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Aims Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesized that elevating MtCK would be beneficial in ischaemia--reperfusion (I/R) injury. Methods and results Mice were created over-expressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localization of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by ¹H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20 min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture, and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55 ± 4% in WT vs. 29 ± 4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Conclusions Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia. [ABSTRACT FROM AUTHOR]

Autologous transplantation of cardiac progenitor cells (CPCs) alleviates myocardial dysfunction in the damaged heart; however, the mechanisms that contribute to their reparative qualities remain poorly understood. In this study, we examined CPC metabolism to elucidate the metabolic pathways that regulate their proliferative capacity. In complete growth medium, undifferentiated CPCs isolated from adult mouse heart proliferated rapidly ( Td = 13.8 hours). CPCs expressed the Glut1 transporter and their glycolytic rate was increased by high extracellular glucose (Glc) concentration, in the absence of insulin. Although high Glc concentrations did not stimulate proliferation, glutamine (Gln) increased CPC doubling time and promoted survival under conditions of oxidative stress. In comparison with Glc, pyruvate (Pyr) or BSA-palmitate, Gln, when provided as the sole metabolic substrate, increased ATP-linked and uncoupled respiration. Although fatty acids were not used as respiratory substrates when present as a sole carbon source, Gln-induced respiration was doubled in the presence of BSA-palmitate, suggesting that Gln stimulates fatty acid oxidation. Additionally, Gln promoted rapid phosphorylation of the mTORC1 substrate, p70S6k, as well as retinoblastoma protein, followed by induction of cyclin D1 and cdk4. Inhibition of either mTORC1 or glutaminolysis was sufficient to diminish CPC proliferation, and provision of cell permeable α-ketoglutarate in the absence of Gln increased both respiration and cell proliferation, indicating a key role of Gln anaplerosis in cell growth. These findings suggest that Gln, by enhancing mitochondrial function and stimulating mTORC1, increases CPC proliferation, and that interventions to increase Gln uptake or oxidation may improve CPC therapy. S tem C ells 2015;33:2613-2627 [ABSTRACT FROM AUTHOR]

Nucleoside reverse transcriptase inhibitors (NRTIs), essential components of combinational therapies used for treatment of human immunodeficiency virus-1, damage heart mitochondria. Here, we have shown mitochondrial compromise in H9c2 rat cardiomyocytes exposed for 16 passages (P) to the NRTIs zidovudine (AZT, 50μM) and didanosine (ddI, 50μM), and we have demonstrated protection from mitochondrial compromise in cells treated with 200μM 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (Tempol) or 200μM 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine (Tempol-H), along with AZT/ddI, for 16P. Exposure to AZT/ddI caused a moderate growth inhibition at P3, P5, P7, and P13, which was not altered by addition of Tempol or Tempol-H. Mitochondrial oxidative phosphorylation capacity was determined as uncoupled oxygen consumption rate (OCR) by Seahorse XF24 Analyzer. At P5, P7, and P13, AZT/ddI-exposed cells showed an OCR reduction of 8.8–57.2% in AZT/ddI-exposed cells, compared with unexposed cells. Addition of Tempol or Tempol-H, along with AZT/ddI, resulted in OCR levels increased by about 300% above the values seen with AZT/ddI alone. The Seahorse data were further supported by electron microscopy (EM) studies in which P16 cells exposed to AZT/ddI/Tempol had less mitochondrial pathology than P16 cells exposed to AZT/ddI. Western blots of P5 cells showed that Tempol and Tempol-H upregulated expression of mitochondrial uncoupling protein-2 (UCP-2). However, Complex I activity that was reduced by AZT/ddI, was not restored in the presence of AZT/ddI/Tempol. Superoxide levels were increased in the presence of AZT/ddI and significantly decreased in cells exposed to AZT/3TC/Tempol at P3, P7, and P10. In conclusion, Tempol protects against NRTI-induced mitochondrial compromise, and UCP-2 plays a role through mild uncoupling. [ABSTRACT FROM PUBLISHER]

The effect of the SH-reagent methyl methanethiosulfonate (MMTS) on the ADP/ATP carrier of bovine heart mitochondria was studied under various conditions. MMTS labeled predominately Cys56 in the first loop facing the matrix (loop Ml), and the labeling inhibited ADP transport via the carrier. The transport inhibition was found to be due to fixation of the carrier in the m-state conformation. MMTS labeling was suggested not to affect ADP binding to its major binding site. These features were the same as those of another commonly used SH-reagent, N-ethylmaleimide (NEM). Although the van der Waals volume of the non-hydrogen-bondable methylthio group of MMTS is much smaller than that of the ethylsuccinimide group of NEM, modification of Cys56 inhibited the interconversion between the m- and c-state conformation. The mechanism by which MMTS inhibited the transport activity is discussed in terms of stabilization of conformation of the loop Ml. [ABSTRACT FROM AUTHOR]

Mitochondria isolated from perfused rat heart preparations were used to study the combined effects of age and physical stress. Age-related declines in oxidative phosphorylation catalyzed by mitochondria from nonperfused hearts were not observed. Low work load perfusion resulted in decreased respiration by mitochondria from 24-month-old hearts ( < .01) but not 10-month old hearts, while high work load perfusion resulted in decreased respiration in both ages. However, the decrease by the 24-month-old hearts was significantly greater than those in the younger hearts ( < .01). Compared to age-matched low work load hearts, 5- and 10-month-old high work load hearts increased mitochondrial protein synthesis by 86% and 93%, respectively, 15-month-old hearts increased by 60%, and 24-month-old hearts by 13%. The results of this study provide evidence that the ability of the heart to respond appropriately so as to adapt to stress decreases with age, becoming apparent in the, laboratory rat between 10 and 15 months of age. [ABSTRACT FROM PUBLISHER]

Purpose: Diazoxide (DZX) is frequently used for induction of effective pharmacological preconditioning (P-PC) of the myocardium in the animal models. This compound was originally classified as an opener of the mitochondrial (MIT) ATP-dependent potassium channels (KATP). But, afterwards it was demonstrated that DZX also i) acts as an inhibitor of the Krebs cycle at the level of succinate dehydrogenase, ii) supports free radicals generation and iii) acts more as a PKC-ε activator than as the activator of KATP channels, which is its secondary effect only. Taking into consideration all these effects of DZX, no satisfactory explanation for the molecular mechanism of its cardioprotective action has been introduced up to now. In the independent studies it was demonstrated that the acute streptozotocin-diabetes (DIA) also confers cardioprotective effect and that the finding of considerably stimulated heart MIT Mg2+-ATPase is intimately involved in this protection. Aim of the present study was to elucidate the role of the MIT Mg2+-ATPase and membrane fluidity in the mechanism of DZX action and, in this way, to bring the preconditioning with DZX closer to practical applicability.Methods: Experiments were performed on 9-11 week old male Wistar rats devided into healthy and DIA groups. Acute DIA (8 days) was induced by a single dose of streptozotocin (65 mg.kg-1, i.p.). Heart mitochondria isolated by differential centrifugation were exposed to DZX (0-7 μmol.l-1). MIT ATP synthase activity, detected in the reaction that proceeded in the opposite direction i.e., as Mg2+-ATPase was assessed by measuring of Pi liberated from ATP splitting. Fluidity of the lipid bilayer of the MIT membrane was estimated by measuring fluorescence anisotropy with the probe 1,6-diphenyl-1,3,5-hexatriene.Results: Starting with the concentration of 3.5 μmol.l-1 DZX induced a significant (p<0.05) increase in Mg2+-ATPase activity in MIT from healthy hearts. A similar trend was observed also in DIA hearts, but it reached no statistical significance even at 7 μmol.l-1. The stimulation of the enzyme activity was not accompanied with any considerable changes in MIT membrane fluidity.Conclusions: Concentrations of DZX equal to or above 5 μmol.l-1 stimulate (p<0.05) the activity of MIT Mg2+-ATPase by a direct interaction with the enzyme molecule as it was revealed by the enzyme kinetics analysis. Stimulation of MIT Mg2+-ATPase plays an essential but not exclusive role in cardioprotective effect provided by both DZX and DIA. Grants: VEGA 2/0101/12, 2/0094/12, APVV 0102-11, KEGA 003 UK-4/2012. [ABSTRACT FROM PUBLISHER]