Nitric oxide (NO) is a highly reactive, free radical signalling molecule that is constitutively released in cardiomyocytes by both the endothelial and neuronal isoforms of nitric oxide synthase (eNOS and nNOS, respectively). There are increasing data indicating that NO modulates various proteins involved in excitation–contraction coupling (ECC), and here we discuss the evidence that NO may modulate the function of the ryanodine receptor Ca2+ release channel (RyR2) on the cardiac sarcoplasmic reticulum (SR). Both constitutive isoforms of NOS have been shown to co-immunoprecipitate with RyR2, suggesting that the channel may be a target protein for NO. eNOS gene deletion has been shown to abolish the increase in spontaneous Ca2+ spark frequency in cardiomyocytes exposed to sustained stretch, whereas the effect of nNOS-derived NO on RyR2 function remains to be investigated. Single channel studies have been performed with RyR2 reconstituted in planar lipid bilayers and exposed to various NO donors and, under these conditions, NO appears to have a dose-dependent, stimulatory effect on channel open probability (Popen). We discuss whether NO has a direct effect on RyR2 via covalent S-nitrosylation of reactive thiol residues within the protein, or whether there are downstream effects via cyclic nucleotides, phosphodiesterases, and protein kinases. Finally, we consider whether the proposed migration of nNOS from the SR to the sarcolemma in the failing heart may have consequences for the nitrosative vs. oxidative balance at the level of the RyR2, and whether this may contribute to an increased diastolic Ca2+ leak, depleted SR Ca2+ store, and reduced contractility in heart failure.
A role for the free radical signalling molecule nitric oxide (NO) in modulating cardiac function has been recognised for 15 years or more. The precise actions of NO, however, remain under investigation. This probably stems from the varied (and often opposing) actions of NO in the heart, which are determined by different subcellular compartmentalization and regulation of the NO synthase (NOS) isoforms.1 Two are now known to be constitutively expressed in cardiomyocytes: ‘endothelial’ NOS (eNOS or NOS3) mainly localized to invaginations of the plasmalemma called caveolae,2 and ‘neuronal’ NOS (nNOS or NOS1), which is mostly found on the sarcoplasmic reticulum (SR)3 (Figure 1). The third isoform is the ‘inducible’ NOS (iNOS or NOS2), whose expression is stimulated by inflammatory mediators.
NOS catalyses the synthesis of NO by the conversion of L-arginine and oxygen to L-citrulline and NO, in the presence of NADPH and tetrahydrobiopterin (BH4).4 eNOS and nNOS are both activated by Ca2+ and produce NO at different rates (16 nmol of NO min−1 mg−1 for eNOS vs. 96 nmol of NO min−1 mg−1 for nNOS).5,6 A further important difference in the regulation of these two NOS isoforms is that under physiological conditions, eNOS activity is predominantly regulated by phosphorylation,7,8 whereas nNOS activity and expression are exquisitely regulated by Ca2+.9 It has been shown that in the left ventricular (LV) myocardium, the concentration of NO changes cyclically and on the same timescale as the heart beat.10 Moreover, increasing or decreasing the LV preload appears to be associated with parallel changes in intramyocardial NO concentration.10 Taken together, these findings support the idea that NO is involved in a fast, autoregulatory mechanism that modulates myocardial contraction on a beat-by-beat basis and assists in matching preload with cardiac output.
During excitation–contraction coupling (ECC), depolarization of the cardiomyocyte plasmalemma opens L-type voltage-gated Ca2+ channels (LTCC) and Ca2+ enters the cell. This Ca2+ influx stimulates a larger release of Ca2+ through the opening of ryanodine receptor Ca2+ release channels (RyR2) on the SR, a process termed Ca2+-induced Ca2+ release (CICR).11 Ca2+ binding to troponin C produces a conformational change in the troponin I—tropomyosin complex, which permits the initiation of myosin—actin cross-bridge cycling and contraction. Relaxation occurs when the concentration of intracellular Ca2+ ([Ca2+]i) is returned to resting levels either by reuptake into the SR via the SR Ca2+ ATPase (SERCA2a) or by extrusion from the cell via the Na+/Ca2+ exchanger (NCX1) and, to a lesser extent, the plasmalemma Ca2+ ATPase (PMCA4b) (for an overview of ECC, see Bers12) (Figure 1).
It is now well established that constitutive NO production modulates the function of many ion channels and transporters involved in cardiac ECC (reviewed in references9,13–15). Here we discuss the evidence suggesting that NO may modulate RyR2 function both through classic cGMP-signalling and via direct redox modification of specific thiol residues in the RyR2 protein.
What are the functional effects of an increase in RyR2 open probability?
The function of the RyR2 is influenced by multiple factors including cytosolic Ca2+ concentration, SR Ca2+ content, and binding of accessory proteins [triadin, junctin, calsequestrin,16 FK-506 binding protein-12.6,17 protein kinase A (PKA),18 protein phosphatases 1 and 2A (PP1 and PP2A),19 and calmodulin20]. NO has been reported to both increase and decrease the open probability (Popen) of the RyR2. To understand the possible functional correlates of these changes, it is useful first to consider the effects of selectively increasing RyR2 Popen. At first sight, one might assume that this would increase the amplitude of the Ca2+ transient. However, experimental work in rat ventricular myocytes shows that the increased Ca2+ transient amplitude produced by low-dose caffeine (a compound that increases the Popen of the RyR2) disappears after a few electrical stimulation pulses and in the steady state the Ca2+ transient is the same size as before adding caffeine.21,22 This occurs because in the steady state, during each heartbeat, the efflux of Ca2+ from the cell (largely via NCX) must exactly equal the influx (largely via LTCC). The amount of Ca2+ pumped out of the cell on NCX depends on both the properties of NCX and the amplitude and duration of the systolic Ca2+ transient. If we assume that neither the properties of NCX nor the duration of the Ca2+ transient is affected, a constant Ca2+ efflux per beat can occur only if the amplitude of the Ca2+ transient is constant. In other words, an increase in RyR2 Popen would not be expected to cause a sustained increase in the amplitude of the systolic Ca2+ transient. Indeed there are two situations in which a decreased Ca2+ transient may result. First, sufficient opening of the RyR2 may decrease the SR Ca2+ content to such a low level that even if all free Ca2+ were released, the Ca2+ transient may still be smaller than in control. Under these conditions, the decay of the Ca2+ transient will be slowed, as the SR would make a smaller contribution to relaxation. Second, if the opening of the RyR2 were increased during diastole, there might be a net diastolic leak of Ca2+ leading to a decrease in SR Ca2+ content and therefore a decrease in the amplitude of the Ca2+ transient. Such a diastolic Ca2+ leak has been suggested to contribute to decreased contractility in heart failure (HF) (Marx et al.17 and see below).
Evidence that eNOS-derived NO modulates RyR2
There is evidence suggesting that stretch-dependent stimulation of NO synthesis by myocardial eNOS may increase the RyR2 Popen. In particular, the stretch-dependent increase in the spontaneous Ca2+ spark rate (which is often used as an indirect measurement of RyR Popen) was abolished in eNOS-deficient cardiomyocytes (Figure 2).23 Furthermore, sustained stretch of wild-type cardiomyocytes was associated with Akt-dependent phosphorylation of eNOS and increased release of NO, which was abolished in eNOS-deficient mice.23 On the basis of these data, the authors suggested that eNOS-derived NO enhanced RyR2 Popen in response to stretch, thereby contributing to the increase in Ca2+ transient amplitude and developed contractile force that accompanies a sustained change in muscle length, known as the Anrep effect. However, it is not clear whether the increased spark frequency and transient amplitude produced by eNOS-derived NO are solely due to effects on RyR2 or whether other components of the Ca2+ handling system are involved. As mentioned above, selective stimulation of RyR2 would not be expected to cause a sustained increase in Ca2+ transient amplitude. The observation that SR Ca2+ content is unchanged under these conditions23 suggests that other Ca2+ fluxes might have been affected by stretch. For instance, previous work has shown that stretch activates the Na+/H+ exchanger (NHE)24 and this activation increases intracellular Na+, which in turn stimulates Ca2+ influx via the reverse mode of NCX.25,26
The pathway by which physical stretch is transduced into Akt activation and eNOS phosphorylation is not well defined. However, it has been proposed that the location of eNOS in plasmalemmal caveolae and its association with the scaffolding protein caveolin-32 may make eNOS particularly well placed to participate in this transduction process. Recent immunoprecipitation and immunofluorescence studies by Martinez-Moreno et al.27 suggest that a fraction of the total eNOS protein (63%) directly associates with RyR2 (Figure 3A), and that they may functionally interact. Evidence for the role of eNOS in modulating RyR function is not restricted to the heart. eNOS-derived NO from endothelial cells has been shown to increase Ca2+ spark frequency in smooth muscle cells from rat cerebral artery.28 Spark frequency decreased by about 50% following inhibition of eNOS or denudation of the endothelium, and increased 3- to 4-fold following application of the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (DEA-NONOate).
Evidence that nNOS-derived NO modulates RyR2
In 1999, Xu et al.3 first demonstrated the presence of a NOS1 gene product in cardiac SR vesicles and suggested that NO produced by this isoform may inhibit SR Ca2+ reuptake by SERCA2a. Subsequent work by Barouch et al.1 showed that murine heart protein extracts immunoprecipitated with the anti-RyR2 antibody exhibited immunoreactivity for nNOS, but not eNOS. A protein–protein link between nNOS and RyR2 has also been observed in the rat29 (Figure 3) and human30 myocardium. It has therefore been postulated1 that NO may achieve specificity of action by being released in the proximity of its target proteins. This concept may be especially pertinent to the myocardium where cytoplasmic diffusion of NO is likely to be limited as a result of scavenging by myoglobin.31 The fact, therefore, that nNOS and RyR2 co-immunoprecipitate suggests that the two proteins may form an interactive complex and that nNOS-derived NO could modulate RyR2 function. Equally, as nNOS is a Ca2+-activated enzyme,5 CICR from RyR2 would be expected to stimulate nNOS production of NO. Pinsky et al.’s10 work using a porphyrinic microsensor to measure the rise and fall of intracellular NO on a beat-by-beat basis supports this idea. Indeed, as NOS quickly desensitizes to sustained elevations in [Ca2+]i, transient increases in [Ca2+]i, such as during ECC, would maintain cyclical NOS activation.
Overall, studies in nNOS−/− mice suggest that, under basal conditions, nNOS-derived NO has negative or neutral inotropic action on the LV myocardium and a positive lusitropic effect.1,32–35 On the other hand, the positive inotropic response to β-adrenergic stimulation has been shown to be impaired in nNOS−/− mice in vivo.1,36 It should be noted, however, that such findings could be complicated by concomitant nNOS gene deletion from the central and peripheral nervous systems37,38 and from blood vessels.39,40 Loss of nNOS from sympathetic nerves, for example, may affect noradrenaline release and thus the β-adrenergic inotropic response, independent from the autocrine actions of nNOS in cardiomyocytes.38 That said, in isolated cardiomyocytes nNOS-derived NO might have a biphasic effect; i.e. to diminish the positive inotropic response to submaximal β-adrenergic stimulation and to enhance or maintain inotropy in response to high concentrations of β-adrenergic receptor agonists.1,32,35 Whether changes in RyR2 function play a role in the myocardial phenotype of mice with nNOS gene deletion or pharmacological inhibition remains to be assessed.
Direct modulation of RyR2 by S-nitrosylation
S-nitrosylation is the direct, covalent attachment of a NO moiety to a reactive thiol side chain on a cysteine amino acid, which can cause conformational and functional changes in proteins. S-nitrosylation is now thought to be involved in dynamic post-translational modification of most or all major classes of protein, including enzymatic proteins (metabolic enzymes, protein kinases and phosphatases, oxidoreductases, and proteases), those involved in cell signalling (receptors, ion channels and transporters, transcription factors, and G-proteins), and structural proteins (cytoskeletal elements); for reviews, see Stamler et al.41,42 Jaffrey et al.43 have identified a population of proteins that are endogenously S-nitrosylated, but which lose this post-translational modification in nNOS−/− mice. These proteins, which have structural, metabolic, and signalling functions, are therefore thought to represent targets for S-nitrosylation by nNOS-derived NO.
In 1997, Stoyanovsky et al.44 found that exogenous NO applied using the NO donor S-nitroso-N-acetyl penicillamine (SNAP) increased the Popen of RyR2 (and of the skeletal isoform, RyR1) reconstituted in synthetic planar lipid bilayers (Figure 4A and B). They found that this was a reversible process and that sulphydryl reducing agents promoted channel closure. Their results must, however, be interpreted with caution because application of NO donors does not confer the subcellular, local signalling of endogenously produced NO as described above. Furthermore, it is difficult to estimate whether the concentrations being applied are comparable to physiological levels. These complicating factors may help to explain the conflicting finding of Zahradnikova et al.45 who showed that addition of L-arginine to stimulate production of NO by eNOS (which co-purified with RyR2) reduced Popen of single RyR2 fused into planar lipid bilayers (Figure 4E and F). It is also possible that exposure of isolated RyR in lipid bilayers to NO donors may over-emphasise direct (cGMP-independent) effects of NO, whereas most of the well-defined actions of NO described to date are cGMP-dependent and require an intact intracellular milieu. Furthermore, the regulatory effects of both cytoplasmic and luminal Ca2+, kinases, phosphatases, phosphodiesterases (PDEs), and FKBP-12.6 may be disrupted in this preparation of RyR, and this in turn may affect the action of NO.
More recently, Stoyanovsky et al.’s44 findings have been supported by quantitative data from Xu et al.,46 who applied the NO donor S-nitrosoglutathione (GSNO) to RyR2 in planar lipid bilayers (Figure 4C and D). They calculated that RyR2 had 364 cysteine residues (89 per monomeric subunit, plus 2 per FKBP-12.6), of which ∼84 (or ∼21 per subunit) were free. They found that RyR2 activation required S-nitrosylation of many thiol residues. Modification of ∼2 sites per RyR2 was associated with minimal activation, while modification of ∼4 sites caused modest (∼50%) activation. Modification of ∼11 thiol sites per RyR2 (∼3 per subunit) was required to generate a 2- to 3-fold increase in channel activation. They concluded that RyR2 Popen was progressively increased as up to ∼3 thiol groups per RyR2 subunit were S-nitrosylated and that this was a reversible process during the cardiac cycle.46 Furthermore, they found that when the reducing agent dithiothreitol (DTT) was eliminated from the standard buffers used in the purification of RyR2 from canine hearts, the isolated channels contained endogenous S-nitrosothiol groups; whereas inclusion of DTT resulted in almost complete loss of S-nitrosylation of the protein.46 These findings strengthen the hypothesis that constitutive S-nitrosylation of RyR2 can occur and lead to modification of channel Popen under physiological conditions.
Subsequent work on skeletal muscle RyRs (RyR1) in synthetic lipid bilayers suggested that NO may have a concentration-dependent effect on RyR Popen.47 Hart and Dulhunty47 found that 10 µM SNAP caused a 2-fold increase in RyR channel Popen, whereas 1 mM SNAP, under the same conditions, resulted in a 2-fold reduction in Popen (but with a 1.5-fold increase in mean open time). Furthermore, Ziolo, et al.48 have shown that the level of PKA activation can also determine the effect of NO on RyR2 activity. Importantly, this was the first time that the action of NO on RyR2 Popen had been assessed in intact ventricular myocytes. They found that application of 300 µM of the NO donor spermine NONOate following low levels of PKA activation induced by 0.01 µM isoprenaline increased the spontaneous Ca2+ spark frequency. Conversely, exactly the same concentration of NO donor following 1 µM isoprenaline (i.e. high PKA activation) caused a reduction of the spontaneous Ca2+ spark frequency.48 The same results were obtained in the presence of a guanylate cyclase inhibitor, indicating that the effects of NO on RyR2 are cGMP-independent, and therefore likely to be via direct S-nitrosylation of the channel protein. The authors speculate that the PKA phosphorylation state of RyR2, together with PKA-dependent changes in SR Ca2+ load, will be important determinants of the functional effect of RyR2 S-nitrosylation. This may also be influenced by other important targets for NO and PKA, including phospholamban and LTCC.
Taken together, these data indicate that NO can have complex and dynamic effects on RyR2 Popen, depending on various other factors, such as the concentration of NO present and the level of β-adrenergic PKA activation. At low levels of PKA activation, NO increases RyR2 Popen and enhances the inotropic response; but at high PKA activation, NO diminishes RyR2 Popen—a mechanism that could be cardioprotective.
Few studies have investigated the action of nitroxyl (HNO) species, the 1-electron reduction product of NO (for review, see Fukuto et al.49). NO and HNO exist in chemical equilibrium, but oxidise thiols by different reaction mechanisms (S-nitrosylation vs. disulphide bond formation, respectively). Recent data indicate that HNO is an even more potent activator of RyR2 than NO (having significant effects at picomolar concentrations), and may contribute in vivo to NO’s effects on RyR2 Popen.50 HNO has also been shown to accelerate Ca2+ reuptake by SERCA2a and therefore to be effective at enhancing Ca2+ cycling with significant positive inotropic effects.51
Nitrosative vs. oxidative modulation of RyR2
In their paper from 1998, Xu et al.46 did not only show that increasing S-nitrosylation of up to 12 thiol sites per RyR2 produced progressive increases in channel Popen; they also demonstrated that oxidation of seven or more thiols per subunit generated irreversible channel activation. The interaction between reactive oxygen species (ROS) and reactive nitrogen species (RNS) has become a recurring theme in recent literature, to the extent that authors have referred to a ROS/RNS signalling axis.52
The enzyme xanthine oxidoreductase (XOR) can be a significant source of ROS, such as superoxide (O2−) and hydrogen peroxide (H2O2), which XOR produces as byproducts of the terminal steps of purine metabolism (reviewed by Harrison53). Scavenging of NO by O2− not only decreases the bioavailability of NO itself, it also generates a potent non-radical oxidant species, peroxynitrite (ONOO−). Recent work has shown that XOR can be a contributory source of NO, especially when hypoxic conditions prevent NOS from generating NO.54 It has been shown that NO production occurs in hypoxic rat myocardium in the presence of NOS inhibitors but not allopurinol (a XOR-selective inhibitor), implicating XOR in the synthesis of NO. XOR can generate NO by the reduction of both organic and inorganic nitrates and nitrites. It should be noted, however, that XOR enzyme activity appears to be much lower in humans than in many other mammals used in experiments (the difference between bovine and human XOR activity, for example, spans three orders of magnitude55). Experiments involving non-human mammals may therefore be at risk of overestimating the importance of XOR as a source of O2− if extrapolated to humans.
In mice, Khan et al.34 showed that XOR and nNOS co-localized in the SR, and furthermore that they co-immunoprecipitated (Figure 5A), suggesting a protein–protein interaction between the two enzymes. O2− production by XOR was ∼60% higher in nNOS−/− mice compared with wild-type controls suggesting that nNOS deficiency (but not eNOS deficiency) may stimulate myocardial O2− production by XOR. An alternative interpretation is that nNOS gene deletion reduces the availability of NO to scavenge XOR-derived O2−. It was also found that aspects of the myocardial positive force-frequency response (FFR) attenuation previously observed in nNOS−/− mice at high pacing frequencies56 could be restored following incubation with allopurinol34 (Figure 5B and C). The authors originally proposed that the absence of nNOS from the SR caused diastolic Ca2+ leak through RyR2. This could be an effect of uninhibited or unscavenged O2− production by XOR causing increased oxidative stress and irreversible activation of RyR2.46 However, this theory does not explain why FFR attenuation in nNOS−/− should increase at higher stimulation frequencies when diastolic interval (and therefore the opportunity for diastolic Ca2+ leak) is shorter. Ziolo and Bers57 suggested that a decrease in SR Ca2+ load at higher stimulation frequencies would be more likely to result from decreased Ca2+ reuptake through SERCA2a, probably secondary to a reduction in phospholamban phosphorylation in the nNOS−/− myocardium.58 Moreover, while the application of allopurinol returned sarcolemma length shortening in nNOS−/− myocytes to wild-type levels, it did not restore the Ca2+ transient amplitude or SR Ca2+ load (Figure 5D).34 Increased cell shortening without a change in systolic Ca2+ suggests that the allopurinol is correcting a XOR-derived, O2−-mediated reduction in myofilament Ca2+ sensitivity. This is in agreement with earlier work in which allopurinol and oxypurinol were shown to improve the force generated by stunned cardiac myofilaments, while at the same time reducing systolic Ca2+ transients, thereby enhancing myofilament Ca2+ sensitivity.59 But again, this does not explain why the FFR attenuation in nNOS−/− mice occurred only at high stimulation frequencies.
Other major sources of redox stress are the nicotinamide-adenine dinucleotides NAD+, NADH, and NADPH. The regulatory effects of these molecules on cardiac and skeletal isoforms of RyR incorporated into planar lipid bilayers were investigated by Zima et al.60 Interestingly, they found that NADH decreased cardiac RyR2 Popen and that this was counteracted by NAD+, but conversely that both NADH and NAD+ increased the skeletal RyR1 Popen. No effect was found for NADPH on either RyR isoform.60 In a subsequent paper, they confirmed the finding that NADH significantly lowered the Ca2+ spark frequency in rat permeabilized cardiomyocytes.61 Interestingly, they also found that application of just 5 µM of spermine NONOate at low PO2 produced 93% inhibition of NADH oxidase activity. Therefore, both RyR2 and NADH oxidase (which co-purifies with SR membrane) appear to be regulated by NO. The direct activating effect of NO on RyR246 is augmented by simultaneous inhibition of NADH oxidase, which relieves the negative modulation of NADH on RyR2.61 Furthermore, the authors have shown that in addition to NADH directly causing an 84% reduction in RyR2 activity, there also appears to be an indirect (O2−-mediated) NADH inhibition of SERCA2a activity.62 Finally, it has recently been shown that local production of O2− by NAD(P)H oxidase on the SR of coronary artery smooth muscle cells is involved in regulating CICR from the RyR.63 It should be noted that the mitochondrion, being an important source of both NO and ROS, is also a key modulator of the myocardial redox state (for a review, see Turrens64).
Indirect, cGMP-dependent modulation of RyR2
So far, direct (poly-)S-nitrosylation has been the main mechanism by which NO has been considered to modulate RyR2 Popen. S-nitrosylation of the skeletal RyR1 isoform is thought to occur at cysteine-3635.65 This residue is localized to the calmodulin-binding domain of RyR1, and the covalent binding of NO to cysteine-3635 reverses the inhibitory effect of calmodulin on the channel.27 However, the classical second messenger pathway involving soluble guanylate cyclase—cGMP—protein kinase G (PKG) should not be discounted. PKG has been shown to be capable of phosphorylating RyR in vitro, 66 but whether this forms part of a physiological pathway in vivo is unknown and likely to be underestimated by experiments in isolated RyR preparations.
Cyclic adenosine diphosphate ribose (cADPR) is thought to be an endogenous modulator of RyR2, and has been shown to increase Popen, and the gain of the CICR process in rat and guinea-pig ventricular myocytes.67 Furthermore, PKG phosphorylates and activates ADP-ribosyl cyclase (the enzyme that synthesises cADPR from β-NAD+in vivo) or a regulator of the enzyme, in a NO- and cGMP-dependent manner.68,69 cGMP can also inhibit or activate PDEs that control the concentration of cAMP70 and thus PKA-mediated phosphorylation. Marx et al. suggested that RyR2 phosphorylation by PKA occurs in response to β-adrenergic stimulation and increases RyR2 Popen. They also proposed that excessive phosphorylation of RyR2 (hyperphosphorylation) contributed to RyR2 dysregulation and diastolic SR Ca2+ leak in HF.17,71–73 However, other groups have disputed these findings. Li et al. showed that changes in Ca2+ spark frequency and amplitude observed after β-adrenergic stimulation were due to an increase in SR Ca2+ content and not to direct stimulation of RyR2.74 Two other groups have failed to detect hyperphosphorylation of RyR2 by PKA in the failing heart.75 In summary, it is unclear whether PKA phosphorylation of RyR2 plays a role in the modulation of RyR2 function and whether this can be affected by cGMP.
Changes in NO modulation of RyR2 in the failing heart
In the failing heart, there is evidence that the physical association between nNOS and RyR2 may decrease. This was first shown in an experimental model of myocardial infarction (MI) in senescent rats.76 Extracts from non-infarcted hearts showed no immunoreactivity between nNOS and caveolin-3, the scaffolding protein localized to caveolae in the sarcolemma and usually associated with eNOS.2 After MI, however, the amount of nNOS/cav-3 complexes was dramatically increased. Migration of nNOS from its association with RyR2 on the SR in the healthy myocardium to a localization with caveolin-3 on the sarcolemma post-MI has subsequently been seen in a rat model of MI-induced HF29 (Figure 3) and in the human failing myocardium.30 Translocation of nNOS from the SR to caveolae may have dual effects on myocardial contractility. First, increased NO production in caveolae may augment feedback inhibition on LTCC,33 whereas secondly, decreased local NO at the SR may disrupt normal regulation of RyR2 or SERCA2A. Loss of nNOS-derived NO from the SR may not only cause decreased reversible activation of RyR2 by S-nitrosylation;46 it may also remove inhibition of XOR, allowing increased O2− production. Furthermore, it has been shown that XOR expression is upregulated in the failing heart,77 and the resulting oxidative stress may enhance the risk of unregulated and irreversible RyR2 activation by O2−, diastolic Ca2+ leak, and depletion of the SR Ca2+ stores. All these changes are likely to depress cardiac contractility and contribute to the phenotype of HF.
In summary, there is increasing evidence suggesting that RyR2 Popen may be regulated by NO derived from both eNOS (under conditions of myocardial stretch)23 and nNOS. RyR2 and nNOS co-immunoprecipitate,29 RyR2 is constitutively S-nitrosylated,46 and NO has been shown to modulate RyR2 Popen (in a PKA activity- and concentration-dependent manner).47,48 It is also increasingly likely that RNS and ROS interact. For a long time NO has been known to scavenge O2− and produce ONOO−. More recently, data have emerged that nNOS and XOR (a major source of O2−) physically interact in the murine LV myocardium34 and that nNOS-derived NO has an inhibitory, and therefore cardioprotective, effect on O2− generation by XOR.52 This may be significant in the failing heart, where it has been shown that nNOS translocates from the SR to the sarcolemma,29 thus potentially removing the inhibition on XOR and allowing irreversible activation of RyR2 by O2−.46 Of course, XOR is not the only relevant source of redox stress in the heart. NO has also been shown to inhibit NADH oxidase and thereby reduce the inhibitory effect of NADH on RyR2.61 The interplay between these proteins is important in order to understand the potential therapeutic benefits of, for example, administering NO donors, HNO donors, or XOR inhibitors in HF.
The authors’ work is supported by the British Heart Foundation.
Conflict of interest: none declared.