Nitric oxide fine-tunes NHE1 to control cardiomyocyte pH

This editorial refers to ‘Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1’, by M.A. Richards et al., pp. 1958–1971.

The maintenance of intracellular pH within a narrow range is of paramount importance for normal cellular function.¹ Under physiological conditions, the extracellular pH is usually ∼7.4 whereas cytosolic pH is ∼7.2. In cardiomyocytes, cellular pH fluctuates with physiological state for example during alterations in cardiac workload and heart rate. During disease states such as myocardial ischaemia, however, a marked intracellular acidosis may occur and have profound detrimental consequences on heart function—including disturbed intracellular Ca²⁺ signalling, depressed contraction, and arrhythmia. Cardiomyocytes (as well as other cells) therefore have endogenous mechanisms capable of rapid and effective extrusion of protons from the cytosol to prevent acid accumulation. The sodium/hydrogen exchanger-1 (NHE1) is the most important pH-regulatory transporter in cardiomyocytes.² A reduction of pH_i activates NHE1 to extrude H⁺ and leads to H⁺-activated Na⁺ influx. The rise in intracellular Na⁺ in turn reduces the driving force for operation of the Na⁺/Ca²⁺ exchanger for Ca²⁺ extrusion and results in increased intracellular Ca²⁺. Therefore, cellular pH regulation by NHE1 intrinsically couples to Ca²⁺ signalling and contraction.³ Physiologically, this mechanism may serve as positively inotropic but persistent or excessive NHE1 activation leads to calcium overload and detrimental consequences, such as ischaemia–reperfusion injury, cardiomyocyte death, and calcium-activated pathological intracellular signalling.⁴

The post-translational regulation of NHE1 activity by different kinases is a crucial aspect of its physiological function to tightly control cardiomyocyte pH and cellular Na⁺/Ca²⁺ balance. Richards et al.⁵ report the impact of nitric oxide (NO) upon NHE1 activity and pH_i in a series of elegant biochemical, microscopic, and functional studies in isolated adult rodent cardiomyocytes. The effects both of endogenously generated NO and exogenous NO, as well as NO-evoked signalling, were studied using a combination of genetic and pharmacological tools. They identify that NO has bifunctional effects on NHE1 activity: low physiological levels of NO (low nM) activate NHE1 whereas high NO levels (>100 nM) lead to inhibition of the exchanger. These actions of NO are related to distinct effects on activatory protein kinase G (PKG) vs. inhibitory protein kinase A (PKA) signalling and the consequent post-translational phosphorylation of NHE1 at Ser703 and Ser648, respectively, in the C-terminus of the exchanger. The authors convincingly demonstrate that the activating effects of low NO involve PKG whereas the inhibitory effects of high NO result from a cGMP-dependent inhibition of cAMP phosphodiesterase (PDE) and consequent rise in cAMP-PKA signalling (Figure 1). This paradigm is consistent with several other settings where biphasic effects of NO in cells have been related to similar cGMP concentration-dependent actions on PKG and PDE.^6,7 Interestingly, the authors found no evidence for nitrosylation of NHE1. At a spatial level, the authors used a dual microperfusion method to provide evidence as to how ‘low’ NO-mediated activatory effects may occur distant to the site of NO production within a cell—owing to the increasing degradation of the diffusible second messenger cGMP by PDE activity the further the distance from its site of production. At a functional level, Richards et al.⁵ show that the biphasic NO-NHE1 interplay produces a similar pattern of opposing effects on the frequency of spontaneous Ca²⁺ waves—a surrogate for diastolic Ca²⁺ overload—although diastolic Ca² concentration and cardiomyocyte contraction were not examined.

Figure 1.

Schematic of effects of NO on NHE1 function. GC, guanylate cyclase; PDE, phosphodiesterase.

NO is generated by a family of NO synthases (NOSs), namely endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS), which are expressed in a cell- and context-specific manner in many tissues. Both eNOS and nNOS are physiologically expressed in the heart whereas iNOS is typically expressed in disease states.^6,7 eNOS expressed in cardiac endothelial cells modulates not only coronary flow but also exerts paracrine effects on cardiomyocytes, for example on relaxation and growth. Both eNOS and nNOS are reported to be expressed in cardiomyocytes but evidence from careful genetic studies and the use of specific inhibitors indicates that the cardiomyocyte-autonomous effects of NO on excitation–contraction coupling are mainly attributable to nNOS.⁷ nNOS expressed in neurones also has important effects on heart rate regulation and coronary microvascular resistance.^8,9 At a whole heart level, therefore, the effects of endogenously generated NO are considerably more complex than might be predicted from studies in isolated cells. Richards et al.⁵ used an nNOS-selective inhibitor and cardiomyocytes from nNOS knockout mice to demonstrate that NO generated by endogenous nNOS has a major role in regulating cardiomyocyte pH by activating NHE1. Although they related this at a functional level to the frequency of calcium waves and discussed the potential effects of low and high NO, from a physiological perspective it may also be useful to consider a paradigm of phasic (=‘physiological’) vs. sustained (=‘pathological’) NO release. Phasic release potentially linked to contractile activity could work as an effective fine-tuning mechanism whereas loss of such synchronicity would be detrimental. In this regard, it would be informative to investigate the physiological regulation of nNOS-dependent NO production. Furthermore, since nNOS-derived NO has other physiological effects on excitation–contraction coupling,⁷ it would be instructive to study how alterations in NHE1 activity integrate with such effects and influence overall cardiomyocyte contractile function. Another interesting question is whether eNOS-derived NO (from coronary microvascular endothelial cells) has similar effects on NHE1 activity in the whole heart context and, if so, how this interacts with the effects of cardiomyocyte-autonomous nNOS-derived NO. The expression, subcellular localization, activity, and contribution of NOS isoforms may change markedly in disease states. It will therefore be of interest in future studies to investigate the impact of the new mechanism identified by Richards et al. in pathophysiological conditions. Additional factors in the disease setting may be the modulation of NHE1 by other factors (e.g. hormones and growth factors) as well as the impact of altered reactive oxygen species (ROS) levels; ROS not only interact with NO but also modulate intracellular signalling.¹⁰

The discovery of the capacity of NO to fine-tune NHE1 activation via biphasic effects significantly increases our understanding of the complexity and flexibility of pH regulation in cardiomyocytes and the complex effects of NO on cardiac physiology. Future studies will need to investigate the relevance of this finding for overall heart function and the therapeutic potential of NHE1 inhibitors.

Conflict of interest: none declared.

Funding

The authors are supported by the British Heart Foundation (PG/17/39/33027 to M.Z. and CH/1999001/11735 to A.M.S.); and the Department of Health via a National Institute for Health Research (NIHR) Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London (IS-BRC-1215-20006).

The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.