We used proteomics to detect regional differences in protein expression levels from mitochondrial fractions of control, ischemia-reperfusion (IR), and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 25 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For three of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included 3-hydroxybutyrate dehydrogenase, prohibitin, 2-oxoglutarate dehydrogenase, adenosine triphosphate synthases, the reduced form of nicotinamide adenine dinucleotide (NADH) oxidoreductase, translation elongation factor, actin alpha, malate dehydrogenase, NADH dehydrogenase, pyruvate dehydrogenase and the voltage-dependent anion channel. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain and energy metabolism. The successful use of multiple techniques, including 2-DE, MALDI-TOF-MS and Western blotting analysis demonstrates that proteomic analysis provides appropriate means for identifying cardiac markers for detection of ischemia-induced cardiac injury.

Significance The concentration of Ca 2+ ions is kept low in cells by specialized ion-pumping proteins at the membrane. We show that in cardiac cells, cytoplasm also has an intrinsic ability to pump Ca 2+ . Histidyl dipeptides and ATP are diffusible cytoplasmic buffer molecules. By exchanging Ca 2+ for H + , they act like local “pumps,” producing uphill Ca 2+ movement within cytoplasm in response to H + ion gradients. Intracellular H + ions are generated locally by metabolism and competitively inhibit many Ca 2+ -activated biochemical processes. Recruiting Ca 2+ to acidic zones facilitates these processes. Cytoplasmic histidyl dipeptides and ATP thus act like a biological pump without a membrane.

Stretch‐activated channels (SACs) were studied in isolated rat atrial myocytes using the whole‐cell and single‐channel patch clamp techniques. Longitudinal stretch was applied by using two patch electrodes. In current clamp configuration, mechanical stretch of 20 % of resting cell length depolarised the resting membrane potential (RMP) from ‐63·6 ± 0·58 mV (n= 19) to ‐54·6 ± 2·4 mV (n= 13) and prolonged the action potential duration (APD) by 32·2 ± 8·8 ms (n= 7). Depolarisation, if strong enough, triggered spontaneous APs. In the voltage clamp configuration, stretch increased membrane conductance in a progressive manner. The current‐voltage (I–V) relationship of the stretch‐activated current (ISAC) was linear and reversed at ‐6·1 ± 3·7 mV (n= 7). The inward component of ISAC was abolished by the replacement of Na+ with NMDG+, but ISAC was hardly altered by the Cl− channel blocker DIDS or removal of external Cl−. The permeability ratio for various cations (PCs:PNa:PLi= 1·05:1:0·98) indicated that the SAC current was a non‐selective cation current (ISAC,NC). The background current was also found to be non‐selective to cations (INSC,b); the permeability ratio (PCs:PNa:PLi= 1·49:1:0·70) was different from that of ISAC,NC. Gadolinium (Gd3+) acted on INSC,b and ISAC,NC differently. Gd3+ inhibited INSC,b in a concentration‐dependent manner with an IC50 value of 46·2 ± 0·8 μM (n= 5). Consistent with this effect, Gd3+ hyperpolarised the resting membrane potential (‐71·1 ± 0·26 mV, n= 9). In the presence of Gd3+ (0·1 mM), stretch still induced ISAC,NC and diastolic depolarisation. Single‐channel activities were recorded in isotonic Na+ and Cs+ solutions using the inside‐out configuration. In NMDG+ solution, outward currents were abolished. Gd3+ (100 μM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 μM) in the pipette solution, an increase in pipette pressure induced an increase in channel opening (21·27 ± 0·24 pS; n= 7), which was distinct from background activity. We concluded from the above results that longitudinal stretch in rat atrial myocytes induces the activation of non‐selective cation channels that can be distinguished from background channels by their different electrophysiology and pharmacology.

BackgroundCardiomyocytes that differentiate from pluripotent stem cells (PSCs) provide a crucial cellular resource for cardiac regeneration. The mechanisms of mitochondrial metabolic and redox regulation for efficient cardiomyocyte differentiation are, however, still poorly understood. Here, we show that inhibition of the mitochondrial permeability transition pore (mPTP) by Cyclosporin A (CsA) promotes cardiomyocyte differentiation from PSCs.Methods and ResultsWe induced cardiomyocyte differentiation from mouse and human PSCs and examined the effect of CsA on the differentiation process. The cardiomyogenic effect of CsA mainly resulted from mPTP inhibition rather than from calcineurin inhibition. The mPTP inhibitor NIM811, which does not have an inhibitory effect on calcineurin, promoted cardiomyocyte differentiation as much as CsA did, but calcineurin inhibitor FK506 only slightly increased cardiomyocyte differentiation. CsA‐treated cells showed an increase in mitochondrial calcium, mitochondrial membrane potential, oxygen consumption rate, ATP level, and expression of genes related to mitochondrial function. Furthermore, inhibition of mitochondrial oxidative metabolism reduced the cardiomyogenic effect of CsA while antioxidant treatment augmented the cardiomyogenic effect of CsA.ConclusionsOur data show that mPTP inhibition by CsA alters mitochondrial oxidative metabolism and redox signaling, which leads to differentiation of functional cardiomyocytes from PSCs.

The pacemaker activity of interstitial cells of Cajal (ICCs) has been known to initiate the propagation of slow waves along the whole gastrointestinal tract through spontaneous and repetitive generation of action potentials. We studied the mechanism of the pacemaker activity of ICCs in the mouse small intestine and tested it using a mathematical model. The model includes ion channels, exchanger, pumps and intracellular machinery for Ca2+ regulation. The model also incorporates inositol 1,4,5-triphosphate (IP3) production and IP3-mediated Ca2+ release activities. Most of the parameters were obtained from the literature and were modified to fit the experimental results of ICCs from mouse small intestine. We were then able to compose a mathematical model that simulates the pacemaker activity of ICCs. The model generates pacemaker potentials regularly and repetitively as long as the simulation continues. The frequency was set at 20 min(-1) and the duration at 50% repolarization was 639 ms. The resting and overshoot potentials were -78 and +1.2 mV, respectively. The reconstructed pacemaker potentials closely matched those obtained from animal experiments. The model supports the idea that cyclic changes in [Ca2+]i and [IP3] play key roles in the generation of ICC pacemaker activity in the mouse small intestine.

. Nitric oxidecGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K ϩ channels in rat hearts. Am J Physiol Heart Circ Physiol 290: H1808 -H1817, 2006. First published December 9, 2005 doi:10.1152/ajpheart.00772.2005.-Nitric oxide (NO) plays an important role in anoxic preconditioning to protect the heart against ischemia-reperfusion injuries. The present work was performed to study better the NO-cGMP-protein kinase G (PKG) signaling pathway in the activation of both sarcolemmal and mitochondrial ATP-sensitive K ϩ (KATP) channels during anoxic preconditioning (APC) and final influence on reducing anoxia-reperfusion (A/R)-induced cardiac damage in rat hearts. The upstream regulating elements controlling NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection were investigated. The involvement of both inducible and endothelial NO synthases (iNOS and eNOS) in the progression of this signaling pathway was followed. Final cellular outcomes of ischemia-induced injury after different preconditioning in the form of lactate dehydrogenase release, DNA strand breaks, and malondialdehyde formation as indexes of cell injury and lipid peroxidation, respectively, were investigated. The lactate dehydrogenase and malondialdehyde values decreased in the groups that underwent preconditioning periods with specific mitochondrial KATP channels opener diazoxide (100 M), nonspecific mitochondrial KATP channels opener pinacidil (50 M), S-nitroso-Nacetylpenicillamine (SNAP, 300 M), or ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclicmonophosphorothioate, Sp-isomer (10 M) before the A/R period. Preconditioning with SNAP significantly reduced the DNA damage. The effect was blocked by glibenclamide (50 M), 5-hydroxydecanoate (100 M), N G -nitro-L-arginine methyl ester (200 M), and ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer (1 M). The results suggest iNOS, rather than eNOS, as the major contributing NO synthase during APC treatment. Moreover, the PKG shows priority over NO as the upstream regulator of NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection during APC treatment.guanosine 3Ј,5Ј-cyclic monophosphate; adenosine 5Ј-triphosphate; oxidative damage ISCHEMIC PRECONDITIONING, in which short-term occlusion and reperfusion of a coronary artery are followed by long-term occlusion, can reduce subsequent ischemia-induced injury to the heart (42). Nitric oxide (NO), protein kinase G (PKG), and ATP-sensitive K ϩ (K ATP ) channels (both the sarcolemmal and mitochondrial subtypes) can mimic the effects of ischemic preconditioning in the heart, and mitochondrial K ATP channels appear to be the end effectors (19,20,25). The activation of these channels may improve the recovery of regional contractility of myocardium by shortening the duration of action potentials and by attenuating membrane depolarization, both of which would decrease myocardial contractility and reduce energy expenditure during ischem...

We have investigated the effect of external H+ concentration ([H+]o) on the human-ether-a-go-go-related gene (HERG) current (IHERG), the molecular equivalent of the cardiac delayed rectifier potassium current (IKr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H+]o was increased, the amplitude of the IHERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pHo) values (the potential required for half-maximum activation, V1/2, was: -41.8 mV, -38.0 mV, -33.7 mV, -26.7 mV in pHo 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (gmax) was also affected by [H+]o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pHo from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca2+ concentration, which is also known to block HERG channels. When the extracellular Ca2+ concentration was increased from 0.5 mM to 5 mM, the shift in V1/2 caused by increasing [H+]o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in gmax caused by increasing [H+]o was not significantly affected by changing external Ca2+ levels. The results indicate that HERG channels are inhibited by [H+]o by two different mechanisms: voltage-dependent blockade (shift of V1/2) and the decrease in gmax. With respect to the voltage-dependent blockade, the interaction between H+ and Ca2+ is competitive, whereas for the decreasing gmax, their interaction is non-competitive.

1 Amitriptyline has been known to induce QT prolongation and torsades de pointes which causes sudden death. We studied the e ects of amitriptyline on the human ether-a-go-go-related gene (HERG) channel expressed in Xenopus oocytes and on the rapidly activating delayed recti®er K + current (I Kr ) in rat atrial myocytes. 2 The amplitudes of steady-state currents and tail currents of HERG were decreased by amitriptyline dose-dependently. The decrease became more pronounced at more positive potential, suggesting that the block of HERG by amitriptyline is voltage dependent. IC 50 for amitriptyline block of HERG current was progressively decreased according to depolarization: IC 50 values at 730, 710, +10 and +30 mV were 23.0, 8.71, 5.96 and 4.66 mM, respectively. 3 Block of HERG by amitriptyline was use dependent: exhibiting a much faster block at higher activation frequency. Subsequent decrease in frequency after high activation frequency resulted in a partial relief of HERG blockade. 4 Steady-state block by amitriptyline was obtained while depolarization to +20 mV for 0.5 s was applied at 0.5 Hz: IC 50 was 3.26 mM in 2 mM [K + ] o . It was increased to 4.78 mM in 4 mM [K + ] o , suggesting that the a nity of amitriptyline on HERG was decreased by external K + . 5 In rat atrial myocytes bathed in 358C, 5 mM amitriptyline blocked I Kr by 55%. However, transient outward K + current (I to ) was not signi®cantly a ected. 6 In summary, the data suggest that the block of HERG currents may contribute to arrhythmogenic side e ects of amitriptyline.