Local control of mitochondrial membrane potential, permeability transition pore and reactive oxygen species by calcium and calmodulin in rat ventricular myocytes

Calmodulin (CaM) and Ca²⁺/CaM-dependent protein kinase II (CaMKII) play important roles in the development of heart failure. In this study, we evaluated the effects of CaM on mitochondrial membrane potential (ΔΨ_m), permeability transition pore (mPTP) and the production of reactive oxygen species (ROS) in permeabilized myocytes; our findings are as follows. (1) CaM depolarized ΔΨ_m dose-dependently, but this was prevented by an inhibitor of CaM (W-7) or CaMKII (autocamtide 2-related inhibitory peptide (AIP)). (2) CaM accelerated calcein leakage from mitochondria, indicating the opening of mPTP, however this was prevented by AIP. (3) Cyclosporin A (an inhibitor of the mPTP) inhibited both CaM-induced ΔΨ_m depolarization and calcein leakage. (4) CaM increased mitochondrial ROS, which was related to ΔΨ_m depolarization and the opening of mPTP. (5) Chelating of cytosolic Ca²⁺ by BAPTA, the depletion of SR Ca²⁺ by thapsigargin (an inhibitor of SERCA) and the inhibition of mitochondrial Ca²⁺ uniporter by Ru360 attenuated the effects of CaM on mitochondrial function. (6) CaM accelerated Ca²⁺ extrusion from mitochondria. We conclude that CaM/CaMKII depolarized ΔΨ_m and opened mPTP by increasing ROS production, and these effects were strictly regulated by the local increase in cytosolic Ca²⁺ concentration, initiated by Ca²⁺ releases from the SR. In addition, CaM was involved in the regulation of mitochondrial Ca²⁺ homeostasis.     

Phenylephrine preconditioning involves modulation of cardiac sarcolemmal K(ATP) current by PKC delta, AMPK and p38 MAPK

Preconditioning of hearts with the α₁-adrenoceptor agonist phenylephrine decreases infarct size and increases the functional recovery of the heart following ischaemia-reperfusion. However, the cellular mechanisms responsible for this protection are not known. We investigated the role of protein kinase C ε and δ (PKCε and PKCδ), AMP-activated protein kinase (AMPK), p38 MAPK (p38) and sarcolemmal ATP-sensitive potassium (sarcK_ATP) channels in phenylephrine preconditioning using isolated rat ventricular myocytes. Preconditioning of ventricular myocytes with phenylephrine increased the recovery of contractile activity following metabolic inhibition and re-energisation from 30.1 ± 1.9% to 66.5 ± 5.2% (P < 0.01) and increased the peak sarcK_ATP current activated during metabolic inhibition from 32.1 ± 1.8 pA/pF to 46.0 ± 5.0 pA/pF (P < 0.05), which was required for protection. Phenylephrine preconditioning resulted in a sustained activation of PKCε and PKCδ, and transient activation of AMPK, which was dependent upon activation of PKCδ but not PKCε. P38 was also activated by phenylephrine preconditioning and this was blocked by inhibitors of PKCε, PKCδ or AMPK. Inhibition of PKCδ, AMPK or p38 was sufficient to prevent the increase in current, suggesting that these kinases are involved in modulation of sarcK_ATP channel current by phenylephrine preconditioning. However, whilst inhibition of AMPK and p38 prevented the protection from phenylephrine preconditioning, PKCδ inhibition paradoxically had no effect. The increase in sarcK_ATP current induced by phenylephrine preconditioning requires PKCδ, AMPK and p38 and may contribute to the observed improvement in contractile recovery.     

Sarcoplasmic ATP-sensitive potassium channel blocker HMR1098 protects the ischemic heart: implication of calcium, complex I, reactive oxygen species and mitochondrial ATP-sensitive potassium channel

The aim of this study was to investigate the effects of HMR1098, a selective blocker of sarcolemmal ATP-sensitive potassium channel (sarcK(ATP)), in Langendorff-perfused rat hearts submitted to ischemia and reperfusion. The recovery of heart hemodynamic and mitochondrial function, studied on skinned fibers, was analyzed after 30-min global ischemia followed by 20-min reperfusion. Infarct size was quantified on a regional ischemia model after 2-h reperfusion. We report that the perfusion of 10 microM HMR1098 before ischemia, delays the onset of ischemic contracture, improves recovery of cardiac function upon reperfusion, preserves the mitochondrial architecture, and finally decreases infarct size. This HMR1098-induced cardioprotection is prevented by 1 mM 2-mercaptopropionylglycine, an antioxidant, and by 100 nM nifedipine, an L-type calcium channel blocker. Concomitantly, it is shown that HMR1098 perfusion induces (i) a transient and specific inhibition of the respiratory chain complex I and, (ii) an increase in the averaged intracellular calcium concentration probed by the in situ measurement of indo-1 fluorescence. Finally, all the beneficial effects of HMR1098 were strongly inhibited by 5-hydroxydecanoate and abolished by glibenclamide, two mitoK(ATP) blockers. This study demonstrates that the HMR1098-induced cardioprotection occurs indirectly through extracellular calcium influx, respiratory chain complex inhibition, reactive oxygen species production and mitoK(ATP) opening. Taken together, these data suggest that a functional interaction between sarcK(ATP) and mitoK(ATP) exists in isolated rat heart ischemia model, which is mediated by extracellular calcium influx.     

Effects of K(ATP) channel openers, P-1075, pinacidil, and diazoxide, on energetics and contractile function in isolated rat hearts

We investigated the metabolic effects of a potent opener of ATP-sensitive K(+) (K(ATP)) channels, P-1075, in perfused rat hearts with the help of(31)P NMR spectroscopy. A 20 min infusion of 5 microm P-1075 depleted phosphocreatine and ATP by approximately 40%, concomitantly with a two-fold increase in inorganic phosphate, while oxygen consumption by the hearts increased by 50%. P-1075 induced a cardiac contracture (left ventricular end diastolic pressure increased from 6 to 60 mmHg) and a cardiac arrest after an infusion of approximately 9 min. The effects were fully reversed by glibenclamide (5 microm), but not by sodium 5-hydroxydecanoate (0.4 m m). A P-1075-related K(ATP) opener, pinacidil (0.3 m m), partially reversed the effects of P-1075, but a structurally unrelated opener, diazoxide (0.5 m m), had no effect. Pinacidil and diazoxide alone did not significantly affect PCr and ATP levels. Bioenergetic and functional effects similar to those of P-1075 were induced by infusion of a classic mitochondrial uncoupler, 2,4-dinitrophenol (50 microm); however, they were not abolished by glibenclamide. In addition, it was shown, using(87)Rb NMR, that both agents, P-1075 and 2,4-dinitrophenol, resulted in a stimulation of Rb(+) efflux from the Rb(+) loaded rat hearts by approximately 130 and 65%, respectively, in a glibenclamide-sensitive manner. An inhibitory effect of P-1075 on ATP synthesis cannot be explained by its well-known action on sarcolemmal K(ATP) channels. We concluded that, unlike an uncoupling effect of 2,4-dinitrophenol, an inhibitory effect of P-1075 is produced by uncoupling of oxidative phosphorylation through the activation of mitochondrial K(ATP) channels.