PKG1α Cysteine-42 Redox State Controls mTORC1 Activation in Pathological Cardiac Hypertrophy.

RATIONALE: Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. OBJECTIVE: We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. METHODS AND RESULTS: Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. CONCLUSIONS: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Adenosine kinase attenuates cardiomyocyte microtubule stabilization and protects against pressure overload-induced hypertrophy and LV dysfunction.

Adenosine exerts numerous protective actions in the heart, including attenuation of cardiac hypertrophy. Adenosine kinase (ADK) converts adenosine to adenosine monophosphate (AMP) and is the major route of myocardial adenosine metabolism, however, the impact of ADK activity on cardiac structure and function is unknown. To examine the role of ADK in cardiac homeostasis and adaptation to stress, conditional cardiomyocyte specific ADK knockout mice (cADK-/-) were produced using the MerCreMer-lox-P system. Within 4 weeks of ADK disruption, cADK-/- mice developed spontaneous hypertrophy and increased ß-Myosin Heavy Chain expression without observable LV dysfunction. In response to 6 weeks moderate left ventricular pressure overload (transverse aortic constriction;TAC), wild type mice (WT) exhibited ~60% increase in ventricular ADK expression and developed LV hypertrophy with preserved LV function. In contrast, cADK-/- mice exhibited significantly greater LV hypertrophy and cardiac stress marker expression (atrial natrurietic peptide and ß-Myosin Heavy Chain), LV dilation, reduced LV ejection fraction and increased pulmonary congestion. ADK disruption did not decrease protein methylation, inhibit AMPK, or worsen fibrosis, but was associated with persistently elevated mTORC1 and p44/42 ERK MAP kinase signaling and a striking increase in microtubule (MT) stabilization/detyrosination. In neonatal cardiomyocytes exposed to hypertrophic stress, 2-chloroadenosine (CADO) or adenosine treatment suppressed MT detyrosination, which was reversed by ADK inhibition with iodotubercidin or ABT-702. Conversely, adenoviral over-expression of ADK augmented CADO destabilization of MTs and potentiated CADO attenuation of cardiomyocyte hypertrophy. Together, these findings indicate a novel adenosine receptor-independent role for ADK-mediated adenosine metabolism in cardiomyocyte microtubule dynamics and protection against maladaptive hypertrophy.

Acute Enhancement of Cardiac Function by Phosphodiesterase Type 1 Inhibition.

BACKGROUND: Phosphodiesterase type-1 (PDE1) hydrolyzes cAMP and cGMP and is constitutively expressed in the heart, although cardiac effects from its acute inhibition in vivo are largely unknown. Existing data are limited to rodents expressing mostly the cGMP-favoring PDE1A isoform. Human heart predominantly expresses PDE1C with balanced selectivity for cAMP and cGMP. Here, we determined the acute effects of PDE1 inhibition in PDE1C-expressing mammals, dogs, and rabbits, in normal and failing hearts, and explored its regulatory pathways. METHODS: Conscious dogs chronically instrumented for pressure-volume relations were studied before and after tachypacing-induced heart failure (HF). A selective PDE1 inhibitor (ITI-214) was administered orally or intravenously±dobutamine. Pressure-volume analysis in anesthetized rabbits tested the role of ß-adrenergic and adenosine receptor signaling on ITI-214 effects. Sarcomere and calcium dynamics were studied in rabbit left ventricular myocytes. RESULTS: In normal and HF dogs, ITI-214 increased load-independent contractility, improved relaxation, and reduced systemic arterial resistance, raising cardiac output without altering systolic blood pressure. Heart rate increased, but less so in HF dogs. ITI-214 effects were additive to ß-adrenergic receptor agonism (dobutamine). Dobutamine but not ITI-214 increased plasma cAMP. ITI-214 induced similar cardiovascular effects in rabbits, whereas mice displayed only mild vasodilation and no contractility effects. In rabbits, ß-adrenergic receptor blockade (esmolol) prevented ITI-214-mediated chronotropy, but inotropy and vasodilation remained unchanged. By contrast, adenosine A2B-receptor blockade (MRS-1754) suppressed ITI-214 cardiovascular effects. Adding fixed-rate atrial pacing did not alter the findings. ITI-214 alone did not affect sarcomere or whole-cell calcium dynamics, whereas ß-adrenergic receptor agonism (isoproterenol) or PDE3 inhibition (cilostamide) increased both. Unlike cilostamide, which further enhanced shortening and peak calcium when combined with isoproterenol, ITI-214 had no impact on these responses. Both PDE1 and PDE3 inhibitors increased shortening and accelerated calcium decay when combined with forskolin, yet only cilostamide increased calcium transients. CONCLUSIONS: PDE1 inhibition by ITI-214 in vivo confers acute inotropic, lusitropic, and arterial vasodilatory effects in PDE1C-expressing mammals with and without HF. The effects appear related to cAMP signaling that is different from that provided via ß-adrenergic receptors or PDE3 modulation. ITI-214, which has completed phase I trials, may provide a novel therapy for HF.

Prevention of PKG-1α Oxidation Suppresses Antihypertrophic/Antifibrotic Effects From PDE5 Inhibition but not sGC Stimulation.

BACKGROUND: Stimulation of sGC (soluble guanylate cyclase) or inhibition of PDE5 (phosphodiesterase type 5) activates PKG (protein kinase G)-1α to counteract cardiac hypertrophy and failure. PKG1α acts within localized intracellular domains; however, its oxidation at cysteine 42, linking homomonomers, alters this localization, impairing suppression of pathological cardiac stress. Because PDE5 and sGC reside in separate microdomains, we speculated that PKG1α oxidation might also differentially influence the effects from their pharmacological modulation. METHODS AND RESULTS: Knock-in mice expressing a redox-dead PKG1α (PKG1αC42S) or littermate controls (PKG1αWT) were subjected to transaortic constriction to induce pressure overload and treated with a PDE5 inhibitor (sildenafil), sGC activator (BAY602770 [BAY]), or vehicle. In PKG1αWT controls, sildenafil and BAY similarly enhanced PKG activity and reduced pathological hypertrophy/fibrosis and cardiac dysfunction after transaortic constriction. However, sildenafil failed to protect the heart in PKG1αC42S, unlike BAY, which activated PKG and thereby facilitated protective effects. This corresponded with minimal PDE5 activation in PKG1αC42S exposed to transaortic constriction versus higher activity in controls and little colocalization of PDE5 with PKG1αC42S (versus colocalization with PKG1αWT) in stressed myocytes. CONCLUSIONS: In the stressed heart and myocytes, PKG1α C42-disulfide formation contributes to PDE5 activation. This augments the pathological role of PDE5 and so in turn enhances the therapeutic impact from its inhibition. PKG1α oxidation does not change the benefits from sGC activation. This finding favors the use of sGC activators regardless of PKG1α oxidation and may help guide precision therapy leveraging the cyclic GMP/PKG pathway to treat heart disease.

Transient receptor potential channel 6 regulates abnormal cardiac S-nitrosylation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked disorder with dystrophin loss that results in skeletal and cardiac muscle weakening and early death. Loss of the dystrophin-sarcoglycan complex delocalizes nitric oxide synthase (NOS) to alter its signaling, and augments mechanosensitive intracellular Ca2+ influx. The latter has been coupled to hyperactivation of the nonselective cation channel, transient receptor potential canonical channel 6 (Trpc6), in isolated myocytes. As Ca2+ also activates NOS, we hypothesized that Trpc6 would help to mediate nitric oxide (NO) dysregulation and that this would be manifest in increased myocardial S-nitrosylation, a posttranslational modification increasingly implicated in neurodegenerative, inflammatory, and muscle disease. Using a recently developed dual-labeling proteomic strategy, we identified 1,276 S-nitrosylated cysteine residues [S-nitrosothiol (SNO)] on 491 proteins in resting hearts from a mouse model of DMD (dmdmdx:utrn+/-). These largely consisted of mitochondrial proteins, metabolic regulators, and sarcomeric proteins, with 80% of them also modified in wild type (WT). S-nitrosylation levels, however, were increased in DMD. Genetic deletion of Trpc6 in this model (dmdmdx:utrn+/-:trpc6-/-) reversed ∼70% of these changes. Trpc6 deletion also ameliorated left ventricular dilation, improved cardiac function, and tended to reduce fibrosis. Furthermore, under catecholamine stimulation, which also increases NO synthesis and intracellular Ca2+ along with cardiac workload, the hypernitrosylated state remained as it did at baseline. However, the impact of Trpc6 deletion on the SNO proteome became less marked. These findings reveal a role for Trpc6-mediated hypernitrosylation in dmdmdx:utrn+/- mice and support accumulating evidence that implicates nitrosative stress in cardiac and muscle disease.

Repetitive ischemia increases myocardial dimethylarginine dimethylaminohydrolase 1 expression.

Pharmacologic inhibition of nitric oxide production inhibits growth of coronary collateral vessels. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the major enzyme that degrades asymmetric dimethylarginine (ADMA), a potent inhibitor of nitric oxide synthase. Here we examined regulation of the ADMA-DDAH1 pathway in a canine model of recurrent myocardial ischemia during the time when coronary collateral growth is known to occur. Under basal conditions, DDAH1 expression was non-uniform across the left ventricular (LV) wall, with expression strongest in the subepicardium. In response to ischemia, DDAH1 expression was up-regulated in the midmyocardium of the ischemic zone, and this was associated with a significant reduction in myocardial interstitial fluid (MIF) ADMA. The decrease in MIF ADMA during ischemia was likely due to increased DDAH1 because myocardial protein arginine N-methyl transferase 1 (PRMT1) and the methylated arginine protein content (the source of ADMA) were unchanged or increased, respectively, at this time. The inflammatory mediators interleukin (IL-1ß) and tumor necrosis factor (TNF-α) were also elevated in the midmyocardium where DDAH1 expression was increased. Both of these factors significantly up-regulated DDAH1 expression in cultured human coronary artery endothelial cells. Taken together, these results suggest that inflammatory factors expressed in response to myocardial ischemia contributed to up-regulation of DDAH1, which was responsible for the decrease in MIF ADMA.

Tetrahydrobiopterin Protects Against Hypertrophic Heart Disease Independent of Myocardial Nitric Oxide Synthase Coupling.

BACKGROUND: Nitric oxide synthase uncoupling occurs under conditions of oxidative stress modifying the enzyme's function so it generates superoxide rather than nitric oxide. Nitric oxide synthase uncoupling occurs with chronic pressure overload, and both are ameliorated by exogenous tetrahydrobiopterin (BH4)-a cofactor required for normal nitric oxide synthase function-supporting a pathophysiological link. Genetically augmenting BH4 synthesis in endothelial cells fails to replicate this benefit, indicating that other cell types dominate the effects of exogenous BH4 administration. We tested whether the primary cellular target of BH4 is the cardiomyocyte or whether other novel mechanisms are invoked. METHODS AND RESULTS: Mice with cardiomyocyte-specific overexpression of GTP cyclohydrolase 1 (mGCH1) and wild-type littermates underwent transverse aortic constriction. The mGCH1 mice had markedly increased myocardial BH4 and, unlike wild type, maintained nitric oxide synthase coupling after transverse aortic constriction; however, the transverse aortic constriction-induced abnormalities in cardiac morphology and function were similar in both groups. In contrast, exogenous BH4 supplementation improved transverse aortic constricted hearts in both groups, suppressed multiple inflammatory cytokines, and attenuated infiltration of inflammatory macrophages into the heart early after transverse aortic constriction. CONCLUSIONS: BH4 protection against adverse remodeling in hypertrophic cardiac disease is not driven by its prevention of myocardial nitric oxide synthase uncoupling, as presumed previously. Instead, benefits from exogenous BH4 are mediated by a protective effect coupled to suppression of inflammatory pathways and myocardial macrophage infiltration.

Soluble guanylate cyclase is required for systemic vasodilation but not positive inotropy induced by nitroxyl in the mouse.

Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-ß subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α(C42S) mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation.

PDE5 inhibitor efficacy is estrogen dependent in female heart disease.

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone's involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS-dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

Oxidative stress regulates left ventricular PDE5 expression in the failing heart.

BACKGROUND: Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF. METHODS AND RESULTS: Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)-induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased approximately equal 4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3'-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (SOD) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase 3 knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF. CONCLUSIONS: Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF.

Adenosine regulation of microtubule dynamics in cardiac hypertrophy.

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.

Adenosine A3 receptor deficiency exerts unanticipated protective effects on the pressure-overloaded left ventricle.

BACKGROUND: Endogenous adenosine can protect the overloaded heart against the development of hypertrophy and heart failure, but the contribution of A(1) receptors (A(1)R) and A(3) receptors (A(3)R) is not known. METHODS AND RESULTS: To test the hypothesis that A(1)R and A(3)R can protect the heart against systolic overload, we exposed A(3)R gene-deficient (A(3)R knockout [KO]) mice and A(1)R KO mice to transverse aortic constriction (TAC). Contrary to our hypothesis, A(3)R KO attenuated 5-week TAC-induced left ventricular hypertrophy (ratio of ventricular mass/body weight increased to 7.6+/-0.3 mg/g in wild-type mice compared with 6.3+/-0.4 mg/g in KO mice), fibrosis, and dysfunction (left ventricular ejection fraction decreased to 43+/-2.5% and 55+/-4.2% in wild-type and KO mice, respectively). A(3)R KO also attenuated the TAC-induced increases of myocardial atrial natriuretic peptide and the oxidative stress markers 3'-nitrotyrosine and 4-hydroxynonenal. In contrast, A(1)R KO increased TAC-induced mortality but did not alter ventricular hypertrophy or dysfunction compared with wild-type mice. In mice in which extracellular adenosine production was impaired by CD73 KO, TAC caused greater hypertrophy and dysfunction and increased myocardial 3'-nitrotyrosine. In neonatal rat cardiomyocytes induced to hypertrophy with phenylephrine, the adenosine analogue 2-chloroadenosine reduced cell area, protein synthesis, atrial natriuretic peptide, and 3'-nitrotyrosine. Antagonism of A(3)R significantly potentiated the antihypertrophic effects of 2-chloroadenosine. CONCLUSIONS: Adenosine exerts protective effects on the overloaded heart, but the A(3)R acts counter to the protective effect of adenosine. The data suggest that selective attenuation of A(3)R activity might be a novel approach to treat pressure overload-induced left ventricular hypertrophy and dysfunction.

Ecto-5'-nucleotidase deficiency exacerbates pressure-overload-induced left ventricular hypertrophy and dysfunction.

This study examined whether endogenous extracellular adenosine acts to facilitate the adaptive response of the heart to chronic systolic overload. To examine whether endogenous extracellular adenosine can protect the heart against pressure-overload-induced heart failure, transverse aortic constriction was performed on mice deficient in extracellular adenosine production as the result of genetic deletion of CD73. Although there was no difference in left ventricular size or function between CD73-deficient mice (knockout [KO] mice) and wild-type mice under unstressed conditions, aortic constriction for 2 or 4 weeks induced significantly more myocardial hypertrophy, left ventricular dilation, and left ventricular dysfunction in KO mice compared with wild-type mice. Thus, after 2 weeks of transverse aortic constriction, left ventricular fractional shortening decreased to 27.4+/-2.5% and 21.9+/-1.7% in wild-type and KO mice, respectively (P<0.05). Consistent with a role of adenosine in reducing tissue remodeling, KO mice displayed increased myocardial fibrosis and myocyte hypertrophy compared with wild-type mice. Furthermore, adenosine treatment reduced phenylephrine-induced cardiac myocyte hypertrophy and collagen production in cultured neonatal rat cardiac myocytes and cardiac fibroblasts, respectively. Consistent with a role for adenosine in modulating cardiomyocyte hypertrophy, KO mice demonstrated increased activation of mammalian target of rapamycin signaling, accompanied by higher expression of the hypertrophy marker atrial natriuretic peptide. Conversely, the adenosine analogue 2-chloro-adenosine significantly reduced cell size, mammalian target of rapamycin/p70 ribosomal S6 kinase activation, and atrial natriuretic peptide expression in cultured neonatal cardiomyocytes. These data demonstrate that CD73 helps to preserve cardiac function during chronic systolic overload by preventing maladaptive tissue remodeling.

Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction.

Extracellular superoxide dismutase (EC-SOD) contributes only a small fraction to total SOD activity in the heart but is strategically located to scavenge free radicals in the extracellular compartment. EC-SOD expression is decreased in myocardial-infarction (MI)-induced heart failure, but whether EC-SOD can abrogate oxidative stress or modify MI-induced ventricular remodeling has not been previously studied. Consequently, the effects of EC-SOD gene deficiency (EC-SOD KO) on left ventricular (LV) oxidative stress, hypertrophy, and fibrosis were studied in EC-SOD KO and wild-type mice under control conditions, and at 4 and 8 weeks after permanent coronary artery ligation. EC-SOD KO had no detectable effect on LV function in normal hearts but caused small but significant increases of LV fibrosis. At 8 weeks after MI, EC-SOD KO mice developed significantly more LV hypertrophy (LV mass increased 1.64-fold in KO mice compared to 1.35-fold in wild-type mice; p<0.01) and more fibrosis and myocyte hypertrophy which was more prominent in the peri-infarct region than in the remote myocardium. EC-SOD KO mice had greater increases of nitrotyrosine in the peri-infarct myocardium, and this was associated with a greater reduction of LV ejection fraction, a greater decrease of sarcoplasmic or endoplasmic reticulum calcium2+ ATPase, and a greater increase of atrial natriuretic peptide in the peri-infarct zone compared to wild-type mice. EC-SOD KO was associated with more increases of phosphorylated p38 (p-p38(Thr180/Tyr182)), p42/44 extracellular signal-regulated kinase (p-Erk(Thr202/Tyr204)), and c-Jun N-terminal kinase (p-JNK(Thr183/Tyr185)) both under control conditions and after MI, indicating that EC-SOD KO increases activation of mitogen-activated protein kinase signaling pathways. These findings demonstrate that EC-SOD plays an important role in protecting the heart against oxidative stress and infarction-induced ventricular hypertrophy.

Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure.

Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload-induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload-induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.