Autocamtide-2-Related Inhibitory Peptide

The release of a fertilizable oocyte from the ovary is dependent upon the expansion of the cumulus cells. The expansion requires cooperation between epidermal growth factor (EGF) family peptide-activated mitogen-activated protein kinase (MAPK)3/1 and oocyte paracrine factor-activated-Sma- and Mad-related protein (SMAD)2/3 signaling in cumulus cells. However, the mechanism underlying (MAPK)3/1 signaling is unclear. In the present study, the EGF-activation of EGF receptor (EGFR) induced cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) phosphorylation in cumulus cells, and the interruption of CREB functional complex formation by naphthol AS-E phosphate (KG-501) completely blocked the EGF-stimulated expansion-related gene expression. EGF-stimulated phosphorylation of CREB was completely inhibited by MAPK3/1 inhibitor U0126, suggesting that EGF-activated MAPK3/1 results in the activation of CREB for cumulus expansion. Also, the role of EGF-stimulated calcium signaling was studied. Calcium-elevating reagents ionomycin and sphingosine-1-phosphate mimicked, but calcium chelators bis-(o'aminophenoxy)-ethane-N,N,N,N-tetraacetic acid, tetra(acetoxymethyl)-ester, and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate abolished the activity of EGF on CREB phosphorylation, cumulus expansion, and expansion-related gene expression. Furthermore, EGF-induced cumulus expansion was inhibited by calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitors, KN-93 and autocamtide-2-related inhibitory peptide. However, the inhibition of SMAD2/3 activity by removal of oocyte from cumulus-oocyte complexes did not affect the EGF-induced CREB phosphorylation, indicating that EGF-activated CREB is independent of oocyte-activated SMAD2/3 signaling. Therefore, EGF-induced CREB activity by MAPK3/1 and Ca2+/CaMKII signaling pathways promotes the expansion-related gene expression and consequent cumulus expansion.

Background:Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of care. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models.Methods:We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2-related inhibitory peptide [AIP], is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell-derived cardiomyocytes and different pathogenic genotypes.Results:AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2R176Q/+) effectively suppressed ventricular arrhythmias induced by either β-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ≈50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell-derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP.Conclusions:This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell-derived cardiomyocyte models with different pathogenic mutations.

Background:In non-diabetic patients with acute coronary syndrome, stress hyperglycemia occasionally occurs and is related to their mortality. Whether transient elevation of glucose affects arrhythmia susceptibility in non-diabetic hearts with non-uniform contraction was examined.Methods and Results:Force, intracellular Ca2+([Ca2+]i), and membrane potential were measured in trabeculae from rat hearts. Non-uniform contraction was produced by a jet of paralyzing solution. Ca2+waves and arrhythmias were induced by electrical stimulation (2.0 mmol/L [Ca2+]o). The activity of Ca2+/calmodulin-dependent protein kinaseII (CaMKII) was measured. An elevation of glucose from 150 to 400 mg/dL increased the velocity of Ca2+waves and the number of spontaneous action potentials triggered by electrical stimulation. Besides, the elevation of glucose increased the CaMKII activity. In the presence of 1 μmol/L KN-93, the elevation of glucose did not increase the velocity of Ca2+waves and the number of triggered action potentials. In addition, in the presence of 1 μmol/L autocamtide-2 related inhibitory peptide or 50 μmol/L diazo-5-oxonorleucine, the elevation of glucose did not increase the number of triggered action potentials. Furthermore, the elevation of glucose by adding L-glucose did not increase their number.Conclusions:In non-diabetic hearts with non-uniform contraction, transient elevation of glucose increases the velocity of Ca2+waves by activating CaMKII,probably through glycosylation with O-linked β-N-acetylglucosamine, thereby increasing arrhythmia susceptibility.