Effect of ischemic preconditioning and a Kv7 channel blocker on cardiac ischemia-reperfusion injury in rats.

Recently, we found cardioprotective effects of ischemic preconditioning (IPC), and from a blocker of KCNQ voltage-gated K+ channels (KV7), XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), in isolated rat hearts. The purpose of the present study was to investigate the cardiovascular effects of IPC and XE991 and whether they are cardioprotective in intact rats. In conscious rats, we measured the effect of the KV7 channel blocker XE991 on heart rate and blood pressure by use of telemetry. In anesthetized rats, cardiac ischemia was induced by occluding the left coronary artery, and the animals received IPC (2 × 5 min of occlusion), XE991, or a combination. After a 2 h reperfusion period, the hearts were excised, and the area at risk and infarct size were determined. In both anesthetized and conscious rats, XE991 increased blood pressure, and the highest dose (7.5 mg/kg) of XE991 also increased heart rate, and 44% of conscious rats died. XE991 induced marked changes in the electrocardiogram (e.g., increased PR interval and prolonged QTC interval) without changing cardiac action potentials. The infarct size to area at risk ratio was reduced from 53 ± 2% (n = 8) in the vehicle compared to 36 ± 3% in the IPC group (P < 0.05, n = 9). XE991 (0.75 mg/kg) treatment alone or on top of IPC failed to reduce myocardial infarct size. Similar to the effect in isolated hearts, locally applied IPC was cardioprotective in intact animals exposed to ischemia-reperfusion. Systemic administration of XE991 failed to protect the heart against ischemia-reperfusion injury suggesting effects on the autonomic nervous system counteracting the cardioprotection in intact animals.

Novel selective PDE type 1 inhibitors cause vasodilatation and lower blood pressure in rats.

BACKGROUND AND PURPOSE: The PDE enzymes (PDE1-11) hydrolyse and thus inactivate cyclic nucleotides and are important in the regulation of the cardiovascular system. Here,we have investigated the effects on the cardiovascular system, of two novel selective PDE1 inhibitors, Lu AF41228 and Lu AF58027. EXPERIMENTAL APPROACH: We used rat mesenteric small arteries (internal diameters of 200-300 µm), RT-PCR and measured isometric wall tension. Effects of Lu AF41228 and Lu AF58027 on heart rate and BP were assessed in both anaesthetized and conscious male rats. KEY RESULTS: Nanomolar concentrations of Lu AF41228 and Lu AF58027 inhibited PDE1A, PDE1B and PDE1C enzyme activity, while micromolar concentrations were required to observe inhibitory effects at other PDEs. RT-PCR revealed expression of PDE1A, PDE1B and PDE1C in rat brain, heart and aorta, but only PDE1A and PDE1B in mesenteric arteries. In rat isolated mesenteric arteries contracted with phenylephrine or U46619, Lu AF41228 and Lu AF58027 induced concentration-dependent relaxations which were markedly reduced by inhibitors of guanylate cyclase, ODQ, and adenylate cyclase, SQ22536, and in preparations without endothelium. In anaesthetized rats, Lu AF41228 and Lu AF58027 dose-dependently lowered mean BP and increased heart rate. In conscious rats with telemetric pressure transducers, repeated dosing with Lu AF41228 lowered mean arterial BP 10-15 mmHg and increased heart rate. CONCLUSIONS AND IMPLICATIONS: These novel PDE1 inhibitors induce vasodilation and lower BP, suggesting a potential use of these vasodilators in the treatment of hypertension and vasospasm.

Involvement of transglutaminase 2 and voltage-gated potassium channels in cystamine vasodilatation in rat mesenteric small arteries.

BACKGROUND AND PURPOSE: Vasodilatation may contribute to the neuroprotective and vascular anti-remodelling effect of the tissue transglutaminase 2 (TG2) inhibitor cystamine. Here, we hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlies cystamine vasodilatation. EXPERIMENTAL APPROACH: We used rat mesenteric small arteries and RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca(2+) ([Ca(2+)]i ), K(+) currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain, in our experiments. KEY RESULTS: RT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited responses to phenylephrine, 5-HT and U46619 and for extracellular potassium. Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of PLC suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca(2+)]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca(2+)]i , and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of Kv 7 channels, XE991 and linopirdine, inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr(855) phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr(855) and myosin regulatory light chain phosphorylation. CONCLUSIONS AND IMPLICATIONS: Cystamine induced vasodilatation by inhibition of receptor-coupled TG2, leading to opening of Kv channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine.

Pulmonary hypertension in wild type mice and animals with genetic deficit in KCa2.3 and KCa3.1 channels.

OBJECTIVE: In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension. APPROACH AND RESULT: Male wild type and KCa3.1-/-/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1-/-/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype. CONCLUSION: Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.