Cardiomyocyte depolarization triggers NOS-dependent NO transient after calcium release, reducing the subsequent calcium transient

Chao Wang,Rainer H A Fink,Matias Mosqueira,Carolin Andresen,Roland Konietzny

doi:10.1007/s00395-021-00860-0

Abstract

Cardiac excitation–contraction coupling and metabolic and signaling activities are centrally modulated by nitric oxide (NO), which is produced by one of three NO synthases (NOSs). Despite the significant role of NO in cardiac Ca2+ homeostasis regulation under different pathophysiological conditions, such as Duchenne muscular dystrophy (DMD), no precise method describes the production, source or effect of NO through two NO signaling pathways: soluble guanylate cyclase-protein kinase G (NO-sGC-PKG) and S-nitrosylation (SNO). Using a novel strategy involving isolated murine cardiomyocytes loaded with a copper-based dye highly specific for NO, we observed a single transient NO production signal after each electrical stimulation event. The NO transient signal started 67.5 ms after the beginning of Rhod-2 Ca2+ transient signal and lasted for approximately 430 ms. Specific NOS isoform blockers or NO scavengers significantly inhibited the NO transient, suggesting that wild-type (WT) cardiomyocytes produce nNOS-dependent NO transients. Conversely, NO transient in mdx cardiomyocyte, a mouse model of DMD, was dependent on inducible NOS (iNOS) and endothelial (eNOS). In a consecutive stimulation protocol, the nNOS-dependent NO transient in WT cardiomyocytes significantly reduced the next Ca2+ transient via NO-sGC-PKG. In mdx cardiomyocytes, this inhibitory effect was iNOS- and eNOS-dependent and occurred through the SNO pathway. Basal NO production was nNOS- and iNOS-dependent in WT cardiomyocytes and eNOS- and iNOS-dependent in mdx cardiomyocytes. These results showed cardiomyocyte produces NO isoform-dependent transients upon membrane depolarization at the millisecond time scale activating a specific signaling pathway to negatively modulate the subsequent Ca2+ transient.

Highlights

Cardiac excitation–contraction coupling (ECC) is the central physiological process of C a2+-mediated contractility in the heart [9]
Line scans were recorded in cardiomyocytes isolated from 12-month-old WT mice that were double-loaded with 10 μM Rhod-2 for Ca2+ transient visualization (Fig. 1a) and 10 μM “nitric oxide (NO)-ON” for NO transients (Fig. 1b). Ca2+ transients were recorded as an internal control to evaluate (a) the viability of the cardiomyocyte, where only those that responded to each electrical stimulation with a single Ca2+ transient were selected for data analyses, and (b) to correlate the temporal NO production
A novel physiological paradigm for intracellular NO production is summarized in Fig. 7, proposing that endogenous NO synthases (NOSs)-dependent NO is transiently produced after the C a2+ transient upon electrical stimulation

Summary

Introduction

Cardiac excitation–contraction coupling (ECC) is the central physiological process of C a2+-mediated contractility in the heart [9]. The cardiac production of NO is mediated by one of three NO synthase isoforms expressed under normal conditions: neuronal. NNOS and eNOS are transiently activated once C a2+ released during the ECC binds to calmodulin ( Ca2+-dependent NOS activation) [69, 101], while iNOS activation is activated through a FAK-PI(3)K-AKT signaling pathway dependent on contractility and independent of C a2+ [16]. The expression levels and activity of three NOS isoforms change altering NO production [34, 55, 112]. Cardiomyocytes from mdx mice exhibit reduced expression and activity levels of the neuronal isoform of NO synthase (nNOS) and reduced production of NO [69, 82, 97]

Methods

Results

Conclusion