Cytoprotection by the NO-Donor SNAP Against Ischemia/Reoxygenation Injury in Mouse Embryonic Stem Cell-Derived Cardiomyocytes

Abstract

Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non-specific NO synthase inhibitor (N-Nitro-L-arginine, L-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by L-NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP-induced cytoprotection. The present mESC-derived cardiomyocyte based screening platform is a useful tool for discovery of cytoprotective molecules.

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

“…Similarly, embryonic stem cells have been used to study potential cytoprotective effects. The longevity of these cells under culture conditions can be prolonged by administration of NO [162, 163, 328]. …”

Protection by the NO-Donor SNAP and BNP against Hypoxia/Reoxygenation in Rat Engineered Heart Tissue

“…Therefore, there is an unmet need to develop cardiomyocyte-based test-beds for at least medium-throughput pharmacological screens. Previously, we have reported the responses of neonatal rat cardiomyocytes [ 7 ] and mouse embryonic stem cell-derived cardiomyocytes [ 9 ] to different concentrations of the NO-donor SNAP and B-type natriuretic peptide. Both test systems show good reproducibility and high-throughput but respond very differently to hypoxia/reoxygenation and drugs than the adult heart.…”

“…Furthermore, administration of SNAP has been shown to mimic preconditioning protection in mouse hearts [ 8 ]. Concentration-dependent protection of exogenous NO against simulated ischemia/reperfusion induced injury was detected in mouse embryonic stem cell-derived cardiomyocytes as well [ 9 ]. The NO-mediated cytoprotection mainly acts via intracellular elevation of cyclic guanosine monophosphate (cGMP), which has a common PKG-dependent downstream signaling pathway with natriuretic peptides [ 7 , 10 , 11 ].…”

ESC Working Group Cellular Biology of the Heart: Position Paper: improving the preclinical assessment of novel cardioprotective therapies

“…The primary adult cardiomyocyte is the most closely related to the adult heart in terms of characteristics and is most often harvested from mice, rats, or rabbits, but can also be obtained from guinea pig, dog, pig, and human. A number of different cells have been used in place of adult primary cardiomyocytes in experimental models of acute I/R injury, including HL-1 cells (derived from murine atrial myoblasts), 25 H9C2 cells (a rat cardiomyoblast cell line), 26 C2C12 cells (a murine myoblast cell line), 27 (embryonic) stem cell-derived cardiomyocytes, 28 and neonatal cardiomyocytes. 29 Although these cells may possess some of the key features of adult cardiomyocytes such as an organized internal structure, cell–cell contacts, high oxygen consumption, and spontaneous beating, several key differences such as the ability to proliferate and signal transduction pathways might affect their use as surrogates for primary adult cardiomyocytes.…”