Structure and function of resistance arteries from BB-creatine kinase and ubiquitous Mt-creatine kinase double knockout mice.

Increasing evidence indicates that the enzyme creatine kinase (CK) is intimately involved in microvascular contractility. The mitochondrial isoenzyme catalyses phosphocreatine synthesis from ATP, while cytoplasmic CK, predominantly the BB isoenzyme in vascular tissue, is tightly bound near myosin ATPase, where it favours ATP production from phosphocreatine to metabolically support vascular contractility. However, the effect of CK gene inactivation on microvascular function is hitherto unknown. We studied functional and structural parameters of mesenteric resistance arteries isolated from 5 adult male mice lacking cytoplasmic BB-CK and ubiquitous mitochondrial CK (CK-/-) vs 6 sex/age-matched controls. Using a Mulvany Halpern myograph, we assessed the acute maximum contractile force with 125 mM K+ and 10-5 M norepinephrine, and the effect of two inhibitors, dinitrofluorobenzene, which inhibits phosphotransfer enzymes (0.1 µM), and the specific adenylate kinase inhibitor P1, P5-di(adenosine 5') pentaphosphate (10-6 to 10-5 M). WT and CK-/- did not significantly differ in media thickness, vascular elasticity parameters, or acute maximum contractile force. CK-/- arteries displayed greater reduction in contractility after dinitrofluorobenzene 38%; vs 14% in WT; and after AK inhibition, 14% vs 5.5% in WT, and displayed abnormal mitochondria, with a partial loss of the inner membrane. Thus, CK-/- mice display a surprisingly mild phenotype in vascular dysfunction. However, the mitochondrial abnormalities and greater effect of inhibitors on contractility may reflect a compromised energy metabolism. In CK-/- mice, compensatory mechanisms salvage energy metabolism, as described for other CK knock-out models.

Exercise-induced reductions in mitochondrial ADP sensitivity contribute to the induction of gene expression and mitochondrial biogenesis through enhanced mitochondrial H2O2 emission.

Acute exercise rapidly induces mitochondrial gene expression, however, the intracellular events regulating this process remain incompletely understood. The purpose of this study was to determine whether reductions in mitochondrial ADP sensitivity during exercise have a biological role in regulating mitochondrial-derived reactive oxygen species (ROS) production and the induction of mitochondrial biogenesis. Mitochondrial creatine kinase wildtype (WT) and knockout (KO) mice have divergent responses in ADP sensitivity during exercise, and we therefore used these mice to determine the relationship between mitochondrial ADP sensitivity, ROS production, and mitochondrial adaptations to exercise. In WT mice, acute exercise reduced mitochondrial ADP respiratory sensitivity and the ability of ADP to suppress ROS production, while increasing mitochondrial gene transcription (PGC-1α, PGC-1ß and PDK4). In stark contrast, in KO mice, exercise increased ADP sensitivity, reduced mitochondrial ROS emission, and did not induce gene transcription. Despite the divergence in mRNA responses, exercise similarly induced calcium/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK) phosphorylation in WT and KO mice, however only WT mice were associated with redox stress (4HNE). These data implicate acute changes in ADP sensitivity in mitochondrial adaptations to exercise. To further examine this we chronically exercise trained mice. While training increased mitochondrial content and reduced ADP sensitivity in WT mice, KO mice did not exhibit adaptations to exercise. Combined, these data suggest that exercise-induced attenuations in mitochondrial ADP sensitivity mediate redox signals that contribute to the induction of acute and chronic mitochondrial adaptations.

Hypothalamic plasticity of neuropeptide Y is lacking in brain-type creatine kinase double knockout mice with defective thermoregulation.

The neural substrate of adaptive thermoregulation in mice lacking both brain-type creatine kinase isoforms is further investigated. The cytosolic brain-type creatine kinase (CK-B) and mitochondrial ubiquitous creatine kinase (UbCKmit) are expressed in neural cells throughout the central and peripheral nervous system, where they have an important role in cellular energy homeostasis. Several integral functions appear altered when creatine kinases are absent in the brain (Jost et al., 2002; Streijger et al., 2004, 2005), which has been explained by inefficient neuronal transmission. The CK--/-- double knockout mice demonstrate every morning a body temperature drop of ~1.0 °C, and they have impaired thermogenesis, as revealed by severe hypothermia upon cold exposure. This defective thermoregulation is not associated with abnormal food intake, decreased locomotive activity, or increased torpor sensitivity. Although white and brown adipose tissue fat pads are diminished in CK--/-- mice, intravenous norepinephrine infusion results in a normal brown adipose tissue response with increasing core body temperatures, indicating that the sympathetic innervation functions correctly (Streijger et al., 2009). This study revealed c-fos changes following a cold challenge, and that neuropeptide Y levels were decreased in the paraventricular nucleus of wildtype, but not CK--/--, mice. A reduction in hypothalamic neuropeptide Y is coupled to increased uncoupling protein 1 expression in brown adipose tissue, resulting in thermogenesis. In CK--/-- mice the neuropeptide Y levels did not change. This lack of hypothalamic plasticity of neuropeptide Y might be the result of inefficient neuronal transmission or can be explained by the previous observation of reduced circulating levels of leptin in CK--/-- mice.

Rearrangement of energetic and substrate utilization networks compensate for chronic myocardial creatine kinase deficiency.

Plasticity of the cellular bioenergetic system is fundamental to every organ function, stress adaptation and disease tolerance. Here, remodelling of phosphotransfer and substrate utilization networks in response to chronic creatine kinase (CK) deficiency, a hallmark of cardiovascular disease, has been revealed in transgenic mouse models lacking either cytosolic M-CK (M-CK(-/-)) or both M-CK and sarcomeric mitochondrial CK (M-CK/ScCKmit(-/-)) isoforms. The dynamic metabolomic signatures of these adaptations have also been defined. Tracking perturbations in metabolic dynamics with (18)O and (13)C isotopes and (31)P NMR and mass spectrometry demonstrate that hearts lacking M-CK have lower phosphocreatine (PCr) turnover but increased glucose-6-phosphate (G-6-P) turnover, glucose utilization and inorganic phosphate compartmentation with normal ATP γ-phosphoryl dynamics. Hearts lacking both M-CK and sarcomeric mitochondrial CK have diminished PCr turnover, total phosphotransfer capacity and intracellular energetic communication but increased dynamics of ß-phosphoryls of ADP/ATP, G-6-P and γ-/ß-phosphoryls of GTP, indicating redistribution of flux through adenylate kinase (AK), glycolytic and guanine nucleotide phosphotransfer circuits. Higher glycolytic and mitochondrial capacities and increased glucose tolerance contributed to metabolic resilience of M-CK/ScCKmit(-/-) mice. Multivariate analysis revealed unique metabolomic signatures for M-CK(-/-) and M-CK/ScCKmit(-/-) hearts suggesting that rearrangements in phosphotransfer and substrate utilization networks provide compensation for genetic CK deficiency. This new information highlights the significance of integrated CK-, AK-, guanine nucleotide- and glycolytic enzyme-catalysed phosphotransfer networks in supporting the adaptivity and robustness of the cellular energetic system.