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We have reported previously that hearts from type 2 diabetic (db/db) mice show decreased cardiac efficiency due to increased work-independent myocardial [O.sub.2] consumption (unloaded MV[o.sub.2]), indicating higher [O.sub.2] use for nonmechanical processes such as basal metabolism (MV[o.sub.2BM]) and excitationcontraction coupling (MV[o.sub.2ECC]). Although alterations in cardiac metabolism and/or [Ca.sup.2+] handling may contribute to increased energy expenditure in diabetic hearts, direct measurements of the [O.sub.2] cost for these individual processes have not been determined. In this study, we 1) validate a procedure for measuring unloaded MV[o.sub.2] directly (MV[o.sub.2unloaded]) and for determining MV[o.sub.2BM] and MV[o.sub.2ECC] separately in isolated perfused mouse hearts and 2) determine 02 cost for these processes in hearts from db/db mice. Unloaded MV[o.sub.2], extrapolated from the relationship between cardiac work (measured as pressure-volume area, PVA) and MV[o.sub.2], was found to correspond with MV[o.sub.2] measured directly in unloaded retrograde perfused hearts (MV[o.sub.2unloaded]). MV[o.sub.2] in [K.sup.+]-arrested hearts was defined as MV[o.sub.2BM]; the difference between MV[o.sub.2unloaded] and MV[o.sub.2BM] represented MV[o.sub.2ECC]. This procedure was validated by demonstrating that elevations in perfusate fatty acid (FA) and/or [Ca.sup.2+] concentrations resulted in changes in either MV[o.sub.2BM] and/or MV[o.sub.2ECC]. The higher MV[o.sub.2unloaded] in db/db mice was due to both a higher MV[o.sub.2BM] and MV[o.sub.2ECC]. Elevation of glucose and insulin decreased FA oxidation and reduced both MV[o.sub.2unloaded] and MV[o.sub.2BM]. In conclusion, this study provides direct evidence that MV[o.sub.2BM] and MV[o.sub.2ECC] are elevated in diabetes and that acute metabolic interventions can have a therapeutic benefit in diabetic hearts due to a MV[o.sub.2]-lowering effect. cardiac efficiency; pressure-volume area; myocardial oxygen consumption; substrate oxidation

Hearts from type 2 diabetic (db/db) mice demonstrate altered substrate utilization with high rates of fatty acid oxidation, decreased functional recovery following ischemia, and reduced cardiac efficiency. Although db/db mice show overall insulin resistance in vivo, we recently reported that insulin induces a marked shift toward glucose oxidation in isolated perfused db/db hearts. We hypothesize that such a shift in metabolism should improve cardiac efficiency and consequently increase functional recovery following low-flow ischemia. Hearts from db/db and nondiabetic (db/+) mice were perfused with 0.7 mM palmitate plus either 5 mM glucose (G), 5 mM glucose and 300 [micro]U/ml insulin (GI), or 33 mM glucose and 900 [micro]M/ml insulin (HGHI). Substrate oxidation and postischemic recovery were only moderately affected by GI and HGHI in db/+ hearts. In contrast, GI and particularly HGHI markedly increased glucose oxidation and improved postischemic functional recovery in db/db hearts. Cardiac efficiency was significantly improved in db/db, but not in db/+ hearts, in the presence of HGHI. In conclusion, insulin and glucose normalize cardiac metabolism, restore efficiency, and improve postischemic recovery in type 2 diabetic mouse hearts. These findings may in part explain the beneficial effect of glucose-insulin-potassium therapy in diabetic patients with cardiac complications. glucose; insulin; myocardial oxygen consumption; pressure-volume area doi:10.1152/ajpendo.00504.2006.

Diabetic (db/ db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult. myocardial metabolism; insulin action; glucose and fatty acid metabolism

Cells subjected to environmental stresses undergo regulated cell death (RCD) when homeostatic programs fail to maintain viability. A major mechanism of RCD is the excessive calcium loading of mitochondria and consequent triggering of the mitochondrial permeability transition (mPT), which is especially important in post‐mitotic cells such as cardiomyocytes and neurons. Here, we show that stress‐induced upregulation of the ROS‐generating protein Nox4 at the ER‐mitochondria contact sites (MAMs) is a pro‐survival mechanism that inhibits calcium transfer through InsP3 receptors (InsP3R). Nox4 mediates redox signaling at the MAM of stressed cells to augment Akt‐dependent phosphorylation of InsP3R, thereby inhibiting calcium flux and mPT‐dependent necrosis. In hearts subjected to ischemia–reperfusion, Nox4 limits infarct size through this mechanism. These results uncover a hitherto unrecognized stress pathway, whereby a ROS‐generating protein mediates pro‐survival effects through spatially confined signaling at the MAM to regulate ER to mitochondria calcium flux and triggering of the mPT. Synopsis: Excessive calcium loading into mitochondria induced by environmental stresses triggers mitochondrial permeability transition (mPT) and regulated cell death (RCD) in mammalian cells. This study shows that NADPH oxidase 4 (Nox4) prevents mitochondrial calcium overload by inhibiting InsP3 receptor at ER‐mitochondria contact sites (MAMs) in cells and infarcted murine hearts. Nox4‐deficient cells show increased mPT‐dependent cell necrosis during serum starvation in cardiac myocytes.Serum starvation induces upregulation of Nox4, which in turn inhibits InsP3R‐mediated calcium transfer from the ER to mitochondria.Nox4 is located at MAMs in multiple cell types and tissues.Nox4‐dependent inhibition of calcium transfer is mediated by redox‐dependent Akt activation and phosphorylation of InsP3R.Nox4 limits infarct size in murine hearts subjected to ischemia‐reperfusion by inhibiting InsP3R. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme implicated in motility, differentiation, and regeneration of skeletal muscle fibers through processing of extracellular substrates. Although MMP-2 has been found to be localized intracellularly in cardiomyocytes where the enzyme is thought to contribute to post-ischemic loss of contractility, little is known about intracellular MMP-2 activity in skeletal muscle fibers. In the present study we demonstrate intracellular MMP-2 in normal skeletal muscle by immunohistochemical staining. Immunogold electron microscopic analyses indicated that the enzyme was concentrated in Z-lines of the sarcomers, in the nuclear membrane, and in mitochondria. By use of in situ zymography, we found that gelatinolytic activity in muscle fibers was co-localized with immunofluorecent staining for MMP-2. Staining for MMP-9, the other member of the gelatinase group of the MMPs, was negative. The broad-spectrum metalloprotease inhibitor EDTA and the selective gelatinase inhibitor CTT2, but not the cysteine inhibitor E64, strongly reduced the gelatinolytic activity. The intracellular gelatinolytic activity was much more prominent in fast twitch type II fibers than in slow twitch type I fibers, and there was a decrease in intracellular gelatinolytic activity and MMP-2 expression in muscles from mice exposed to high intensity interval training. Together our results indicate that MMP-2 is part of the intracellular proteolytic network in normal skeletal muscle, especially in fast twitch type II fibers. Further, the results suggest that intracellular MMP-2 in skeletal muscle fibers is active during normal homeostasis, and affected by the level of physical activity. J. Cell. Physiol. 229: 160-169, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]