeostasis and LV mechanics under metabolic stress conditions.     

Myofibrillar or mitochondrial creatine kinase deficiency alone does not impair mouse diaphragm isotonic function.

Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. The diaphragm (Dia) expresses both M-CK and ScCKmit in abundance. We compared the power and work output of 1) control CK-sufficient (Ctl), 2) M-CK-deficient [M-CK(-/-)], 3) ScCKmit-deficient [ScCKmit(-/-)], and 4) combined M-CK/ScCKmit-deficient null mutant [CK(-/-)] Dia during repetitive isotonic activations to determine the effect of CK phenotype on Dia function. Maximum power was obtained at approximately 0.4 tetanic force in all groups. M-CK(-/-) and ScCKmit(-/-) Dia were able to sustain power and work output at Ctl levels during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load), and the duration of sustained Dia shortening was 67 +/- 4 s in M-CK(-/-), 60 +/- 4 s in ScCKmit(-/-), and 62 +/- 5 s in Ctl Dia. In contrast, CK(-/-) Dia power and work declined acutely and failed to sustain shortening altogether by 40 +/- 6 s. We conclude that Dia power and work output are not absolutely dependent on the presence of either M-CK or ScCKmit, whereas the complete absence of CK acutely impairs Dia shortening capacity during repetitive activation.     

Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart

Creatine kinase (CK) exists as a family of isoenzymes in excitable tissue. We studied isolated perfused hearts from mice lacking genes for either the main muscle isoform of CK (M-CK) or both M-CK and the main mitochondrial isoform (Mt-CK) to determine 1) the biological significance of CK isoenzyme shifts, 2) the necessity of maintaining a high CK reaction rate, and 3) the role of CK isoenzymes in establishing the thermodynamics of ATP hydrolysis. (31)P NMR was used to measure [ATP], [PCr], [P(i)], [ADP], pH, as well as the unidirectional reaction rate of PCr--> [gamma-P]ATP. Developmental changes in the main fetal isoform of CK (BB-CK) were unaffected by loss of other CK isoenzymes. In hearts lacking both M- and Mt-CK, the rate of ATP synthesis from PCr was only 9% of the rate of ATP synthesis from oxidative phosphorylation demonstrating a lack of any high energy phosphate shuttle. We also found that the intrinsic activities of the BB-CK and the MM-CK isoenzymes were equivalent. Finally, combined loss of M- and Mt-CK (but not loss of only M-CK) prevented the amount of free energy released from ATP hydrolysis from increasing when pyruvate was provided as a substrate for oxidative phosphorylation.     

Impaired muscular contractile performance and adenine nucleotide handling in creatine kinase-deficient mice

Creatine kinase (CK) forms a small family of isoenzymes playing an important role in maintaining the concentration of ATP and ADP in muscle cells. To delineate the impact of a lack of CK activity, we studied contractile performance during a single maximal tetanic contraction and during 12 repeated tetanic contractions of intact dorsal flexors of CK knockout (CK(-/-)) mice. To investigate the effect on ATP regeneration, muscular high-energy phosphate content was determined at rest, immediately after the contraction series, and after a 60-s recovery period. Maximal torque of the dorsal flexors was significantly lower in CK(-/-) mice than in wild-type animals, i.e., 23.7 +/- 5.1 and 33.3 +/- 6.8 mN. m. g(-1) wet wt, respectively. Lower muscle ATP (20.1 +/- 1.4 in CK(-/-) vs. 28.0 +/- 2.1 micromol/g dry wt in controls) and higher IMP (1.2 +/- 0.5 in CK(-/-) vs. 0.3 +/- 0.1 micromol/g dry wt in controls) levels at the onset of contraction may contribute to the declined contractility in CK(-/-) mice. In contrast to wild-type muscles, ATP levels could not be maintained during the series of 12 tetanic contractions of dorsal flexors of CK(-/-) mice and dropped to 15.5 +/- 2.4 micromol/g dry wt. The significant increase in tissue IMP (2.4 +/- 1.1 micromol/g dry wt) content after the contraction series indicates that ATP regeneration through adenylate kinase was not capable of fully compensating for the lack of CK. ATP regeneration via the adenylate kinase pathway is a likely cause of reduced basal adenine nucleotide levels in CK(-/-) mice.     

Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa

We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired intracellular energetic communication in muscles from creatine kinase and adenylate kinase (M-CK/AK1) double knock-out mice

Previously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency.     

Xanthine oxidase inhibitors improve energetics and function after infarction in failing mouse hearts

After myocardial infarction, ventricular geometry and function, as well as energy metabolism, change markedly. In nonischemic heart failure, inhibition of xanthine oxidase (XO) improves mechanoenergetic coupling by improving contractile performance relative to a reduced energetic demand. However, the metabolic and contractile effects of XO inhibitors (XOIs) have not been characterized in failing hearts after infarction. After undergoing permanent coronary ligation, mice received a XOI (allopurinol or oxypurinol) or matching placebo in the daily drinking water. Four weeks later, 1H MRI and 31P magnetic resonance spectroscopy (MRS) were used to quantify in vivo functional and metabolic changes in postinfarction remodeled mouse myocardium and the effects of XOIs on that process. End-systolic (ESV) and end-diastolic volumes (EDV) were increased by more than sixfold after infarction, left ventricle (LV) mass doubled (P < 0.005), and the LV ejection fraction (EF) decreased (14 +/- 9%) compared with control hearts (59 +/- 8%, P < 0.005) at 1 mo. The myocardial phosphocreatine (PCr)-to-ATP ratio (PCr/ATP) was also significantly decreased in infarct remodeled hearts (1.4 +/- 0.6) compared with control animals (2.1 +/- 0.5, P < 0.02), in agreement with prior studies in larger animals. The XOIs allopurinol and oxypurinol did not change LV mass but limited the increase in ESV and EDV of infarct hearts by 50%, increased EF (23 +/- 9%, P = 0.01), and normalized cardiac PCr/ATP (2.0 +/- 0.5, P < 0.04). We conclude that XOIs improve ventricular function after infarction and normalize high-energy phosphate ratio in heart failure. Thus XOI therapy offers a new and potentially complementary approach to limit the adverse contractile and metabolic consequences after infarction.     

Interstitial purine metabolites in hearts with LV remodeling

The myocardial ATP concentration is significantly decreased in failing hearts, which may be related to the progressive loss of the myocardial total adenine nucleotide pool. The total myocardial interstitial purine metabolites (IPM) in the dialysate of interstitial fluid could reflect the tissue ATP depletion. In rats, postmyocardial infarction (MI) left ventricular (LV) remodeling was induced by ligation of the coronary artery. Cardiac microdialysis was employed to assess changes of IPM in response to graded beta-adrenergic stimulation with isoproterenol (Iso) in myocardium of hearts with post-MI LV remodeling (MI group) or hearts with sham operation (sham group). The dialysate samples were analyzed for adenosine, inosine, hypoxanthine, xanthine, and uric acid. LV volume was greater in the MI group (2.2 +/- 0.2 ml/kg) compared with the sham group (1.3 +/- 0.2 ml/kg, P < 0.05). Infarct size was 28 +/- 4%. The baseline dialysate level of uric acid was higher in the MI group (18.9 +/- 3.4 micromol) compared with the sham group (4.6 +/- 0.7 micromol, P < 0.01). During and after Iso infusion, the dialysate levels of adenosine, xanthine, and uric acid were all significantly higher in the MI group. Thus the level of IPM is increased in hearts with postinfarction LV remodeling both at baseline and during Iso infusion. These results suggest that the decreased myocardial ATP level in hearts with post-MI LV remodeling may be caused by the chronic depletion of the total adenine nucleotide pool.     

Multimodal functional cardiac MRI in creatine kinase-deficient mice reveals subtle abnormalities in myocardial perfusion and mechanics

A decrease in the supply of ATP from the creatine kinase (CK) system is thought to contribute to the evolution of heart failure. However, previous studies on mice with a combined knockout of the mitochondrial and cytosolic CK (CK(-/-)) have not revealed overt left ventricular dysfunction. The aim of this study was to employ novel MRI techniques to measure maximal myocardial velocity (V(max)) and myocardial perfusion and thus determine whether abnormalities in the myocardial phenotype existed in CK(-/-) mice, both at baseline and 4 wk after myocardial infarction (MI). As a result, myocardial hypertrophy was seen in all CK(-/-) mice, but ejection fraction (EF) remained normal. V(max), however, was significantly reduced in the CK(-/-) mice [wild-type, 2.32 +/- 0.09 vs. CK(-/-), 1.43 +/- 0.16 cm/s, P < 0.05; and wild-type MI, 1.53 +/- 0.11 vs. CK(-/-) MI, 1.26 +/- 0.11 cm/s, P = not significant (NS), P < 0.05 vs. baseline]. Myocardial perfusion was also lower in the CK(-/-) mice (wild-type, 6.68 +/- 0.27 vs. CK(-/-), 4.12 +/- 0.63 ml/g.min, P < 0.05; and wild-type MI, 3.97 +/- 0.65 vs. CK(-/-) MI, 3.71 +/- 0.57 ml/g.min, P = NS, P < 0.05 vs. baseline), paralleled by a significantly reduced capillary density (histology). In conclusion, myocardial function in transgenic mice may appear normal when only gross indexes of performance such as EF are assessed. However, the use of a combination of novel MRI techniques to measure myocardial perfusion and mechanics allowed the abnormalities in the CK(-/-) phenotype to be detected. The myocardium in CK-deficient mice is characterized by reduced perfusion and reduced maximal contraction velocity, suggesting that the myocardial hypertrophy seen in these mice cannot fully compensate for the absence of the CK system.     

Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics

Although ischemic preconditioning induces bioenergetic tolerance and thereby remodels energy metabolism that is crucial for postischemic recovery of the heart, the molecular components associated with preservation of cellular energy production, transfer, and utilization are not fully understood. Here myocardial bioenergetic dynamics were assessed by (18)O-assisted (31)P-NMR spectroscopy in control or preconditioned hearts from wild-type (WT) or Kir6.2-knockout (Kir6.2-KO) mice that lack metabolism-sensing sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 +/- 20 vs. 155 +/- 15 nmol x mg protein(-1) x min(-1)), ATP synthesis rate (58 +/- 3 vs. 46 +/- 3% (18)O labeling of gamma-ATP), and ATP consumption rate (51 +/- 4 vs. 31 +/- 4% (18)O labeling of P(i)) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 +/- 26 nmol x mg protein(-1) x min(-1)) but not Kir6.2-KO (133 +/- 18 nmol x mg protein(-1) x min(-1)) hearts. In contrast with WT hearts, preconditioning failed to preserve contractile recovery in Kir6.2-KO hearts, as tight coupling between postischemic performance and high-energy phosphoryl transfer was compromised in the K(ATP)-channel-deficient myocardium. Thus intact K(ATP) channels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.     

Glycolytic buffering affects cardiac bioenergetic signaling and contractile reserve similar to creatine kinase

Creatine kinase (CK) and glycolysis represent important energy-buffering processes in the cardiac myocyte. Although the role of compartmentalized CK in energy transfer has been investigated intensely, similar duties for intracellular glycolysis have not been demonstrated. By measuring the response time of mitochondrial oxygen consumption to dynamic workload jumps (tmito) in isolated rabbit hearts, we studied the effect of inhibiting energetic systems (CK and/or glycolysis) on transcytosolic signal transduction that couples cytosolic ATP hydrolysis to activation of oxidative phosphorylation. Tyrode-perfused hearts were exposed to 15 min of the following: 1) 0.4 mM iodoacetamide (IA; n = 6) to block CK (CK activity <3% vs. control), 2) 0.3 mM iodoacetic acid (IAA; n = 5) to inhibit glycolysis (GAPDH activity <3% vs. control), or 3) vehicle (control, n = 7) at 37 degrees C. Pretreatment tmito was similar across groups at 4.3 +/- 0.3 s (means +/- SE). No change in tmito was observed in control hearts; however, in IAA- and IA-treated hearts, tmito decreased by 15 +/- 3% and 40 +/- 5%, respectively (P < 0.05 vs. control), indicating quicker energy supply-demand signaling in the absence of ADP/ATP buffering by CK or glycolysis. The faster response times in IAA and IA groups were independent of the size of the workload jump, and the increase in myocardial oxygen consumption during workload steps was unaffected by CK or glycolysis blockade. Contractile function was compromised by IAA and IA treatment versus control, with contractile reserve (defined as increase in rate-pressure product during a standard heart rate jump) reduced to 80 +/- 8% and 80 +/- 10% of baseline, respectively (P < 0.05 vs. control), and significant elevations in end-diastolic pressure, suggesting raised ADP concentration. These results demonstrate that buffering of phosphate metabolites by glycolysis in the cytosol contributes appreciably to slower mitochondrial activation and may enhance contractile efficiency during increased cardiac workloads. Glycolysis may therefore play a role similar to CK in heart muscle.     

Bioenergetic and functional consequences of stem cell-based VEGF delivery in pressure-overloaded swine hearts

In an established swine model of severe left ventricular (LV) hypertrophy (LVH), the bioenergetic and functional consequences of transplanting autologous mesenchymal stem cells (MSCs) overexpressing vascular endothelial growth factor (VEGF-MSCs) into the LV were evaluated; transplantation was accomplished by infusion of VEGF-MSCs into the interventricular cardiac vein. Specifically, the hypertrophic response to aortic banding was compared in seven pigs treated with 30 million VEGF-MSCs, eight pigs treated with 30 million MSCs without VEGF modification, and 19 untreated LVH pigs. Eight pigs without banding or cell transplantation (normal) were also studied. Four weeks postbanding, LV wall thickening (MRI), myocardial blood flow (MBF), high-energy phosphate levels ((31)P magnetic resonance spectroscopy), and hemodynamic measurements were obtained under basal conditions and during a catecholamine-induced high cardiac workstate (HCW). Although 9 of 19 untreated banded pigs developed clinical evidence of biventricular failure, no MSCs-treated animal developed heart failure. MSCs engraftment was present in both cell transplant groups, and both baseline and HCW MBF values were significantly increased in hearts receiving VEGF-MSCs compared with other groups (P < 0.05). During HCW, cardiac inotropic reserve (defined as the percent increase of rate pressure product at HCW relative to baseline) was normal in the VEGF-MSCs group and significantly decreased in all other banded groups. Additionally, during HCW, the myocardial energetic state [reflected by the phosphocreatine-to-ATP ratio (PCr/ATP)] of VEGF-MSCs-treated hearts remained stable, whereas in all other groups, PCr/ATP decreased significantly from baseline values (P < 0.05, each group). Myocardial von Willebrand factor and VEGF mRNA expressions and myocardial capillary density were significantly increased in VEGF-MSCs-treated hearts (P < 0.05). Hence, in the pressure-overloaded LV, transplantation of VEGF-MSCs prevents LV decompensation, induces neovascularization, attenuates hypertrophy, and improves MBF, myocardial bioenergetic characteristics, and contractile performance.     

Kinetic characterization of human heart and skeletal muscle CK isoenzymes

The purpose of this study was to investigate the kinetic properties of human creatine kinase (CK) isoenzymes partially purified from heart and skeletal muscle. Utilizing the backward CK-catalyzed reaction of creatine phosphate + ADP in equilibrium creatine + ATP, Km values for heart and skeletal muscle CK MM (3.7 mmol/l) were significantly (p less than 0.05) greater than CK MB (2.1 mmol/l) which were significantly (p less than 0.05) greater than mitochondrial CK (1.8 mmol/l) at variable creatine phosphate and fixed ADP concentrations. However, Km values for similar isoenzymes from the two different tissues, i.e., CK MB from heart vs. skeletal muscle, were not different. These results show that kinetic analysis of CK isoenzymes cannot differentiate the tissue source of elevated blood CK isoenzymes after the acute stress of long distance running or after acute myocardial infarction.     

Stress kinase phosphorylation is increased in pacing-induced heart failure in rabbits

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.     

Bioenergetic effect of liposomal coenzyme Q10 on myocardial ischemia reperfusion injury.

The antioxidant and bioenergetic effects of CoQ10 are well known but its clinical utility is limited by the requirement for enteral administration. A newly developed liposomal CoQ10 (CoQ) is water soluble and capable of intravenous administration. The purpose of this study is to determine the mechanism by which acute administration CoQ protects myocardium from reperfusion (Rp) injury. Rats were pretreated with CoQ 10 mg/kg i.v. 30 min prior to the experiment. Control rats were pretreated with liposome only. Hearts were excised and subjected to equilibration, 25 min of normothermic ischemia and 40 min of Rp on a Langendorff apparatus. At end Rp, CoQ hearts recovered 74 +/- 5% of their DP vs. 50 +/- 9% in control (p < 0.05). Aerobic efficiency was maintained (0.66 +/- 0.02 vs. control, 0.5 +/- 0.04, p < 0.003) and CoQ hearts lost less CK activity vs. control (p < 0.02). PCr and ATP were higher than control (p < 0.05, 0.02, respectively). Results show that i.v. CoQ improves recovery of function, aerobic efficiency, CK activity, and recovery of PCr and ATP after Rp. This suggests that acute administration of liposomal CoQ improves myocardial tolerance to I/R via its role as an antioxidant as well as improving oxygen utilization and high energy phosphate production.     

Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.     

Advantages of perfluorochemical perfusion in the isolated working rabbit heart preparation using 31P-NMR

Quantitative 31P-NMR and enzymatic analysis of high-energy phosphates were used to characterize an isolated perfused working rabbit heart preparation. In this model, the left side of the heart works against a physiological after-load. Two perfusates, Krebs-Henseleit saline and the perfluorocarbon emulsion FC-43 (perfluorotributylamine), were evaluated in their ability to maintain cardiac function and high-energy phosphate metabolites over a period of 2-3 h. Adenine nucleotides ATP, ADP, phosphocreatine and inorganic phosphate (Pi) were measured by 31P-NMR while monitoring cardiac output and coronary flow. Intracellular pH was determined using the chemical shift of Pi. At the end of each experiment, hearts were freeze clamped and enzymatically assayed for adenine nucleotides, phosphocreatine and Pi. In every experiment, hearts perfused with FC-43 emulsion maintained the same rate of cardiac output as hearts perfused with Krebs-Henseleit saline, but with half the coronary flow rate: FC-43, 22 +/- 2.5 (n = 5), Krebs-Henseleit saline 42 +/- 2.7 (n = 6) ml/min, P less than 0.001. Hearts perfused with FC-43 emulsion showed higher [phosphocreatine] and [ATP] measured by 31P-NMR. For [phosphocreatine]: FC-43 3.2 +/- 0.7 (n = 5), Krebs-Henseleit saline 1.7 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.01. For [ATP]: FC-43 1.8 +/- 0.7 (n = 5), Krebs-Henseleit saline 0.9 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.02. [phosphocreatine] and [ATP] determined by 31P-NMR values were identical within experimental error to those values obtained by enzymatic analysis. Comparing [Pi] determined by both methods, 36% of Pi in FC-43-perfused hearts, and only 24% of Pi in Krebs-Henseleit saline-perfused hearts were visible by NMR, indicating that a large proportion of Pi is bound in the intact functioning heart. Similar results were obtained for [ADP]. Using the combined techniques of 31P-NMR and enzymatic assay, we have shown in this model of the isolated working rabbit heart preparation, that FC-43 emulsion maintains significantly better function and high-energy phosphate levels than Krebs-Henseleit saline.     

Creatine Kinase-Overexpression Improves Myocardial Energetics,Contractile Dysfunction and Survival in Murine Doxorubicin Cardiotoxicity

Doxorubicin (DOX) is a commonly used life-saving antineoplastic agent that also causes dose-dependent cardiotoxicity. Because ATP is absolutely required to sustain normal cardiac contractile function and because impaired ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve reaction, may contribute to contractile dysfunction in heart failure, we hypothesized that impaired CK energy metabolism contributes to DOX-induced cardiotoxicity. We therefore overexpressed the myofibrillar isoform of CK (CK-M) in the heart and determined the energetic, contractile and survival effects of CK-M following weekly DOX (5mg/kg) administration using in vivo ³¹P MRS and ¹H MRI. In control animals, in vivo cardiac energetics were reduced at 7 weeks of DOX protocol and this was followed by a mild but significant reduction in left ventricular ejection fraction (EF) at 8 weeks of DOX, as compared to baseline. At baseline, CK-M overexpression (CK-M-OE) increased rates of ATP synthesis through cardiac CK (CK flux) but did not affect contractile function. Following DOX however, CK-M-OE hearts had better preservation of creatine phosphate and higher CK flux and higher EF as compared to control DOX hearts. Survival after DOX administration was significantly better in CK-M-OE than in control animals (p<0.02). Thus CK-M-OE attenuates the early decline in myocardial high-energy phosphates and contractile function caused by chronic DOX administration and increases survival. These findings suggest that CK impairment plays an energetic and functional role in this DOX-cardiotoxicity model and suggests that metabolic strategies, particularly those targeting CK, offer an appealing new strategy for limiting DOX-associated cardiotoxicity.