Molecular mechanisms of neuronal nitric oxide synthase in cardiac function and pathophysiology

Abstract

Neuronal nitric oxide synthase (nNOS or NOS1) is the major endogenous source of myocardial nitric oxide (NO), which facilitates cardiac relaxation and modulates contraction. In the healthy heart it regulates intracellular Ca(2+), signalling pathways and oxidative homeostasis and is upregulated from early phases upon pathogenic insult. nNOS plays pivotal roles in protecting the myocardium from increased oxidative stress, systolic/diastolic dysfunction, adverse structural remodelling and arrhythmias in the failing heart. Here, we show that the downstream target proteins of nNOS and underlying post-transcriptional modifications are shifted during disease progression from Ca(2+)-handling proteins [e.g. PKA-dependent phospholamban phosphorylation (PLN-Ser(16))] in the healthy heart to cGMP/PKG-dependent PLN-Ser(16) with acute angiotensin II (Ang II) treatment. In early hypertension, nNOS-derived NO is involved in increases of cGMP/PKG-dependent troponin I (TnI-Ser(23/24)) and cardiac myosin binding protein C (cMBP-C-Ser(273)). However, nNOS-derived NO is shown to increase S-nitrosylation of various Ca(2+)-handling proteins in failing myocardium. The spatial compartmentation of nNOS and its translocation for diverse binding partners in the diseased heart or various nNOS splicing variants and regulation in response to pathological stress may be responsible for varied underlying mechanisms and functions. In this review, we endeavour to outline recent advances in knowledge of the molecular mechanisms mediating the functions of nNOS in the myocardium in both normal and diseased hearts. Insights into nNOS gene regulation in various tissues are discussed. Overall, nNOS is an important cardiac protector in the diseased heart. The dynamic localization and various mediating mechanisms of nNOS ensure that it is able to regulate functions effectively in the heart under stress.

Figure 1

Distribution and structure of neuronal nitric oxide synthase (nNOS) protein in the heart A, expression of nNOS protein in cardiac myocytes, coronary artery smooth muscle cells, cardiac ganglia, and sympathetic and parasympathetic nerves. B, structure of nNOS. Each monomer of nNOS contains an oxygenase domain (–COOH terminal) and a reductase domain (–NH2 terminal). Electrons from NADPH transfer from the reductase domain [via flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)] to the oxygenase domain (heme iron), enable nNOS to catalyse the oxidation of l-arginine to l-citrulline and release NO. These two domains are linked and the enzyme is activated by Ca²⁺–CaM.

Figure 2

Neuronal nitric oxide synthase (nNOS) protein expression, compartmentation and function in healthy cardiac myocytes The diagram illustrates the localization of nNOS and target proteins and regulation. nNOS is located in the sarcoplasmic reticulum (SR), mitochondria, plasma membrane and possibly nucleus. nNOS interacts with ryanodine receptor (RyR), xanthine oxidoreductase (XOR), α-syntrophin [forming a macrocomplex with Na⁺ channel (SCN5a) and plasma membrane Ca²⁺-ATPase (PMCA4b)]. nNOS modifies the activities of various proteins either through S-nitrosylation (-SNO) or through PKA-dependent, cGMP/PKG-dependent phosphorylations of downstream proteins.

Figure 3

Cardiac neuronal nitric oxide synthase (nNOS) during disease progression nNOS is functionally expressed in healthy myocardium. From the early stages of disease progression (pre-disease) to hypertension, cardiac nNOS is upregulated and facilitates myocyte relaxation. The mechanisms mediating nNOS shift from PKA-dependent PLN-Ser¹⁶ to PKG-dependent PLN-Ser¹⁶ at pre-disease and PKG-dependent cMBP-C Ser²⁷³ and cTnI Ser^23/24 at hypertension. Myocyte contraction is not affected by nNOS.

Figure 4

Neuronal nitric oxide synthase (nNOS) compartmentation, function and mechanism in failing cardiac myocyte nNOS protein expression and activity are increased in failing myocardium. nNOS translocates to plasma membrane and interacts with caveolin-3 (Cav3) but dissociates from ryanodine receptors (RyR). nNOS-derived NO changes the activities of Ca²⁺ handling proteins via S-nitrosylation. Cardiac-specific overexpression of nNOS is associated with increased nNOS localization in mitochondria and moderates mitochondria activity.