Binding of 17b-estradiol (E 2 ) to novel G-protein coupled receptor, Gper1, promotes intra-oocyte adenylyl cyclase activity and transactivates epidermal growth factor receptor to ensure prophase-I arrest. Although involvement of either membrane progestin receptor (mPR) or Igf system has been implicated in regulation of meiosis resumption, possibility of concurrent activation and potential synergism between 17a,20b-dihydroxy-4-pregnen-3-one (DHP)-and Igf-mediated signalling cascades in alleviating E 2 inhibition of oocyte maturation (OM) has not been investigated. Here using zebrafish (Danio rerio) defolliculated oocytes, we examined the effect of DHP and Igf1, either alone or in combination, in presence or absence of E 2 , on OM in vitro. While priming of denuded oocytes with E 2 blocked spontaneous maturation, co-treatment with DHP (3 nM) and Igf1 (10 nM), but not alone, reversed E 2 inhibition and promoted a robust increase in germinal vesicle breakdown (GVBD). Although stimulation with either Igf1 or DHP promoted Akt phosphorylation, pharmacological inhibition of PI3K/Akt signalling prevented Igf1-induced GVBD but delayed DHP action till 4-5 h of incubation. Moreover, high intra-oocyte cAMP attenuates both DHP and Igf1-mediated OM and co-stimulation with DHP and Igf1 could effectively reverse E 2 action on PKA phosphorylation. Interestingly, data from in vivo studies reveal that heightened expression of igf1, igf3 transcripts in intact follicles corresponded well with elevated phosphorylation of Igf1r and Akt, mPRa immunoreactivity, PKA inhibition and accelerated GVBD response just prior to ovulation. This indicates potential synergism between maturational steroid and Igf1 which might have physiological relevance in overcoming E 2 inhibition of meiosis resumption in zebrafish oocytes.

Aims The cardiac natriuretic peptides (atrial natriuretic peptide [ANP] and B-type natriuretic peptide [BNP]) are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities. Methods and Results We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP deficient (Nppa-/-) or BNP deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human derived induced pluripotent stem cell cardiomyocytes (iPS-CM). In the unstressed state, both ANP and BNP deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodeling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild type hearts to stress induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen activated protein kinase (p38 MAPK) signaling cascade. Conclusions Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signaling which sensitizes the heart to stress induced ventricular arrhythmias. Translational Perspective Our study found that ANP or BNP deficiency leads to increased sudden death and ventricular arrhythmias after acute myocardial stress in murine models. We discovered that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels and uncovered a unique role for these peptides in regulating cardiomyocyte p38 MAPK and CREB signaling through a cGMP-PKG1 pathway. Importantly, this signaling pathway was conserved in human cardiomyocytes. This study provides mechanistic insight into how modulating natriuretic peptide levels in human heart failure patients reduces sudden death and mortality.

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