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The goal of the study was to determine whether defects in intracellular Ca2+ signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca2+ concentration ([Ca2+]i). The sarcoplasmic reticulum (SR) Ca2+ store and rates of Ca2+ release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca2+ efflux via sarcolemmal Na+/Ca2+ exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca2+ channel current that triggers Ca2+ release from the SR. The depression in SR function was associated with decreased SR Ca2+-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na+/Ca2+ exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca2+ signaling caused by alteration of expression and function of the proteins that regulate [Ca2+]i contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na+/Ca2+ exchanger, and unchanged L-type Ca2+ channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.

The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc. 115, 11799-11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nM) than ruthenium red (IC50 = 6.85 nM) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (Kd = 0.34 nM, Bmax = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nM, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 microM, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 microM) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.