Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1.
Richards MA., Simon JN., Ma R., Loonat AA., Crabtree MJ., Paterson DJ., Fahlman RP., Casadei B., Fliegel L., Swietach P.
AIMS: When activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically-important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear. METHODS AND RESULTS: NHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium prepulse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by low [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yl)ation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling. CONCLUSIONS: Physiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Na+-driven cellular Ca2+ loading. These responses are positively inotropic but also increase the likelihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive. TRANSLATIONAL PERSPECTIVE: NHE1 regulates intracellular [H+] and [Na+], but its over-activation can drive ionic imbalances that affect cardiac contractility and rhythm. Pharmacological control of NHE1 (e.g. with cariporide) has been proposed as cardioprotective in conditions such as ischemia/reperfusion injury, but trials (e.g. GUARDIAN) failed to demonstrate overall clinical benefit. A confounding factor in these analyses is NHE1 modulation by endogenous factors. We demonstrate that NO signalling fine-tunes NHE1, producing stimulation at low levels, turning into inhibition as the signal grows stronger. Thus, evaluations of the therapeutic efficacy of NHE1-blocking drugs should consider NO signalling, a pathway known to undergo changes in disease.