SUMMARY Notch receptor signaling is implicated in controlling smooth muscle cell proliferation and maintaining smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension is a disease characterized by excessive vascular smooth muscle cell proliferation in small pulmonary arteries, leading to elevation of pulmonary vascular resistance with consequent right ventricular failure and death. Here we show that human pulmonary hypertension is characterized by overexpression of NOTCH3 in small pulmonary artery smooth muscle cells, and that severity of disease in humans and rodents correlates with the amount of Notch3 protein in the lung. We further demonstrate that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation. We report that pulmonary hypertension can be successfully treated in rodents by administration of DAPT, a γ-secretase inhibitor that blocks activation of Notch3 in smooth muscle cells. We demonstrate a mechanistic link between NOTCH3 receptor signaling through HES5 and smooth muscle cell proliferation and a shift to an undifferentiated smooth muscle cell phenotype. These data suggest that the NOTCH3-HES5 signaling pathway is crucial for the development of pulmonary arterial hypertension and provides a target pathway for therapeutic intervention.

Abstract-Recent studies have suggested that inositol-1,4,5-trisphosphate-receptor (IP 3 R)-mediated Ca 2ϩ release plays an important role in the modulation of excitation-contraction coupling (ECC) in atrial tissue and the generation of arrhythmias, specifically chronic atrial fibrillation (AF). IP 3 R type-2 (IP 3 R2) is the predominant IP 3 R isoform expressed in atrial myocytes. To determine the role of IP 3 R2 in atrial arrhythmogenesis and ECC, we generated IP 3 R2-deficient mice. Our results revealed that endothelin-1 (ET-1) stimulation of wild-type (WT) atrial myocytes caused an increase in basal [Ca 2ϩ ] i , an enhancement of action potential (AP)-induced [Ca 2ϩ ] i transients, an improvement of the efficacy of ECC (increased fractional SR Ca 2ϩ release), and the occurrence of spontaneous arrhythmogenic Ca 2ϩ release events as the result of activation of IP 3 R-dependent Ca 2ϩ release. In contrast, ET-1 did not alter diastolic [Ca 2ϩ ] i or cause spontaneous Ca 2ϩ release events in IP 3 R2-deficient atrial myocytes. Under basal conditions the spatio-temporal properties (amplitude, rise-time, decay kinetics, and spatial spread) of [Ca 2ϩ ] i transients and fractional SR Ca 2ϩ release were not different in WT and IP 3 R2-deficient atrial myocytes. WT and IP 3 R2-deficient atrial myocytes also showed a significant and very similar increase in the amplitude of AP-dependent [Ca 2ϩ ] i transients and Ca 2ϩ spark frequency in response to isoproterenol stimulation, suggesting that both cell types maintained a strong inotropic reserve. No compensatory changes in Ca 2ϩ regulatory protein expression (IP 3 R1, IP 3 R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP 3 R2-deficient mice. These results show that lack of IP 3 R2 abolishes the positive inotropic effect of neurohumoral stimulation with ET-1 and protects from its arrhythmogenic effects. (Circ Res. 2005;96:1274-1281.)Key Words: IP 3 receptor Ⅲ intracellular calcium Ⅲ atrial arrhythmias Ⅲ excitation-contraction coupling Ⅲ endothelin C hronic atrial fibrillation (AF) is the most common sustained form of cardiac arrhythmia. AF is characterized by an atrial activation rate of typically Ͼ400 beats per minute, and is associated with 2 major complications including cardiac dysfunction and thrombus formation, resulting in an increased risk of morbidity because of heart failure and stroke. 1,2 In recent years, considerable attention has focused on the cellular and molecular mechanisms involved in AF (for review see Nattel 3,4 activates the RyR which leads to massive Ca 2ϩ release from the SR by a mechanism known as Ca 2ϩ -induced Ca 2ϩ release (CICR 12 ), which is required for inducing contraction.Cardiac myocytes also contain IP 3 R channels, however their functional importance in the heart has remained controversial. IP 3 Rs release Ca 2ϩ from intracellular Ca 2ϩ stores when activated by IP 3 , a product generated by phospholipase C (PLC) metabolism of phosphoinositol-4,5-bisphosphate (PIP 2 ) in response to G-protein-c...

Carvedilol is one of the most effective beta-blockers for preventing ventricular tachyarrhythmias (VTs) in heart failure (HF), but the mechanisms underlying its favorable anti-arrhythmic benefits remain unclear. Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), are known to evoke VTs in patients with HF. Here we show that carvedilol is the only beta-blocker that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol combined with its beta-blocking activity likely contributes to its favorable anti-arrhythmic effect. To allow individual and optimal titration of these beneficial activities, we developed a novel SOICR-inhibiting, minimally-beta-blocking carvedilol analogue VK-II-86. We found that VK-II-86 alone prevented stress-induced VTs in RyR2 mutant mice, and was more effective when combined with a selective beta-blocker metoprolol or bisoprolol. Thus, SOICR inhibition combined with optimal beta-blockade presents a new, promising and potentially patient-tailorable anti-arrhythmic approach.