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.

Abstract-Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads. Key Words: mitochondria Ⅲ sarcoplasmic reticulum Ⅲ myofibrils Ⅲ creatine kinase Ⅲ knockout mice D ifferentiation and maturation of adult mammalian muscle cells lead to complex specialization and organization. In cardiac cells, specialized cellular functions are highly organized within structural and functional compartments. Energy-consuming processes are localized to the sarcoplasmic reticulum (SR) and myofibrillar compartments, while energy production occurs mainly within mitochondria. Muscle cells contain complex and specialized energy transfer systems, which efficiently link energy production and utilization. One such system is the family of creatine kinase (CK) isoenzymes that catalyze the reversible transfer of a phosphate moiety between creatine (Cr) and ATP. The mitochondrial sarcomeric isoenzyme (mi-CK) is bound to the outer surface of the inner mitochondrial membrane so that ATP generated by oxidative phosphorylation is transphosphorylated to phosphocreatine (PCr). 1-3 On the other hand, the cytosolic isoenzyme (MM-CK) that is structurally associated with myofibrils and SR membranes can use PCr to rephosphorylate all of the ADP produced by the ATPases and thus provide enough energy for normal contractile kinetics or SR calcium uptake. 4 ...

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The physiological role of mitochondrial uncoupling proteins (UCPs) in heart and skeletal muscle is unknown, as is whether mitochondrial uncoupling of oxidative phosphorylation by fatty acids occurs in vivo. In this study, we found that UCP2 and UCP3 protein content, determined using Western blotting, was increased by 32 and 48%, respectively, in hyperthyroid rat heart mitochondria. Oligomycin-insensitive respiration rate, a measure of mitochondrial uncoupling, was increased in all mitochondria in the presence of palmitate: 36% in controls and 71 and 100% with 0.8 and 0.9 mM palmitate, respectively, in hyperthyroid rat heart mitochondria. In the isolated working heart, 0.4 mM palmitate significantly lowered cardiac output by 36% and cardiac efficiency by 38% in the hyperthyroid rat heart. Thus increased mitochondrial UCPs in the hyperthyroid rat heart were associated with increased uncoupling and decreased myocardial efficiency in the presence of palmitate. In conclusion, a physiological effect of UCPs on fatty acid oxidation has been found in heart at the mitochondrial and whole organ level.

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We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.

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.

Creatine kinase-deficient hearts exhibit increased susceptibility to ischemia-reperfusion injury and impaired calcium homeostasis. Am J Physiol Heart Circ Physiol 287: H1039 -H1045, 2004. First published April 22, 2004 10.1152/ajpheart.01016.2003.-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 Ϫ/Ϫ ) show unchanged basal left ventricular (LV) performance but reduced myocardial high-energy phosphate concentrations. Moreover, skeletal muscle from M/Mito-CK Ϫ/Ϫ mice demonstrates altered Ca 2ϩ 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 Ca 2ϩ homeostasis. We simultaneously studied LV performance and myocardial Ca 2ϩ metabolism in isolated, perfused hearts of M/Mito-CK Ϫ/Ϫ (n ϭ 6) and wild-type (WT, n ϭ 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 Ϯ 72 vs. 678 Ϯ 54 s) and to a greater extent (50 Ϯ 2 vs. 36 Ϯ 3 mmHg) in M/Mito-CK Ϫ/Ϫ mice. During reperfusion, recovery of diastolic function was impaired (LV end-diastolic pressure: 22 Ϯ 3 vs. 10 Ϯ 2 mmHg), whereas recovery of systolic performance was delayed, in M/Mito-CK Ϫ/Ϫ mice. In parallel, Ca 2ϩ transients were similar during baseline conditions; however, M/Mito-CK Ϫ/Ϫ mice showed a greater increase in diastolic Ca 2ϩ concentration ([Ca 2ϩ ]) during ischemia (237 Ϯ 54% vs. 167 Ϯ 25% of basal [Ca 2ϩ ]) compared with WT mice. In conclusion, CK-deficient hearts show an increased susceptibility of LV performance and Ca 2ϩ homeostasis to ischemic injury, associated with a blunted postischemic recovery. This demonstrates a key function of an intact CK system for maintenance of Ca 2ϩ homeostasis and LV mechanics under metabolic stress conditions. aequorin bioluminescence; transgenic mouse THE CREATINE KINASE (CK) system comprises a family of mitochondrial (Mito-CK) and cytosolic (MM-, MB-, and BB-CK) isoenzymes that are critically involved in intracellular energy homeostasis. The primary role of CK is to catalyze the reversible transfer of a high-energy phosphoryl group between ATP and phosphocreatine (PCr; PCr ϩ ADP ϩ H ϩ 7 ATP ϩ creatine). The functional and physical coupling of certain members of the CK isoenzyme family to the sites of energy production and utilization has underscored the integrated properties of this important enzyme system in excitable tissue, particularly in muscle cells (26). MM-CK, for example, is present in membrane vesicles of the sarcoplasmic reticulum (SR) isolated from skeletal muscle (15), suggesting that an efficient and fast energy replenishing system is necessary for op...