Neuronal NO Synthase–Derived NO
A Novel Relaxing Factor in Myocardium?
See related article, pages 242–249
Nitric oxide (NO) is generated by a family of NO synthases (NOSs) and is involved in a number of physiological and pathological processes in the cardiovascular system, most notably in the vasculature.1 The classic and, therefore, best-established action is its paracrine action in blood vessels, maintaining homeostasis, in that NO released from the endothelium reduces the tone of the vascular smooth muscle cells via activation of cGMP-dependent protein kinase (PKG).2 In the heart, 2 distinct types of NOSs, known as endothelial NOS (eNOS) and neuronal NOS (nNOS), are expressed in physiological settings (see elsewhere3,4 and references therein). Although both eNOS and nNOS are expressed in cardiomyocytes, they are localized to distinct subcellular compartments; that is, eNOS is localized to caveolae in the sarcolemma,5 whereas nNOS is localized predominantly to the sarcoplasmic reticulum (SR).6
In this issue of Circulation Research, Casadei and colleagues have refined our understanding of the role of nNOS-derived NO in the regulation of cardiac contractility, providing evidence that nNOS-derived NO accelerates ventricular relaxation via a cGMP/PKG-independent mechanism.7 Before discussing their novel findings, we would like to briefly summarize the mechanisms of cardiac muscle relaxation (Figure 1), based on the literature.8
On stimulation of cardiomyocytes, Ca2+ enters the myocyte via sarcolemmal L-type Ca2+ channels and induces release of Ca2+ from the SR, resulting in Ca2+ binding to troponin (Tn)C and subsequent formation of cross-bridges, hence, the onset of contraction. Unlike in skeletal muscle, cardiac myofilaments are not normally fully activated even during the peak of contraction, allowing production of greater active force with increasing preload (ie, the Frank–Starling mechanism; see elsewhere9 for review). Generally, 4 transport systems are involved in the removal of Ca2+ from the myoplasm, causing relaxation: (1) sequestration into the SR by the Ca2+-ATPase pump (ie, SERCA2a protein); (2) efflux via the sarcolemmal Na+/Ca2+ exchanger; (3) extrusion by the sarcolemmal Ca2+-ATPase pump; and (4) uptake into mitochondria via the Ca2+ uniporter. Of these 4 processes, the first process has the greatest contribution to Ca2+ removal, at least under physiological conditions, in both large and small mammals. Myofilament relaxation takes place following Ca2+ removal from the myoplasm, initiated by the dissociation of Ca2+ from TnC and, hence, subsequent cessation of actomyosin interaction. Under isometric conditions, because of coupling between twitch force and the affinity of TnC for Ca2+, myocardial relaxation time is prolonged (shortened) with an increase (a decrease) in the force level.10 Another important player in myocardial relaxation is the giant elastic protein titin (connectin); the titin-based “restoring force” (that is produced at sarcomere lengths below the slack length [≈1.9 μm], as in unloaded myocyte shortening) compresses the thick filaments, leading to immediate deactivation of cross-bridges (ie, length-dependent deactivation).11 nNOS-derived NO can influence myocardial relaxation by modulating [Ca2+]i- or myofilament-based control (Figure 1), as discussed below.
Myocardial relaxation is accelerated by β-adrenergic stimulation (a phenomenon called the “positive lusinotropic effect”).8 The importance of positive lusinotropy is highlighted in preventing incomplete ventricular filling because of the shortened diastolic interval during β-adrenergic stimulation. Positive lusinotropy is generally attributed to the increased Ca2+ uptake rate by the SR, secondary to the phosphorylation of phospholamban (PLB) (a protein associated with SERCA2a) via activation of protein kinase A (PKA).8 There is also myofilament-based control in the positive lusinotropy during β-adrenergic stimulation (Figure 1), albeit to a relatively small magnitude, in that phosphorylation of TnI reduces the interaction with TnC, enhancing dissociation of Ca2+ from TnC.8
A number of recent findings indicate that nNOS-derived NO is involved in the regulation of cardiac contractility under physiological conditions via multiple pathways (see elsewhere3,4 and references therein), unlike eNOS-derived NO (eg, see elsewhere12). nNOS has initially been reported to be localized to the SR, showing its inhibitory effect on Ca2+ uptake by SERCA2a.6 This unique localization of nNOS suggests that ion channels and transporters involved in the regulation of Ca2+ handling may be targets for NO-based signaling. Indeed, the Casadei group reported in prior publications that nNOS-derived NO reduces the open probability of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels of the SR, coupled possibly with S-nitrosylation (see elsewhere4 and references therein). In their mouse model, nNOS−/− myocytes indeed exhibit an augmentation of the Ca2+ transient and cell shortening (both seen in Zhang et al7 as well). Therefore, nNOS-derived NO likely plays as a negative inotropic factor in myocardium (see elsewhere4 and references therein). These effects of nNOS-derived NO on myocardial contractility, however, may vary depending on the experimental condition, such as stimulation frequency (eg, see elsewhere13).
Zhang et al7 demonstrated that nNOS-derived NO promotes ventricular relaxation by regulating the PKA-mediated phosphorylation of PLB via inhibition of protein phosphatases (hence, independent of cGMP/PKG activation; see Figure 2). The slowing of the rate of decay of the Ca2+ transient in nNOS−/− myocytes suggests that a mechanism coupled with Ca2+ sequestration by the SR primarily underlies the impaired relaxation. Zhang et al7 indeed found that the phosphorylation level of PLB was reduced in nNOS−/− myocytes. β-Adrenergic stimulation elevated PLB phosphorylation and accelerated relaxation in both nNOS−/− and nNOS+/+ myocytes, but more markedly in nNOS−/− myocytes, abolishing the differences between groups. An interesting and important finding is that although the expression level of type 1 (PP1) or 2A (PP2A) protein phosphatases was similar in nNOS−/− and nNOS+/+ hearts, their activities, especially that of PP1, were elevated in the former. Pharmacological inhibition of PP1 or PP2A indeed increased PLB phosphorylation and accelerated the rate of relaxation in nNOS−/− but not in nNOS+/+ myocytes. Therefore, the PLB phosphorylation level resulting from the balance of PKA and PP1 or PP2A activation likely plays a key role in these findings under basal conditions. However, as shown in figure 4 of the article by Zhang et al, strong β-adrenergic stimulation can easily overcome this baseline physiological regulation via pronounced PKA activation and subsequent acceleration of Ca2+ uptake into the SR, through overriding the relaxing effect of nNOS-derived NO.
A few concerns should be mentioned. First, it is unclear how nNOS-derived NO reduces phosphatase activity, although the authors claim that this mechanism may involve the protein phosphatase inhibitor-1 (I-1) and/or -2 (I-2). There could be other mechanisms as well. Using various approaches, the intracellular signal transduction mechanisms should be clarified for better understanding of the findings of the authors. Second, considering greater enhancement of cell shortening in nNOS−/− myocytes, compared with the Ca2+ transient (as shown in the tracings in their Figure 1), the impaired relaxation may result in part from a factor that is not directly related to the intracellular Ca2+ regulation (ie, myofilament-based control; cf, Figure 1). Importantly, TnI phosphorylation is less in nNOS−/− myocytes (as acknowledged by the authors), and this in turn suggests an involvement of TnI dephosphorylation as a possible mechanism for impaired relaxation. Likewise, yet not examined, titin degradation, phosphorylation, or isoform switching (from a stiff to more compliant isoform; eg, from N2B to N2BA isoform) and resultant reduction in myocyte restoring force may also tend to delay myofilament deactivation.11,14 A thorough investigation of sarcomere proteins in nNOS−/− mice, focusing especially on the relations with myocardial relaxation, would enhance the outstanding findings by Zhang et al.7
In summary, the present work shows that nNOS-derived NO acts as a unique relaxing factor in myocardium under physiological conditions, independent of the cGMP/PKG-based mechanism. The results imply that this process involves a reduction of protein phosphatase activity. Future studies should be directed toward elucidating the molecular mechanism(s) by which nNOS-derived NO regulates phosphatase activity under various experimental conditions, aiming at developing a novel approach for the treatment of diastolic dysfunction, which is frequently associated with heart failure.
Sources of Funding
The work performed by the authors was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (N.F. and S.K.) and grants from the Japan Science and Technology Agency (CREST) (N.F.) and the Vehicle Racing Commemorative Foundation (S.K.).
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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