Abstract
Introduction and Aim. Nitric oxide (NO) can trigger cardiac differentiation of embryonic stem cells (ESCs), indicating a cardiogenic function of the NO synthetizing enzyme(s) (NOS). However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC) and cGMP activated protein kinase I (PKG-I) is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. Methods. Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs) were evaluated as an index of cardiogenesis. Results and Discussion. Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. Conclusion. We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.
Highlights
Introduction and AimNitric oxide (NO) can trigger cardiac differentiation of embryonic stem cells (ESCs), indicating a cardiogenic function of the NO synthetizing enzyme(s) (NOS)
NO is produced by three different enzymes and acts directly or indirectly through the stimulation of the soluble guanylate cyclase, a heterodimeric enzyme, and the subsequent formation of cyclic GMP
Assessment of cardiac differentiation in vitro is a prerequisite to identify factors and drugs able to increase the cardiogenic potential of stem cells during organogenesis, and during the healing and reparation after degenerative processes of the heart
Summary
Nitric oxide (NO), a biological active free radical, is an intracellular and intercellular messenger in mammalian cells [1, 2], where its signaling cascade plays an important modulatory role in many physiological and pathological conditions [1, 3]. On myocardial tissue low activation level of NO/NOS/sGC/PKG-I signaling increases contractility, while high levels exert a negative inotropic effect [7]. In this setting many functional proteins are substrates for PKG-I, including L-type calcium channels and phospholamban (PLB), which in the phosphorylated form relieves its inhibitory effect on sarcoendoplasmic Ca-ATPasi type 2 (SERCA2), enhancing calcium uptake into the sarcoendoplasmic reticulum (SR) and reducing cytosolic free calcium [10, 11]. The NO-donor Snitrosocysteine proved to enhance cardiac differentiation and spontaneous contraction of D3 mESC [20], corroborating the hypothesis of a cardiogenic function of NO/sGC pathway and that of the downstream effector PKG-I. These data have been partially presented as an abstract at the Congress of the European Society of Cardiology, Council on Basic Cardiovascular Science, held in Florence on 8–10 July 2016 [21]
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