Mitochondrial creatine kinase is critically necessary for normal myocardial high-energy phosphate metabolism

The individual functional significance of the various creatine kinase (CK) isoenzymes for myocardial energy homeostasis is poorly understood. Whereas transgenic hearts lacking the M subunit of CK (M-CK) show unaltered cardiac energetics and left ventricular (LV) performance, deletion of M-CK in combination with loss of sarcomeric mitochondrial CK (ScCKmit) leads to significant alterations in myocardial high-energy phosphate metabolites. To address the question as to whether this alteration is due to a decrease in total CK activity below a critical threshold or due to the specific loss of ScCKmit, we studied isolated perfused hearts with selective loss of ScCKmit (ScCKmit(-/-), remaining total CK activity approximately 70%) using (31)P NMR spectroscopy at two different workloads. LV performance in ScCKmit(-/-) hearts (n = 11) was similar compared with wild-type hearts (n = 9). Phosphocreatine/ATP, however, was significantly reduced in ScCKmit(-/-) compared with wild-type hearts (1.02 +/- 0.05 vs. 1.54 +/- 0.07, P < 0.05). In parallel, free [ADP] was higher (144 +/- 11 vs. 67 +/- 7 microM, P < 0.01) and free energy release for ATP hydrolysis (DeltaG(ATP)) was lower (-55.8 +/- 0.5 vs. -58.5 +/- 0.5 kJ/mol, P < 0.01) in ScCKmit(-/-) compared with wild-type hearts. These results demonstrate that M- and B-CK containing isoenzymes are unable to fully substitute for the loss of ScCKmit. We conclude that ScCKmit, in contrast to M-CK, is critically necessary to maintain normal high-energy phosphate metabolite levels in the heart.     

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.     

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.     

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.     

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.     

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.     

Similar mitochondrial activation kinetics in wild-type and creatine kinase-deficient fast-twitch muscle indicate significant Pi control of respiration

Past simulations of oxidative ATP metabolism in skeletal muscle have predicted that elimination of the creatine kinase (CK) reaction should result in dramatically faster oxygen consumption dynamics during transitions in ATP turnover rate. This hypothesis was investigated. Oxygen consumption of fast-twitch (FT) muscle isolated from wild-type (WT) and transgenic mice deficient in the myoplasmic (M) and mitochondrial (Mi) CK isoforms (MiM CK(-/-)) were measured at 20°C at rest and during electrical stimulation. MiM CK(-/-) muscle oxygen consumption activation kinetics during a step change in contraction rate were 30% faster than WT (time constant 53 ± 3 vs. 69 ± 4 s, respectively; mean ± SE, n = 8 and 6, respectively). MiM CK(-/-) muscle oxygen consumption deactivation kinetics were 380% faster than WT (time constant 74 ± 4 s vs. 264 ± 4 s, respectively). Next, the experiments were simulated using a computational model of the oxidative ATP metabolic network in FT muscle featuring ADP and Pi feedback control of mitochondrial respiration (J. A. L. Jeneson, J. P. Schmitz, N. A. van den Broek, N. A. van Riel, P. A. Hilbers, K. Nicolay, J. J. Prompers. Am J Physiol Endocrinol Metab 297: E774-E784, 2009) that was reparameterized for 20°C. Elimination of Pi control via clamping of the mitochondrial Pi concentration at 10 mM reproduced past simulation results of dramatically faster kinetics in CK(-/-) muscle, while inclusion of Pi control qualitatively explained the experimental observations. On this basis, it was concluded that previous studies of the CK-deficient FT muscle phenotype underestimated the contribution of Pi to mitochondrial respiratory control.     

Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.     

Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Sustained overexpression of IGF-1 prevents age-dependent decrease in charge movement and intracellular Ca(2+) in mouse skeletal muscle

In this work we tested the hypothesis that transgenic sustained overexpression of IGF-1 prevents age-dependent decreases in charge movement and intracellular Ca(2+) in skeletal muscle fibers. To this end, short flexor digitorum brevis (FDB) muscle fibers from 5-7- and 21-24-month-old FVB (wild-type) and S1S2 (IGF-1 transgenic) mice were studied. Fibers were voltage-clamped in the whole-cell configuration of the patch-clamp technique according to described procedures (Wang, Z. M., M. L. Messi, and O. Delbono. 1999. Biophys. J. 77:2709-2716). Charge movement and intracellular Ca(2+) concentration were recorded simultaneously. The maximum charge movement (Q(max)) recorded in young wild-type and transgenic mice was (mean +/- SEM, in nC microF(-1)): 52 +/- 2.1 (n = 46) and 54 +/- 1.9 (n = 38) (non-significant, ns), respectively, whereas in old wild-type and old transgenic mice the values were 36 +/- 2.1 (n = 32) and 49 +/- 2.3 (n = 35), respectively (p < 0.01). The peak intracellular calcium [Ca(2+)](i) recorded in young wild-type and transgenic mice was (in muM): 14.5 +/- 0.9 and 16 +/- 2.1 (ns), whereas in old wild-type and transgenic mice the values were 9.9 +/- 0.1 and 14 +/- 1.1 (p < 0.01), respectively. No significant changes in the voltage distribution or steepness of the Q-V or [Ca(2+)]-V relationship were found. These data support the concept that overexpression of IGF-1 in skeletal muscle prevents age-dependent reduction in charge movement and peak [Ca(2+)](i).     

Novel cobalt complex inhibitors of mitochondrial calcium uptake.

Reperfusion of the ischaemic myocardium leads to intracellular calcium overload followed by mitochondrial dysfunction, resulting in insufficient energy supply and ultimately myocardial necrosis. Ruthenium red (RR), a potent mitochondrial calcium uptake inhibitor, prevents this disruption to mitochondrial metabolism and improves post reperfusion recovery. This therefore suggested that mitochondrial calcium influx is an attractive target for the treatment of reperfusion injury. However, RR is unsuitable for therapeutic use, so we undertook a search for novel compounds which inhibit mitochondrial calcium uptake. The most potent compounds discovered were simple tris(ethylenediamine) transition metal complexes and dinuclear Co complexes. The structure-activity relationship (SAR) of these small molecules has helped to define the structural requirements for inhibition of calcium transport by outlining the size and charge dependency of the interactive site on the mitochondrial calcium uniporter.     

Mitochondrial affinity for ADP is twofold lower in creatine kinase knock-out muscles. Possible role in rescuing cellular energy homeostasis

Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and/or mitochondrial (Mi) creatine kinase (CK) isoforms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK-/-), mitochondrial (Mi-CK-/-) and double knock-out (MiM-CK-/-), respectively). Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2 x mg mitochondrial protein(-1) x min(-1)) and ADP affinity (K50ADP; microM) were determined by respirometry. State 3 Vmax and of M-CK-/- and MiM-CK-/- gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK-/-. For mutant cardiac mitochondria, only the of mitochondria isolated from the MiM-CK-/- phenotype was different (i.e. twofold higher) than that of WT. The implications of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentrations in MiM-CK-/- muscle genotypes may contribute to linear mitochondrial control of the reduced cytosolic ATP free energy potentials in these phenotypes.     

In vivo effects of uncoupling protein-3 gene disruption on mitochondrial energy metabolism

To clarify the role of uncoupling protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using (31)P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of (13)C enrichment in C-4 and C-2 of glutamate during an infusion of [2-(13)C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from P(i) more than 4-fold under fasting conditions (wild type, 2.2 +/- 0.6 versus knockout, 9.1 +/- 1.4 micromol/g of muscle/min, p < 0.001) with no change in TCA cycle flux rate (wild type, 0.74 +/- 0.04 versus knockout, 0.71 +/- 0.03 micromol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05) increase in the ratio of ATP to ADP in the muscle of UCP3KO mice (5.9 +/- 0.3) compared with controls (4.5 +/- 0.4). The data presented here provide the first evidence of uncoupling activity by UCP3 in skeletal muscle in vivo.     

Regulation of murine myocardial energy metabolism during adrenergic stress studied by in vivo 31P NMR spectroscopy

Image-guided, spatially localized 31P magnetic resonance spectroscopy (MRS) was used to study in vivo murine cardiac metabolism under resting and dobutamine-induced stress conditions. Intravenous dobutamine infusion (24 mug. min-1. kg body wt-1) increased the mean heart rate by approximately 39% from 482 +/- 46 per min at baseline to 669 +/- 77 per min in adult mice. The myocardial phosphocreatine (PCr)-to-ATP (PCr/ATP) ratio remained unchanged at 2.1 +/- 0.5 during dobutamine stress, compared with baseline conditions. Therefore, we conclude that a significant increase in heart rate does not result in a decline in the in vivo murine cardiac PCr/ATP ratio. These observations in very small mammals, viz., mice, at extremely high heart rates are consistent with studies in large animals demonstrating that global levels of high-energy phosphate metabolites do not regulate in vivo myocardial metabolism during physiologically relevant increases in cardiac work.     

Reduced creatine kinase activity in transgenic amyotrophic lateral sclerosis mice

Creatine (Cr), the substrate of the creatine kinase (CK) isoenzymes, was shown to be neuroprotective in several models of neurodegeneration, including amyotrophic lateral sclerosis (ALS). In order to investigate the mechanism of this beneficial effect, we determined CK activities and mitochondrial respiration rates in tissues from G93A transgenic mice, which overexpress a mutant form of human superoxide dismutase associated with familial ALS (FALS). While respiration rates of mitochondria from G93A transgenic or wild-type control mice isolated from spinal cord showed no difference, a significant and dramatic loss of CK activity could be detected in these tissues. In homogenates from spinal cord of G93A transgenic mice, CK activity decreased to 49% and in mitochondrial fractions to 67% compared to CK activities in wild-type control mice. Feeding the G93A transgenic mice with 2% Cr, the same tissues showed no statistically significant increase of CK activity compared to regular fed G93A transgenic mice. Experiments with isolated mitochondria, however, showed that Cr and adenosine triphosphate (ATP) protected mitochondrial CK activity against peroxynitrite-induced inactivation, which may play a role in tissue damage in neurodegeneration. Our data provide evidence for oxidative damage to the CK system in ALS, which may contribute to impaired energy metabolism and neurodegeneration.     

Role of interleukin-6 in cardiac inflammation and dysfunction after burn complicated by sepsis

To examine the role of myocardial interleukin-6 (IL-6) in myocardial inflammation and dysfunction after burn complicated by sepsis, we performed 40% total body surface area contact burn followed by late (7 days) Streptococcus pneumoniae pneumonia sepsis in wild-type (WT) mice, IL-6 knockout (IL-6 KO) mice, and transgenic mice overexpressing IL-6 in the myocardium (TG). Twenty-four hours after sepsis was induced, isolated cardiomyocytes were harvested and cultured in vitro, and supernatant concentrations of IL-6 and tumor necrosis factor (TNF)-alpha were measured. Cardiomyocyte intracellular calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) concentrations were also determined. Separate mice in each group underwent in vivo global hemodynamic and cardiac function assessment by cannulation of the carotid artery and insertion of a left ventricular pressure volume conductance catheter. Hearts from these mice were collected for histopathological assessment of inflammatory response, fibrosis, and apoptosis. In the WT group, there was an increase in cardiomyocyte TNF-alpha, [Ca(2+)](i), and [Na(+)](i) after burn plus sepsis, along with cardiac contractile dysfunction, inflammation, and apoptosis. These changes were attenuated in the IL-6 KO group but accentuated in the TG group. We conclude myocardial IL-6 mediates cardiac inflammation and contractile dysfunction after burn plus sepsis.     

Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.     

(99m)Tc-sestamibi uptake in rat skeletal muscle and heart: physiological determinants and correlations

The lipophilic cationic radiotracer (99m)Tc-sestamibi, known to be concentrated within mitochondria, is widely used for myocardial perfusion and to a lesser extent for muscle metabolism imaging. However, the exact distribution pattern in skeletal muscle has not been yet studied in detail. The present study aims to investigate the (99m)Tc-sestamibi uptake in rat skeletal muscle and myocardium in relation to their metabolic characteristics. (99m)Tc-sestamibi was i.v. administered in twenty adult male Wistar rats and uptake, as percent of injected dose per tissue gram (%ID/g), in the myocardium, soleus, extensor digitorum longus and gastrocnemius muscles was assessed 2 h after the injection. Muscle uptake was also correlated with myocardial uptake, muscle weight and body weight. Skeletal muscle (99m)Tc-sestamibi uptake was a small (9-16 %) fraction of that found in myocardium (1.71+/-0.63 %ID/g). Among the three hindlimb muscles considered, the slow-oxidative soleus muscle showed the highest uptake (0.28+/-0.16 %ID/g). Metabolically diverse parts of the gastrocnemius muscle showed different uptake. Skeletal muscle uptake was positively correlated with myocardial uptake and both were negatively correlated with tissue and body weight. Skeletal muscle and myocardium (99m)Tc-sestamibi uptake is related to their metabolic profile. Myocardium, with an exceptional rich mitochondrial concentration, shows much higher (99m)Tc-sestamibi uptake compared to skeletal muscles. Among muscles, uptake is dependent on their mitochondrial content. Evidence of matching exists between myocardial and muscle uptake, and both are size-dependent.