Nitroxyl (HNO) Stimulates Soluble Guanylyl Cyclase to Suppress Cardiomyocyte Hypertrophy and Superoxide Generation

Background New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated. Methods Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli's salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. Results We now demonstrate that Angeli's salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli's salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli's salt were mimicked by BNP. We also demonstrate that the effects of Angeli's salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP). Conclusions Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.


Introduction
Cardiac hypertrophy is strongly implicated in the development of heart failure of almost all etiologies. In addition to heart failure, it remains an independent risk factor for myocardial infarction and sudden death [1][2][3]. Cardiac hypertrophy initially develops in vivo as an adaptive response to maintain myocardial function, for example in hypertension when cardiac workload is chronically elevated [4]. Individual cardiomyocytes hypertrophy, accompanied by re-expression of embryonic genes, a switch in prevalence of contractile protein expression from ato b-myosin heavy chain and sarcomeric organization [2,3]. Ultimately, hypertrophy may progress to a maladaptive state, with progressive decline in ventricular contractility and diastolic function, with adverse outcomes [4,5]. Current therapies (e.g. renin-angiotensin system inhibition) slow progression of cardiac hypertrophy, but patients still die with enlarged hearts. Identification of new therapeutic targets to prevent or reverse cardiac hypertrophy is essential [3].
We and other have shown that the nitroxyl anion, NO 2 , the one electron reduction product of NO N , is a novel regulator of cardiovascular function [3,[6][7][8][9][10][11][12][13][14][15][16]. At physiological pH, nitroxyl exists predominantly in the protonated form as HNO [12]. Similar to NO N , HNO mediates potent vasodilatation, largely via sGC activation and an elevation in cGMP [6,7]. In direct contrast to NO N however, HNO also elicits a marked inotropic effect (independent of cGMP), that persists even in failing myocardium in vivo [3,8,9]. Other distinct advantages offered by HNO include its lack of reactivity with reactive oxygen species (ROS) [14,[17][18][19], an absence of tolerance development [15,20] and a direct interaction with thiols [10][11][12]. Much of this evidence has been obtained using the HNO donor, Angeli's salt (sodium trioxodinitrate, Na 2 N 2 O 3 ), which releases both HNO and nitrite [3,12]. Cardiac HNO actions (in contrast to those of NO N ) may thus well be preserved under conditions of oxidative stress (e.g. cardiac hypertrophy and heart failure) [3,13]. With these therapeutic advantages, HNO donors are now in development for clinical management of acute heart failure events.
cGMP-dependent signaling is a powerful antihypertrophic and ROS-suppressing mechanism in the heart; much of this work has emanated from our own studies [3,[21][22][23][24][25][26][27][28]. Exploiting cGMP for the treatment of hypertrophy and heart failure via conventional NO N donors is limited however by the rapid reaction of ROS with NO N to form peroxynitrite and impairing NO N bioavailability [3]. Given the ability of HNO to stimulate sGC even in settings of elevated ROS, we now test the hypothesis that HNO elicits cGMP-dependent antihypertrophic effects in neonatal rat cardiomyocytes. Further, given that HNO elicits antioxidant actions in yeast, cell-free systems and in vascular tissues [13,29], the impact on cardiomyocyte NADPH oxidase was also determined. BNP, a cGMP-elevating agent with known antihypertrophic efficacy [21,24,26], was used for comparison. Our results provide the first evidence that the HNO donor Angeli's salt prevents cardiomyocyte hypertrophy, and that cGMP-dependent suppression of cardiomyocyte NADPH oxidase contributes to these antihypertrophic actions.

Materials and Methods
This investigation conforms with both the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996) and the National Health and Medical Research Council of Australia guidelines, and was approved by the Alfred Medical, Research and Education Precinct (AMREP) Animal Ethics Committee (approval E/0698/2008/B). All materials were purchased from Sigma-Aldrich (St. Louis, USA) except where indicated, and were of analytic grade or higher.

Hypertrophic responses in primary neonatal rat cardiomyocytes
Hearts were collected from 1-to 2-day-old neonatal rat pups, promptly after euthanasia by decapitation. Cardiomyocytes were then isolated, and plated at a density of 1610 3 cells/mm 2 for determination of all measures except two-dimensional (2D) cardiomyocyte size, in which cells were plated at a density of 2610 2 cells/mm 2 (to permit delineation of defined single cells, as previously described) [21]. All materials used for cardiomyocyte isolation were of tissue culture grade. Following 48 h incubation under serum-free conditions, cardiomyocytes were incubated for 48 h in the presence and absence of the hypertrophic stimuli angiotensin II (Ang II, 1 mmol/L, Auspep, Parkville, Australia) or endothelin-1 (ET 1 , 60 nmol/L) [21,26,30], and/or the HNO donor Angeli's salt (sodium trioxodinitrate, 1 mmol/L unless otherwise stated [6], added 46/day to compensate for its shorter half-life, Cayman chemicals, Michigan, USA). BNP (1 mmol/L, Auspep) and the stable cGMP analog 8-bromo-cGMP (8BrcGMP, 1 mmol/L) were used for comparison [21,26]. Concentrations of all drugs studied were based on those previously reported, as indicated. The vehicle control for Angeli's salt, 0.01 mol/L NaOH [15], was incorporated into the study design, and was also added 46/day. Markers of cardiomyocyte hypertrophy included 2D area (mm 2 ) of live cells (30 individual myocytes measured per treatment), de novo protein synthesis (determined via incorporation of [ 3 H]phenylalanine, Amersham Biosciences, Castle Hill, Australia), 4 replicates per treatment), and expression of the prohypertrophic gene, b-myosin heavy chain, as previously described [21,22,26,30]. Real time PCR reagents were all of molecular biology grade, and included TaqmanH reverse transcription reagents, TaqmanH Universal PCR master mix, DNase treatment kits, fluorogenic probes (Applied Biosystems, Scoresby, Australia), as well as forward and reverse primers for real-time PCR (Geneworks, Thebarton, Australia).

Triggers of cardiomyocyte hypertrophy
The impact of Angeli's salt on key triggers of pathological hypertrophy included cardiomyocyte expression of the Nox2 . This is evident on A cardiomyocyte area (n = 10 myocyte preparations); B de novo protein synthesis (on [ 3 H]phenylalanine incorporation, n = 9 myocyte preparations); and C hypertrophic gene expression (using the fetal isoform of the contractile protein, b-myosin heavy chain, n = 6 myocyte preparations). *P,0.05 and ***P,0.001 vs control; # P,0.05 and ### P,0.001 vs Ang II alone. doi:10.1371/journal.pone.0034892.g001 subunit of NADPH oxidase, superoxide generation, and phosphorylation of p38MAPK, as previously described [21,31]. In addition, phosphorylation of the cell survival kinase Akt and its downstream target glycogen synthase kinase-3b (GSK-3b, as well as of the mitogen-activated protein kinase ERK1/2, were also determined [30]. For determination of Nox2 expression, cells were incubated for 48 h in the presence and absence of Ang II or ET 1 , and/or Angeli's salt (replenished 46/day). Relative quantification of changes in cardiomyocyte expression of the Nox2 subunit of NADPH oxidase (a major source of ROS), was determined using real time PCR analysis, with 18S as the endogenous control, as previously described [21,31]. Cardiomyocyte superoxide generation was determined using NADPH-driven lucigenin-enhanced chemiluminescence, an estimate of NADPH oxidase activity, as previously described [21,31,32]. Cells were incubated for 48 h in the presence or absence of Angeli's salt, BNP, 8BrcGMP, with Ang II or ET 1 , added for the final 24 h. Each measurement was expressed as relative light units per second (RLU/sec). Background luminescence (in the absence of cells) was subtracted from the average of 8 readings. Each experiment was studied with at least 4 replicates, and the average result was taken. In a separate series of experiments, cardiomyocyte activation of the mitogen-activated protein kinases ERK1/2 and p38MAPK, as well as phosphorylation of Akt and glycogen synthase kinase-3b (GSK-3b, were determined in the presence or absence of Angeli's salt for 48 h; Ang II was added only for the final 10 min. Western analyses used phospho-specific antibodies (Cell Signaling Technology, Danvers, MA), as previously described [30,32].

HNO/sGC/cGMP signaling
The role of sGC and cGK-I in mediating the actions of Angeli's salt in cardiomyocytes was determined using the selective inhibitors, ODQ (1 mmol/L) [15] and KT5823 (250 nmol/L, Calbiochem-Novabiochem, La Jolla, CA) [21,26], respectively. The vehicle control for KT5823 and ODQ (0.01% DMSO) was also incorporated into study design. The impact of Angeli's salt on cardiomyocyte protein levels of cGK-I and sGC (48 h incubation), and phosphorylation of VASP (10 min incubation, a biomarker of cGK-I signaling) were determined, via Western analysis, using primary antibodies from Cell Signaling Technology. Cell-free purified sGC activity was determined by conversion of GTP (40 mmol/L, Sigma) to cGMP by sGC (34 ng, Alexis Biochemicals, San Diego, CA) over 10 min, in the presence and absence of Angeli's salt [33]. Cardiomyocyte cGMP generation was also, determined via enzyme immunoassay (Cayman Chemical) following 5 and 15 min incubation with either Angeli's salt or BNP, as previously described [21].
The relative roles of HNO and NO N in the actions of Angeli's salt were determined firstly on generation of NO N using an NO N -sensing electrode (World Precision Instruments, Sarasota, FL) in the presence and absence of Angeli's salt, and results compared to the pure NO N donor, DEA/NO (both 0.1-30 mmol/L, Cayman Chemical) [15]. Subsequent studies used the selective scavengers, L-cysteine (3 mmol/L, for HNO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(carboxy-PTIO, 200 mmol/L, for NO N ) [15]. In addition, the potential cGMP-elevating and antihypertrophic effects of both sodium nitrite (1 mmol/L) and degraded Angeli's salt (1 mmol/L, replenished 46/day, obtained by storing Angeli's salt solution at room temperature for 48 h, followed by 2 h at 37uC, prior to use), was determined.

Statistical analysis
All results were expressed as mean 6 standard error for each treatment group, with the number of myocyte preparations studied denoted by ''n''. Changes in [ 3 H]phenylalanine incorporation, 2D cardiomyocyte size, superoxide generation and cGMP content were expressed as a percentage of paired control cardiomyocytes from the same preparation. For changes in both gene expression (b-myosin heavy chain, Nox2) and protein (ERK1/2, p38MAPK, Akt, GSK-3b, sGC, cGK-I, P-VASP), results were expressed as a fold of paired control. Statistical comparison of $3 different experimental groups was performed using one way repeated measures analysis of variance to compare the effect of Ang II with paired control, or of antihypertrophic interventions (e.g. Angeli's salt in the presence of Ang II) with Ang II alone, where n$4. The Student-Newman-Keuls correction for pairwise multiple comparisons was applied where required. Where the experiment only used 2 groups, the effect of Angeli's salt alone versus control was compared using paired ttests. Results with P values,0.05 were considered statistically significant.

Antihypertrophic actions of Angeli's salt in neonatal cardiomyocytes
The hypertrophic stimulus, Ang II (1 mmol/L), induces hypertrophic responses in neonatal rat cardiomyocytes, increasing 2D area to 184612% of control ( Figure 1A, P,0.001), de novo protein synthesis ([ 3 H]phenylalanine incorporation) to 14669% ( Figure 1B, P,0.001), and b-myosin heavy chain expression to 2.360.4-fold of paired control myocytes ( Figure 1C, P,0.05). The HNO donor Angeli's salt (1 mmol/L, added 46/day over 48 h) exerts marked anti-hypertrophic actions, virtually abolishing the Ang II-induced increases in 2D area ( Figure 1A, P,0.001 vs Ang II alone), protein synthesis ( Figure 1B, P,0.001 vs Ang II alone) and hypertrophic gene expression ( Figure 1C, P,0.05 vs Ang II alone). Angeli's salt alone does not significantly affect 2D area, protein synthesis or b-myosin heavy chain in neonatal cardiomyocytes. Further, as shown in Table 1, the NaOH vehicle used for Angeli's salt does not significantly affect cardiomyocyte responses, either alone or in the presence of Ang II (Table 1). Angeli's salt suppresses ROS generation in neonatal cardiomyocytes Ang II significantly increases NADPH-driven cardiomyocyte superoxide generation 2.560.4 fold paired control (Figure 2A, P,0.05). Angeli's salt completely prevents Ang II induction of cardiomyocyte superoxide (Figure 2A, P,0.05 vs Ang II alone). Ang II-induced increases in Nox2 expression (to 3.860.5-fold of control) are also completely abolished by Angeli's salt ( Figure 2B, P,0.001 vs Ang II alone). Angeli's salt alone does not significantly affect either parameter.

The actions of Angeli's salt are mediated via HNO
To confirm that the actions of Angeli's salt are mediated via HNO rather than nitrite (the other metabolite of Angeli's salt) or extracellular oxidation of HNO to NO N , we demonstrate that neither sodium nitrite nor degraded Angeli's salt (both 1 mmol/L) elicit significant inhibition of Ang II-stimulated cardiomyocyte hypertrophy ( Figure 7A). The cardiomyocyte actions of intact Angeli's salt are completely prevented by the HNO-selective scavenger, L-cysteine (3 mmol/L, Figure 7B, P,0.05) but are unaffected by the NO N -selective scavenger, carboxy-PTIO (200 mmol/L). Furthermore, neither sodium nitrite nor degraded Angeli's salt (both 1 mmol/L) elicit significant impact on cardiomyocyte cGMP levels after 15 min, in direct contrast to paired cardiomyocytes treated with Angeli's salt ( Figure 7C, both n = 5 cardiomyocyte preparations and P = NS vs control). Lastly, under our cell culture conditions, Angeli's salt fails to generate NO N , even at concentrations 30-fold higher than that used in the present study ( Figure 7D). By contrast, DEA/NO (0.1-30 mmol/ L) releases significant amounts of NO N in a concentrationdependent manner ( Figure 7D). Together these data indicate that

Discussion
The major finding to emerge from this study is the first evidence that an HNO donor potently blunts cardiomyocyte hypertrophy. Angeli's salt prevents all of the hypertrophic actions of Ang II in neonatal cardiomyocytes in vitro, including Ang II-induced increases in cell area, de novo protein synthesis and hypertrophic gene expression on b-myosin heavy chain analysis. Ang II-induced increases in cardiomyocyte NADPH oxidase expression (of the sarcolemmal Nox2 subunit) and activity (superoxide generation), as well as activation of p38MAPK, both implicated as triggers of the cardiomyocyte hypertrophic response in vitro, are also blunted by the HNO donor. The HNO donor is equally effective at blunting prohypertrophic and pro-oxidant responses, regardless of the hypertrophic stimulus (Ang II vs ET 1 ). Further, no role for extracellular oxidation of HNO to NO N , or of nitrite, in these actions was evident. The cGMP system is a powerful antihypertrophic mechanism in the heart [3,[23][24][25][26][27][28], and like NO N , the vascular actions of HNO appear to be mediated predominantly via the activation of sGC and a subsequent increase in cGMP [6][7][8]12]. We now provide evidence that the HNO donor Angeli's salt elevates cardiomyocyte cGMP and directly activates sGC activity. Both the antihypertrophic and superoxide-suppressing effects of Angeli's salt are sensitive to both sGC and cGK-I inhibition. These findings confirm cGMPdependence of these cardiac actions of HNO.
Angeli's salt is considered a classical HNO donor [3,12]. It dissociates at physiological pH and temperature to yield HNO and nitrite (NO 2 2 ) [12]. Although nitrite is capable of stimulating sGC-dependent vasorelaxation [34], it is at least 15,000-fold less potent a vasodilator as Angeli's salt [6,16], and only lowers blood pressure in rats in vivo at high concentrations (0.3-1.0 g/kg body weight) [34]. Based on our studies with sodium nitrite and degraded Angeli's salt; we now report that nitrite has negligible effects in cardiomyocytes and thus is unlikely to mediate the antihypertrophic actions of Angeli's salt. Under certain conditions (cell-free, in the absence of oxygen), higher concentrations of Angeli's salt than utilized in the present study (10 mmol/L) has been reported to also result in some generation of NO N [16], likely via oxidation of HNO to NO N by Cu 2+ or Cu 2+ -containing enzymes (intracellular or extracellular) [12,34,35]. Whilst we cannot exclude the possibility of intra-cardiomyocyte oxidation of HNO to NO N in our studies, we demonstrate that extracellular oxidation of HNO does not occur under our experimental conditions, as even at 30 mmol/L, no detectable NO N is generated, in accordance with previous observations in the vasculature [6]. In addition, we show that cardiomyocyte responses to Angeli's salt are significantly attenuated by the selective HNO scavenger Lcysteine, but are completely unaffected by the NO N scavenger carboxy-PTIO, analogous to its vasorelaxation responses [6,7]. The sensitivity of Angeli's salt to the HNO scavenger lends further support to HNO (rather than nitrite or NO N ) being the responsible entity for cardiomyocyte effects. Given that HNO (in contrast to NO N ) is resistant to scavenging by ROS [12,[17][18][19], Angeli's salt retains its advantage over NO N donors for limiting cardiomyocyte hypertrophy, particularly in settings of elevated ROS generation.
In the present study, we demonstrate that an HNO donor prevents cardiomyocyte hypertrophy via cGMP-dependent mechanisms that included suppression of NADPH oxidase. Such findings provide the first evidence that HNO exerts actions in the myocardium via the cGMP signaling pathway. As superoxide plays a pivotal role in triggering the hypertrophic response in the intact heart in vivo, and in cardiomyocytes in vitro [4], HNO/cGMP are an attractive antihypertrophic strategy [3,21]. Our finding that the HNO donor Angeli's salt suppresses superoxide production and NADPH oxidase induction, is further evidence of HNO superoxide-suppressing actions in mammalian cells. Given that this action was mimicked by BNP and 8-BrcGMP, cGMP-generating agents thus appear to mediate their actions, at least in part, by suppressing cardiomyocyte NADPH oxidase expression and/or activity. The potential mechanism(s) of this action (e.g. cGK-mediated phosphorylation of Nox2) warrant further investigation.
Downstream of ROS, p38MAPK activation is a critical mediator of pathological cardiomyocyte hypertrophy induced by neurohumoral activation [2,3,36,37]; cardiomyopathy often results. In contrast, Akt promotes physiological hypertrophy and prevents apoptosis [2,38]. A similar role for ERK1/2 in cardiomyocyte survival and physiological hypertrophy has been proposed [2,39]. Although Ang II-and ET 1 -induced cardiomyocyte ERK1/2 activation is often evident in vitro [2,3,30,39], ERK1/2 does not contribute to pathological hypertrophy in vivo [40]. Interestingly, Angeli's salt selectively blunts Ang II-induced phosphorylation of p38MAPK. As shown in Figure 4, the actions of Angeli's salt are dependent on cGMP/cGK-I. MAPK phosphatase-1 (MKP-1) dephosphorylation of p38MAPK lies immediately downstream of cGK-I [3,21]. It is thus possible that Angeli's salt enhances MKP-1 activity, to mediate the reduced p38MAPK phosphorylation observed here. Ang II-induced phosphorylation of both Akt and ERK1/2 however remained elevated after treatment with the HNO donor, despite normalization of three distinct parameters of cardiomyocyte hypertrophy (cardiomyocyte size, protein synthesis and hypertrophic gene expression). Given the cardioprotective properties of Akt [3,38], the ability of HNO to inhibit cardiomyocyte hypertrophy in the face of preserved Akt signaling is a desirable trait.
The antihypertrophic effects of HNO are markedly attenuated by KT5823 or ODQ. Further, cardiomyocyte superoxide upregulation, a key trigger of cardiomyocyte hypertrophy [3,21,36], is completely prevented by both KT5823 and ODQ, leaving no residual cGMP-independent HNO actions. Although it is possible that the modest, apparently residual, component of the antihypertrophic actions of Angeli's salt that are not accounted for by KT5823 or ODQ could be due to calcitonin gene-related peptide (CGRP), this is probably unlikely. CGRP has been identified as a mediator of a component of HNO vasorelaxation [7]. The effects of CGRP on cardiac hypertrophy remain to be resolved however, with pro-hypertrophic effects observed in vitro [41] and antihypertrophic effects in vivo [42]. Further, superoxide is a key trigger of cardiomyocyte hypertrophy [3,21], yet there was no parallel residual component of cardiomyocyte superoxide levels in Ang II-treated myocytes not prevented by KT5823 or ODQ. Robust sGC-independent cardiovascular actions of HNO linked to its reactivity with thiols include ryanodine receptors, protein Nnitrosation and S-glutathiolation, and activation of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) [10][11][12]43]. Indeed, acute supra-pharmacological concentrations of Angeli's salt (up to 500fold of those used here) directly activate SERCA, via Sglutathiolation at cysteine residue 674 [43] and disulfide bond formation on phospholamban (preventing its inhibition of SERCA) [44]. Thus, HNO-selective, thiol-mediated interactions independent of cGMP likely explain the inotropic and lusitropic actions of Angeli's salt [11,43]. Our findings show in contrast that the antihypertrophic actions of HNO in contrast are critically dependent on cGMP. Exploiting the cGMP antihypertrophic mechanism with chronic clinical use of traditional nitrovasodilators in the management of patients suffering hypertrophy/failing cardiac pathologies is limited, firstly by the phenomenon of ''nitrate tolerance'' [45]. In addition superoxide, generated in excess amounts in cardiac hypertrophy and failure, rapidly reacts with NO [3]. HNO donors offer considerable advantage over traditional NO N donors as the redox siblings exhibit quite distinct pharmacology, both in vitro and in vivo. HNO donors neither exhibit cross-tolerance with organic nitrates (e.g. glyceryl trinitrate), nor do they induce tolerance to their own actions [20]. In addition, unlike NO N , HNO is resistant to scavenging by ROS [17][18][19]. Whilst the preference of HNO for Fe 3+ -versus Fe 2+ -heme groups [46] was initially thought to potentially permit HNO activation of the NO N -insensitive, oxidized form of sGC; this concept has now been refuted [35,47]. Importantly, HNO elicits hemodynamic effects favorable in settings of cardiac remodeling and failure. This includes a marked positive inotropic effect that is both load-and reflexindependent, and persists even in failing myocardium in vivo. Moreover, HNO potentiates b-adrenergic inotropic responses in the failing heart [8,9,48]. These observations are all in direct contrast to conventional nitrovasodilators [9,10,17,20].

Limitations of the study
Our detailed investigation of the antihypertrophic actions of Angeli's salt and BNP, and the insights obtained into their mechanisms of action, were performed in a single cardiomyocyte strain and phenotype. The large majority of in vitro studies addressing cardiomyocyte hypertrophy similarly use neonatal rat cardiomyocyte preparations [21,22,28,49,50]. The antihypertrophic actions of Angeli's salt in adult cardiomyocytes may warrant further investigation. Cardiomyocytes isolated from adult mouse hearts are obtained in too few numbers, with too limited a timeframe of viability. We have previously demonstrated however that the antihypertrophic actions of BNP, like those of other cGMP-dependent antihypertrophic interventions, are observed in adult rat cardiomyocytes and/or the intact heart [22][23][24][25][26]. Hypertrophic responses in these settings are studied over a much shorter time-frame (2 h) and thus preclude assessment of changes in cell size.

Concluding remarks
We now propose that stimulators of sGC that are not susceptible to ROS-mediated inactivation, and indeed suppress cardiomyocyte ROS generation, represent a superior approach to exploiting the antihypertrophic actions of the sGC/cGMP system in the heart. In conclusion, the present study suggests that HNO prevents acute cardiac hypertrophic responses (up to 48 h); cGMPdependent suppression of cardiomyocyte NADPH oxidase and p38MAPK (key triggers of the hypertrophic response) likely contribute to these antihypertrophic actions (illustrated in Figure 8). Our findings indicate that longer-term studies of the antihypertrophic effects of this new class of agent in vivo are warranted. Given these potent antihypertrophic and superoxidesuppressing actions shown here, together with their established positive inotropic and vasodilatory actions, HNO donors may hence form the basis of more effective therapeutics for the clinical management of cardiac hypertrophy, alone or in combination with standard care.