Aims: The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S -nitrosoglutathione (GSNO), a protein S -nitrosylating agent. Although S -nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S -nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments. Results: Incubation of intact mouse cardiomyocytes with S -nitrosocysteine (CysNO, a cell-permeable low-molecular-weight nitrosothiol) significantly decreased myofilament Ca ²⁺ sensitivity. In demembranated (skinned) fibers, S -nitrosylation with 1 μ M GSNO also decreased Ca ²⁺ sensitivity of contraction and 10 μ M reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥100 μ M ). Reducing S -nitrosylation with ascorbate partially reversed the effects on Ca ²⁺ sensitivity and ATPase activity. In live cardiomyocytes treated with CysNO, resin-assisted capture of S -nitrosylated protein thiols was combined with label-free liquid chromatography–tandem mass spectrometry to quantify S -nitrosylation and determine the susceptible cysteine sites on myosin, actin, myosin-binding protein C, troponin C and I, tropomyosin, and titin. The ability of sarcomere proteins to form S -NO from 10–500 μ M CysNO in intact cardiomyocytes was further determined by immunoblot, with actin, myosin, myosin-binding protein C, and troponin C being the more susceptible sarcomeric proteins. Innovation and Conclusions: Thus, specific physiological effects are associated with S -nitrosylation of a limited number of cysteine residues in sarcomeric proteins, which also offer potential targets for interventions in pathophysiological situations. Antioxid. Redox Signal. 23, 1017–1034.

To test the hypothesis that the activation of the growth hormone-releasing hormone (GHRH) receptor signalling pathway within the myocardium both prevents and reverses diastolic dysfunction and pathophysiologic features consistent with heart failure with preserved ejection fraction (HFpEF). Impaired myocardial relaxation, fibrosis, and ventricular stiffness, among other multi-organ morbidities, characterize the phenotype underlying the HFpEF syndrome. Despite the rapidly increasing prevalence of HFpEF, few effective therapies have emerged. Synthetic agonists of the GHRH receptors reduce myocardial fibrosis, cardiomyocyte hypertrophy, and improve performance in animal models of ischaemic cardiomyopathy, independently of the growth hormone axis. CD1 mice received 4- or 8-week continuous infusion of angiotensin-II (Ang-II) to generate a phenotype with several features consistent with HFpEF. Mice were administered either vehicle or a potent synthetic agonist of GHRH, MR-356 for 4-weeks beginning concurrently or 4-weeks following the initiation of Ang-II infusion. Ang-II-treated animals exhibited diastolic dysfunction, ventricular hypertrophy, interstitial fibrosis, and normal ejection fraction. Cardiomyocytes isolated from these animals exhibited incomplete relaxation, depressed contractile responses, altered myofibrillar protein phosphorylation, and disturbed calcium handling mechanisms (ex vivo). MR-356 both prevented and reversed the development of the pathological phenotype in vivo and ex vivo. Activation of the GHRH receptors increased cAMP and cGMP in cardiomyocytes isolated from control animals but only cAMP in cardiac fibroblasts, suggesting that GHRH-A exert differential effects on cardiomyocytes and fibroblasts. These findings indicate that the GHRH receptor signalling pathway(s) represents a new molecular target to counteract dysfunctional cardiomyocyte relaxation by targeting myofilament phosphorylation and fibrosis. Accordingly, activation of GHRH receptors with potent, synthetic GHRH agonists may provide a novel therapeutic approach to management of the myocardial alterations associated with the HFpEF syndrome. Proposed mechanism of action for GHRH-As to modulate the Excitation-Contraction Coupling (ECC). It is well known that activation of GHRHRs (GPCR) induces the adenylyl cyclase (AC)/cyclic AMP/protein kinase A (PKA) pathway. PKA can both phosphorylate sarcomeric and calcium handling proteins and activate soluble guanylyl cyclase (sGC). This pathway also promotes reparative mechanisms at transcriptional level. On the other hand, has been well demonstrated that GHRH-As also promote activation of AKT (PKB) among other kinases. Endothelial Nitric Oxide Synthase eNOS) is also activated by GHRH. The generated nitric oxide (NO) in turn activates sGC to produce the second messenger cGMP, an activator of the protein kinase G (PKG). This kinase is also able to phosphorylate the above-mentioned proteins. This pathway has been proposed to be activated downstream GHRHRs through activation of PI3K by the Gβγ proteins or β-arrestins (among other possible mechanisms).

Therapies for heart failure with preserved ejection fraction (HFpEF) are lacking. Growth hormone-releasing hormone agonists (GHRH-As) have salutary effects in ischemic and nonischemic heart failure animal models. Accordingly, we hypothesized that GHRH-A treatment ameliorates chronic kidney disease (CKD)-induced HFpEF in a large-animal model. Female Yorkshire pigs (n = 16) underwent 5/6 nephrectomy via renal artery embolization and 12 wk later were randomized to receive daily subcutaneous injections of GHRH-A (MR-409; n = 8; 30 µg/kg) or placebo (n = 8) for 4 to 6 wk. Renal and cardiac structure and function were serially assessed postembolization. Animals with 5/6 nephrectomy exhibited CKD (elevated blood urea nitrogen [BUN] and creatinine) and faithfully recapitulated the hemodynamic features of HFpEF. HFpEF was demonstrated at 12 wk by maintenance of ejection fraction associated with increased left ventricular mass, relative wall thickness, end-diastolic pressure (EDP), end-diastolic pressure/end-diastolic volume (EDP/EDV) ratio, and tau, the time constant of isovolumic diastolic relaxation. After 4 to 6 wk of treatment, the GHRH-A group exhibited normalization of EDP (P = 0.03), reduced EDP/EDV ratio (P = 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte [Ca²⁺] transient amplitude (P = 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (P = 0.0022). GHRH-A exerted a beneficial effect on diastolic function in a CKD large-animal model as demonstrated by improving hemodynamic, structural, and molecular characteristics of HFpEF. These findings have important therapeutic implications for the HFpEF syndrome. Significance This randomized, blinded study used rigorous hemodynamic tools to test the efficacy of a synthetic growth hormone-releasing hormone agonist (GHRH‐A) on the restoration of diastolic function in a large animal model of chronic kidney disease (CKD)-induced heart failure with preserved ejection fraction (HFpEF). CKD was produced in Yorkshire swine by performing a 5/6 nephrectomy via catheter-based renal artery embolization. HFpEF was evident at 12 wk postembolization. Daily injection of GHRH-A improved cardiac diastolic hemodynamics, including end-diastolic pressure, end-diastolic pressure–volume ratio, and stroke work, compared to placebo. Development of this swine model and therapeutic approach has important implications because of the high prevalence of CKD-induced HFpEF and cardiorenal syndromes, lack of effective therapies, and few large-animal models available for investigation.

Background: Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S‐nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results: Here, we show that mice lacking S‐nitrosoglutathione reductase ( GSNOR ^−⁄− ), a denitrosylase regulating protein S‐nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S‐nitrosylated amino acid residues in GSNOR ^−⁄− mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S‐nitrosoproteome, and identifies a unique set of S‐nitrosylated proteins in GSNOR ^−⁄− mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions: Therefore, deficiency of GSNOR creates dysregulation of placental S‐nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

This randomized study used rigorous hemodynamic tools to test the efficacy of a synthetic GHRH agonist to improve cardiac performance in a cardiometabolic HFpEF. Daily injection of the GHRH agonist, MR-356, reduced the HFpEF-like effects as evidenced by improved diastolic dysfunction, reduced cardiac hypertrophy, fibrosis, and pulmonary congestion. Notably, end-diastolic pressure and end-diastolic pressure-volume relationship were reset to control levels. Moreover, treatment with MR-356 increased exercise capacity and reduced myocardial stress associated with metabolic inflammation in HFpEF.

Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca²⁺]_i) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca²⁺]_i and contractility. We tested the hypothesis that in the aged heart, [Ca²⁺]_i handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20–24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca²⁺]_i, isolated cardiac myocytes were field-stimulated and [Ca²⁺]_i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca²⁺]_i in aged myocytes (p < 0.05). Time-50 [Ca²⁺]_i decay was increased in aged myocytes (p < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca²⁺ sensitivity was reduced in aged myocytes (p < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment (p < 0.05). In conclusion, β-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca²⁺ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca²⁺ handling proteins and myofilaments.