Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major unmet medical need owing to its diverse pathophysiology and lack of effective therapies. Potent synthetic, agonists ...(MR-356 and MR-409) of growth hormone-releasing hormone (GHRH) improve the phenotype of models of HF with reduced ejection fraction (HFrEF) and in cardiorenal models of HFpEF. Endogenous GHRH exhibits a broad range of regulatory influences in the cardiovascular (CV) system and aging and plays a role in several cardiometabolic conditions including obesity and diabetes. Whether agonists of GHRH can improve the phenotype of cardiometabolic HFpEF remains untested and unknown. Here we tested the hypothesis that MR-356 can mitigate/reverse the cardiometabolic HFpEF phenotype. C57BL6N mice received a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (l-NAME) for 9 wk. After 5 wk of HFD + l-NAME regimen, animals were randomized to receive daily injections of MR-356 or placebo during a 4-wk period. Control animals received no HFD + l-NAME or agonist treatment. Our results showed the unique potential of MR-356 to treat several HFpEF-like features including cardiac hypertrophy, fibrosis, capillary rarefaction, and pulmonary congestion. MR-356 improved cardiac performance by improving diastolic function, global longitudinal strain (GLS), and exercise capacity. Importantly, the increased expression of cardiac pro-brain natriuretic peptide (pro-BNP), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor-A (VEGF-A) was restored to normal levels suggesting that MR-356 reduced myocardial stress associated with metabolic inflammation in HFpEF. Thus, agonists of GHRH may be an effective therapeutic strategy for the treatment of cardiometabolic HFpEF phenotype.
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.
RATIONALE:The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow–derived ...mesenchymal stem cells (MSCs) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes.
OBJECTIVE:Here, we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire.
METHODS AND RESULTS:Female Yorkshire pigs (n=31) underwent experimental myocardial infarction (MI), and 3 days later, received transendocardial injections of allogeneic male bone marrow–derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit CSCs increased 20-fold in MSC-treated animals versus controls (P<0.001), there was a 6-fold increase in GATA-4 CSCs in MSC versus control (P<0.001), and mitotic myocytes increased 4-fold (P=0.005). Porcine endomyocardial biopsies were harvested and plated as organotypic cultures in the presence or absence of MSC feeder layers. In vitro, MSCs stimulated c-kit CSCs proliferation into enriched populations of adult cardioblasts that expressed Nkx2–5 and troponin I.
CONCLUSIONS:MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics.
S-Nitrosylation is a ubiquitous post-translational modification that regulates diverse biologic processes. In skeletal muscle, hypernitrosylation of the ryanodine receptor (RyR) causes sarcoplasmic ...reticulum (SR) calcium leak, but whether abnormalities of cardiac RyR nitrosylation contribute to dysfunction of cardiac excitation-contraction coupling remains controversial. In this study, we tested the hypothesis that cardiac RyR2 is hyponitrosylated in heart failure, because of nitroso-redox imbalance. We evaluated excitation-contraction coupling and nitroso-redox balance in spontaneously hypertensive heart failure rats with dilated cardiomyopathy and age-matched Wistar-Kyoto rats. Spontaneously hypertensive heart failure myocytes were characterized by depressed contractility, increased diastolic Ca2+ leak, hyponitrosylation of RyR2, and enhanced xanthine oxidase derived superoxide. Global S-nitrosylation was decreased in failing hearts compared with nonfailing. Xanthine oxidase inhibition restored global and RyR2 nitrosylation and reversed the diastolic SR Ca2+ leak, improving Ca2+ handling and contractility. Together these findings demonstrate that nitroso-redox imbalance causes RyR2 oxidation, hyponitrosylation, and SR Ca2+ leak, a hallmark of cardiac dysfunction. The reversal of this phenotype by inhibition of xanthine oxidase has important pathophysiologic and therapeutic implications.
Although nitric oxide (NO) signaling promotes differentiation and maturation of endothelial progenitor cells, its role in the differentiation of mesenchymal stem cells (MSCs) into endothelial cells ...remains controversial. We tested the role of NO signaling in MSCs derived from WT mice and mice homozygous for a deletion of S -nitrosoglutathione reductase (GSNOR ⁻/⁻), a denitrosylase that regulates S -nitrosylation. GSNOR ⁻/⁻ MSCs exhibited markedly diminished capacity for vasculogenesis in an in vitro Matrigel tube–forming assay and in vivo relative to WT MSCs. This decrease was associated with down-regulation of the PDGF receptorα (PDGFRα) in GSNOR ⁻/⁻ MSCs, a receptor essential for VEGF-A action in MSCs. Pharmacologic inhibition of NO synthase with L-N ᴳ-nitroarginine methyl ester (L -NAME) and stimulation of growth hormone–releasing hormone receptor (GHRHR) with GHRH agonists augmented VEGF-A production and normalized tube formation in GSNOR ⁻/⁻ MSCs, whereas NO donors or PDGFR antagonist reduced tube formation ∼50% by murine and human MSCs. The antagonist also blocked the rescue of tube formation in GSNOR ⁻/⁻ MSCs by L -NAME or the GHRH agonists JI-38, MR-409, and MR-356. Therefore, GSNOR ⁻/⁻ MSCs have a deficient capacity for endothelial differentiation due to downregulation of PDGFRα related to NO/GSNOR imbalance. These findings unravel important aspects of modulation of MSCs by VEGF-A activation of the PDGFR and illustrate a paradoxical inhibitory role of S -nitrosylation signaling in MSC vasculogenesis. Accordingly, disease states characterized by NO deficiency may trigger MSC-mediated vasculogenesis. These findings have important implications for therapeutic application of GHRH agonists to ischemic disorders.
Cardiac aging is characterized by alterations in contractility and intracellular calcium (Ca
) 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 Ca
and contractility. We tested the hypothesis that in the aged heart, Ca
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 Ca
, isolated cardiac myocytes were field-stimulated and Ca
was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (
< 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of Ca
in aged myocytes (
< 0.05). Time-50 Ca
decay was increased in aged myocytes (
< 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca
sensitivity was reduced in aged myocytes (
< 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 (
< 0.05). In conclusion, β-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca
handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca
handling proteins and myofilaments.
Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that ...S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and β-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed β-adrenergic inotropic responses that were associated with impaired β-agonist–induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.
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.
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 (
), 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
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
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.
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 (
= 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;
= 8; 30 µg/kg) or placebo (
= 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 (
= 0.03), reduced EDP/EDV ratio (
= 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte Ca
transient amplitude (
= 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (
= 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.
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.
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 μM GSNO also decreased Ca(2+) sensitivity of contraction and 10 μM reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥ 100 μM). 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 μM 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.
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.
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