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Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.

Extracellular purines and pyrimidines have major effects on cardiac rhythm and contraction. ATP/UTP are released during various physiopathological conditions, such as ischemia, and despite degradation by ectonucleotidases, their interstitial concentrations can markedly increase, a fact that is clearly associated with arrhythmia. In the present whole cell patch-clamp analysis on ventricular cardiomyocytes isolated from various mammalian species, ATP and UTP elicited a sustained, nonselective cationic current, I(ATP). UDP was ineffective, whereas 2'(3')-O-(4-benzoylbenzoyl)-ATP was active, suggesting that P2Y(2) receptors are involved. I(ATP) resulted from the binding of ATP(4-) to P2Y(2) purinoceptors. I(ATP) was maintained after ATP removal in the presence of guanosine 5'-[gamma-thio]triphosphate and was inhibited by U-73122, a PLC inhibitor. Single-channel openings are rather infrequent under basal conditions. ATP markedly increased opening probability, an effect prevented by U-73122. Two main conductance levels of 14 and 23 pS were easily distinguished. Similarly, in fura-2-loaded cardiomyocytes, Mn(2+) quenching and Ba(2+) influx were significant only in the presence of ATP or UTP. Adult rat ventricular cardiomyocytes expressed transient receptor potential channel TRPC1, -3, -4, and -7 mRNA and the TRPC3 and TRPC7 proteins that coimmunoprecipitated. Finally, the anti-TRPC3 antibody added to the patch pipette solution inhibited I(ATP). In conclusion, activation of P2Y(2) receptors, via a G protein and stimulation of PLCbeta, induces the opening of heteromeric TRPC3/7 channels, leading to a sustained, nonspecific cationic current. Such a depolarizing current could induce cell automaticity and trigger the arrhythmic events during an early infarct when ATP/UTP release occurs. These results emphasize a new, potentially deleterious role of TRPC channel activation.

Objectives: We investigated the inward rectifier potassium current (I(K1)), which can be blocked by intracellular Ca(2+), in heart failure (HF)., Methods: We used the whole-cell patch-clamp technique to record I(K1) from single rat ventricular myocytes in voltage-clamp conditions. Fluorescence measurements of diastolic Ca(2+) were performed with Indo-1 AM. HF was examined 8 weeks after myocardial infarction (coronary artery ligation)., Results: I(K1) was reduced and diastolic Ca(2+) was increased in HF cells. The reduction of I(K1) was attenuated when EGTA was elevated from 0.5 to 10 mM in the patch pipette and prevented with high BAPTA (20 mM). Ryanodine (100 nM) and FK506 (10 microM), both of which promote spontaneous SR Ca(2+) release from ryanodine receptor (RyR2) during diastole, reproduced the effect of HF on I(K1) in normal cells but had no effect in HF cells. The effects of ryanodine and FK506 were not additive and were prevented by BAPTA. Rapamycin (10 microM), which removes FKBP binding proteins from RyR2 with no effect on calcineurin, mimicked the effect of FK506 on I(K1). Cyclosporine A (10 microM), which inhibits calcineurin via cyclophilins, had no effect. In both HF cells and normal cells treated by FK506, the protein kinase C (PKC) inhibitor staurosporine totally restored the inward component of I(K1), but only partially restored its outward component at potentials corresponding to the late repolarizing phase of the action potential (-80 to -40 mV)., Conclusions: I(K1) is reduced by elevated diastolic Ca(2+)in HF, which involves in parallel PKC-dependent and PKC-independent mechanisms. This regulation provides a novel paradigm for Ca(2+)-dependent modulation of membrane potential in HF. Since enhanced RyR2-mediated Ca(2+)release also reduces I(K1), this paradigm might be relevant for arrhythmias related to acquired or inherited RyR2 dysfunction.

FK-506, a widely used immunosuppressant, has caused a few clinical cases with QT prolongation and torsades de pointe at high blood concentration. The proarrhytmogenic potential of FK-506 was investigated in single rat ventricular cells using the whole cell clamp method to record action potentials (APs) and ionic currents. Fluorescence measurements of Ca2+ transients were performed with indo-1 AM using a multiphotonic microscope. FK-506 (25 micromol/l) hyperpolarized the resting membrane potential (RMP; -3 mV) and prolonged APs (AP duration at 90% repolarization increased by 21%) at 0.1 Hz. Prolongation was enhanced by threefold at 3.3 Hz, and early afterdepolarizations (EADs) occurred in 59% of cells. EADs were prevented by stronger intracellular Ca2+ buffering (EGTA: 10 vs. 0.5 mmol/l in the patch pipette) or replacement of extracellular Na+ by Li+, which abolishes Na+/Ca2+ exchange [Na+/Ca2+ exchanger current (INaCa)]. In indo-1-loaded cells, FK-506 generated doublets of Ca(2+) transients associated with increased diastolic Ca2+ in one-half of the cells. FK-506 reversibly decreased the L-type Ca2+ current (ICaL) by 25%, although high-frequency-dependent facilitation of ICaL persisted, and decreased three distinct K+ currents: delayed rectifier K+ current (IK; >80%), transient outward K+ current (<20%), and inward rectifier K+ current (IK1; >40%). A shift in the reversal potential of IK1 (-5 mV) accounted for RMP hyperpolarization. Numerical simulations, reproducing all experimental effects of FK-506, and the use of nifedipine showed that frequency-dependent facilitation of ICaL plays a role in the occurrence of EADs. In conclusion, the effects of FK-506 on the cardiac AP are more complex than previously reported and include inhibitions of IK1 and ICaL. Alterations in Ca2+ release and INaCa may contribute to FK-506-induced AP prolongation and EADs in addition to the permissive role of ICaL facilitation at high rates of stimulation.

Living cells exhibit multiple K(+) channel proteins; among these is the recently reported atypical two-pore domain K(+) channel protein TREK-1. Most K(+) currents are modulated by neurohormones and under various pathological conditions. Here, in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique, we characterize for the first time a native TREK-1-like current (I(TREK)) that is activated by ATP, a purine agonist applied at a micromolar range. This current is sensitive to arachidonic acid, intracellular acidosis, and various K(+) current inhibitors. Reverse transcription-polymerase chain reaction reveals the presence of a TREK-1-like mRNA in rat cardiomyocytes that shows 93% identity with mouse TREK-1. ATP effects are greatly attenuated in the presence of arachidonic acid or HCO(-)(3)-induced intracellular acidosis. Using a series of inhibitors, we further demonstrate that the ATP-induced stimulation of I(TREK) implies the activation of cytosolic phospholipase A(2) and the release of arachidonic acid. These events require the simultaneous involvement of p38 MAPK and p42/44 MAPK, respectively, via a cAMP-dependent protein kinase and a tyrosine kinase pathway, whereas the two MAPKs conjugate to activate a mitogen- and stress-activated protein kinase (MSK-1). Our results thus demonstrate the occurrence of a TREK-1-like current in cardiac cells whose activation by purine agonists implies a dual-MAPK cytosolic pathway.

Objective: Deleterious electrical abnormalities evolve during myocardial infarction. The goal of this study was to analyse current changes during the late decompensated phase of heart disease induced by coronary ligation and to compare them in various heart regions., Methods: Young rats were submitted to left coronary ligature. After 4-6 months, cells were enzymatically dissociated and isolated from the upper part basal region of the left ventricle, as well as from the septum, apex and the right ventricle before being studied under whole-cell patch-clamp., Results: Basal L-type Ca2+ current, ICaL elicited at +10 mV did not exhibit regional dependence neither in control nor after post-myocardial infarction (PMI). ICaL showed both a significantly reduced peak amplitude (17.1 +/- 2.8 pA/pF versus 9.9 +/- 1.4 pA/pF in seven control and seven PMI hearts, n = 32 and 40, respectively) and a slower inactivation, such that the amount of inward charges during a 200 ms-depolarizing pulse was nearly unchanged. beta-Adrenergic stimulation was less effective in increasing ICaL in PMI cells but it slowed inactivation further. Significant differences in the K+ currents were observed. A regional distribution was seen for Ito only, with the largest amplitude in the right ventricle (in pA/pF: 23.1 +/- 2.4, 18.2 +/- 3.9, 14.8 +/- 2.4, 8.3 +/- 1.7 in the right ventricle, apex, septum and left ventricle, respectively n = 8, 7, 8 and 9). This was also true in failing heart cells despite Ito being halved in each of the four regions (in pA/pF: 12.2 +/- 2.5, 11.2 +/- 1.9, 5.1 +/- 1.0 and 4.8 +/- 1.0, respectively n = 12, 12, 11 and 13). IK1 was also significantly reduced by 20% in the PMI cells. Two-way analyses of variance demonstrated the absence of interaction between the topographical origin of the cells and the physiological state of the rats. The alpha 1-adrenergic agonist, methoxamine significantly reduced Ito and IK1 to the same extent in both sham and PMI cells, by about 35% and 20% respectively., Conclusions: Long-term left coronary occlusion induces significant alterations in both Ca2+ and K+ currents that occur with similar amplitude in both ventricles. They include a marked reduction in Ito amplitude as well as a slowing of ICaL inactivation. Both factors could contribute to the disturbances in cellular electrical behaviour and the occurrence of arrhythmias in the post-myocardial infarcted heart.