Investigation of muscle bioenergetics in the Marfan syndrome indicates reduced metabolic efficiency.

BACKGROUND: The Marfan syndrome is an inherited multisystem disorder caused by mutations in fibrillin 1, with cardiovascular involvement being the most important feature of the phenoptype. Affected individuals have impaired flow-mediated dilatation (FMD) of large arteries of a similar severity to patients with chronic heart failure (CHF). AIMS: Skeletal muscle bioenergetics were studied in patients with the Marfan syndrome in order to evaluate the impact of impaired flow-mediated dilatation on skeletal muscle metabolism. Skeletal muscle metabolism is abnormal in CHF and the aetiology is unclear. METHODS: Thirteen patients and 12 controls were studied by phosphorus Magnetic Resonance spectroscopy of the calf muscle using an incremental exercise protocol and by Magnetic Resonance imaging. RESULTS: Metabolic variables measured at rest were normal in Marfan patients. For a similar total work output measured at end of the standardized incremental exercise, the total rate of energy consumption (EC) was significantly increased in patients (21.2 +/- 2.3 mM ATP/min/W vs 13.6 +/- 1.4 mM ATP/min/W in controls). Similarly, both PCr and pH time-dependent changes were significantly different between groups. The absolute contributions of aerobic and glycolytic pathways to energy production were significantly higher in patients whereas they were similar when expressed relative to EC. CONCLUSIONS: The higher EC measured in patients with Marfan syndrome was supported by both oxidative and anaerobic metabolic pathways, thereby suggesting a decrease in muscle efficiency and/or muscle mass, as previously described in other diseases affecting skeletal muscle function such as heart failure and peripheral vascular disease.

Mechanisms of creatine depletion in chronically failing rat heart.

The failing myocardium is characterised by energetic imbalance, reflected by reduced phosphocreatine and creatine content. These changes may contribute to cardiac dysfunction, yet mechanisms of creatine and phosphocreatine depletion are poorly understood. Creatine is taken up by the heart via the creatine transporter. We investigated the mechanisms leading to myocardial creatine depletion in heart failure. Therefore rats were subjected to chronic left coronary artery ligation (MI; n = 36) or to sham operation (sham; n = 25). After 8 weeks, hearts were perfused with 14C-creatine buffer to determine creatine uptake rates via the creatine transporter. Total creatine content was determined by HPLC. Creatine transport in sham hearts followed Michaelis-Menten kinetics with a V(max) of 5.9 +/- 0.5 nmol/min per gww. Heart failure led to a significant 30% decrease in intracellular creatine content and to a significant 26% reduction in creatine uptake (V(max) in MI 4.3 +/- 0.4 nmol/min per gww; P < 0.001 vs. sham). We conclude that depletion of creatine/phosphocreatine content in the failing heart is due to reduced sarcolemmal creatine uptake. The creatine transporter may be a potential therapeutic target to prevent energetic imbalance in heart failure.

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.

Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency.

We generated a knockout mouse model for guanidinoacetate N-methyltransferase (GAMT) deficiency (MIM 601240), the first discovered human creatine deficiency syndrome, by gene targeting in embryonic stem cells. Disruption of the open reading frame of the murine GAMT gene in the first exon resulted in the elimination of 210 of the 237 amino acids present in mGAMT. The creation of an mGAMT null allele was verified at the genetic, RNA and protein levels. GAMT knockout mice have markedly increased guanidinoacetate (GAA) and reduced creatine and creatinine levels in brain, serum and urine, which are key findings in human GAMT patients. In vivo (31)P magnetic resonance spectroscopy showed high levels of PGAA and reduced levels of creatine phosphate in heart, skeletal muscle and brain. These biochemical alterations were comparable to those found in human GAMT patients and can be attributed to the very similar GAMT expression patterns found by us in human and mouse tissues. We provide evidence that GAMT deficiency in mice causes biochemical adaptations in brain and skeletal muscle. It is associated with increased neonatal mortality, muscular hypotonia, decreased male fertility and a non-leptin-mediated life-long reduction in body weight due to reduced body fat mass. Therefore, GAMT knockout mice are a valuable creatine deficiency model for studying the effects of high-energy phosphate depletion in brain, heart, skeletal muscle and other organs.

Hypertrophic cardiomyopathy due to sarcomeric gene mutations is characterized by impaired energy metabolism irrespective of the degree of hypertrophy.

OBJECTIVES: We investigated cardiac energetics in subjects with mutations in three different familial hypertrophic cardiomyopathy (HCM) disease genes, some of whom were nonpenetrant carriers without hypertrophy, using phosphorus-31 magnetic resonance spectroscopy. BACKGROUND: Familial hypertrophic cardiomyopathy is caused by mutations in sarcomeric protein genes. The mechanism by which these mutant proteins cause disease is uncertain. A defect of myocyte contractility had been proposed, but in vitro studies of force generation have subsequently shown opposing results in different classes of mutation. An alternative hypothesis of "energy compromise" resulting from inefficient utilization of adenosine triphosphate (ATP) has been suggested, but in vivo data in humans with genotyped HCM are lacking. METHODS: The cardiac phosphocreatine (PCr) to ATP ratio was determined at rest in 31 patients harboring mutations in the genes for either beta-myosin heavy chain, cardiac troponin T, or myosin-binding protein C, and in 24 controls. Transthoracic echocardiography was used to measure left ventricular (LV) dimensions and maximal wall thickness. RESULTS: The PCr/ATP was reduced in the HCM subjects by 30% relative to controls (1.70 +/- 0.43 vs. 2.44 +/- 0.30; p < 0.001), and the reduction was of a similar magnitude in all three disease-gene groups. The PCr/ATP was equally reduced in subjects with (n = 24) and without (n = 7) LV hypertrophy. CONCLUSIONS: Our data provide evidence of a bioenergetic deficit in genotype-confirmed HCM, which is present to a similar degree in three disease-gene groups. The presence of energetic abnormalities, even in those without hypertrophy, supports a proposed link between altered cardiac energetics and development of the disease phenotype.

From energy store to energy flux: a study in creatine kinase-deficient fast skeletal muscle.

Fast-twitch skeletal muscle of mice deficient in cytosolic and mitochondrial creatine kinase isoforms (CK-/-) lack burst activity but can sustain prolonged contractile activity, suggesting that adaptive mechanisms can regulate local adenine nucleotide turnover. We investigated whether direct energy and signal channeling between mitochondria and sarcoplasmic reticulum (SR) or myofilaments may exist that compensate for the lack of CK isoenzymes. Oxidative capacity of fast-twitch muscle was increased twofold in CK-/- mice. Energy cross talk between organelles was studied in muscle fibers with permeabilized sarcolemma. Energy supply to SR was estimated by analyzing the tension transient induced by caffeine and energy supply to myofilaments was estimated by the relaxation of rigor tension, both under different conditions of energy supply. In normal mice, ATP directly produced by mitochondria was not able to sustain calcium uptake and to relax rigor tension as efficiently as ATP produced by bound CK. However, in CK-/- mice, mitochondria ability to provide ATP for calcium uptake and relaxation of rigor tension was dramatically enhanced, suggesting a direct ATP/ADP channeling between sites of energy production mitochondria) and energy utilization in CK-/- mice. These results demonstrate two possible patterns of energy transport in muscle cells: energy store with phosphocreatine and energy flux through mitochondria.

Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine.

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta-guanidinopropionic acid (beta-GPA). Creatine transport in control hearts followed saturation kinetics with a K(m) of 70 +/- 13 mM and a V(max) of 3.7 +/- 0.07 nmol x min(-1) x g wet wt(-1). Creatine supplementation significantly decreased the V(max) of the CrT (2.7 +/- 0.17 nmol x min(-1) x g wet wt(-1)). This was matched by an approximately 35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta-GPA exhibited a >80% decrease in tissue creatine and increase in beta-GPA(total). The V(max) of the CrT was increased (6.0 +/- 0.25 nmol x min(-1) x g wet wt(-1)) and the K(m) decreased (39.8 +/- 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.

Role of creatine kinase in cardiac excitation-contraction coupling: studies in creatine kinase-deficient mice.

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.