New therapeutic approach to heart failure due to myocardial infarction based on targeting growth hormone-releasing hormone receptor

Background We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. Methods and Results H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-? compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of bone morphogenetic proteins. Conclusions Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remod eling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists

Growth Hormone Releasing Hormone Agonist Improves Cardiometabolic Parameters In A Murine Model Of Heart Failure With Preserved Ejection Fraction

The incidence of heart failure (HF) with preserved ejection fraction (HFpEF) is steadily rising, becoming the predominant form of HF. To date, no effective therapies reverse HFpEF symptoms. Several preclinical and clinical studies demonstrate that GHRH plays a role in the cardiovascular system, aging and obesity. We hypothesized that a GHRH-agonist (GHRH-A) can mitigate/reverse the HFpEF phenotype. C57BL6N mice received a high fat diet (HFD) plus the nitric oxide synthase inhibitor (L-NAME) for 9 weeks (HFD+L-NAME). After 5 weeks of the HFD+L-NAME regimen, animals were randomized to receive daily subcutaneous injections of GHRH-A (200 μg/Kg/day) or placebo (DMSO+ propylene glycol) for 4-weeks. Control animals received neither HFD+L-NAME nor treatment. Evaluation of cardiac performance was assessed by serial echocardiography. Blood pressure, glucose tolerance and exercise exhaustion were also evaluated. After 9 weeks of HFD+L-NAME there was no difference in EF, but the E/E’ ratio, global longitudinal strain (GLS), and exercise tolerance test revealed significantly impaired cardiac performance in HFpEF mice compared to control (Fig. 1A-D). Increased glucose levels were seen in the placebo group compared to control, suggesting glucose intolerance (Fig 1 E-F). GHRH-A administration improved most of these parameters (one-way ANOVA with Tukey's multiple comparisons test, *p<0.05, **p<0.01, ***p<0.001, and ***p<0.0001. Unpaired Student's t-test, *p<0.05). Our results reveal that GHRH-A treatment reduces HFpEF-like effects associated with HFD+ L-NAME treatment, suggesting that activation of GHRH receptor signaling as an effective therapeutic strategy for the treatment of cardiometabolic HFpEF phenotype

Pharmacologic and genetic strategies to enhance cell therapy for cardiac regeneration

Cell-based therapy is emerging as an exciting potential therapeutic approach for cardiac regeneration following myocardial infarction (MI). As heart failure (HF) prevalence increases over time, development of new interventions designed to aid cardiac recovery from injury are crucial and should be considered more broadly. In this regard, substantial efforts to enhance the efficacy and safety of cell therapy are continuously growing along several fronts, including modifications to improve the reprogramming efficiency of inducible pluripotent stem cells (iPS), genetic engineering of adult stem cells, and administration of growth factors or small molecules to activate regenerative pathways in the injured heart. These interventions are emerging as potential therapeutic alternatives and/or adjuncts based on their potential to promote stem cell homing, proliferation, differentiation, and/or survival. Given the promise of therapeutic interventions to enhance the regenerative capacity of multipotent stem cells as well as specifically guide endogenous or exogenous stem cells into a cardiac lineage, their application in cardiac regenerative medicine should be the focus of future clinical research. This article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure.” ► We review small molecules to activate cardiac regenerative pathways. ► Chemokines may improve stem cell engraftment, survival and differentiation. ► Modulation of genes or cell signaling pathways offers new therapeutic targets. ► Enhancing cell therapy with small molecules will transform cardiac repair potential

Abstract 104: Sex-specific Impact Of S-nitrosoglutathione Reductase (gsnor) On Ventricular Remodeling And Function Following Myocardial Infarction In Mice

Introduction: Female hearts are less susceptible to myocardial injury following myocardial infarction (MI) and estrogen working through the nitric oxide (NO) pathway is thought to play a role. S-nitrosylation of cysteine thiols is a major signaling pathway through which NO exerts its action and mice with targeted deletion of S-nitrosoglutathione reductase (GSNOR), a denitrosylase that regulates S-nitrosylation, show increased levels of nitrosylated proteins. Male GSNOR -/- mice show a more favorable outcome after MI as compared to wild-type (WT), including reduced myocardial infarct size, improved ejection fraction and preserved left ventricular volumes. Whether female GSNOR -/- mice show gender-related cardiac protection following MI was not known and thus investigated. Methods & Results: MI was induced in male and female GSNOR -/- mice and their respective controls, C57Bl/6J, at 3-5 months via left anterior descending coronary artery occlusion. Serial echocardiography was performed prior to MI and after 1- and 4-weeks post-MI to assess ejection fraction (EF) and left ventricular volume both at diastole (end-diastolic volume, EDV) and systole (end-systolic volume, ESV). Compared to WT, GSNOR -/- males showed less dilation in both EDV (98.6 ± 9.3 mm vs. 140.6 ± 7.4 mm in WT, P<0.001) and ESV (73.1 ± 9.2 vs. 112.7 ± 7.3 mm in WT, P<0.05) at 4 weeks post-MI. Whereas, GSNOR -/- females showed greater dilation in both EDV (162.2 ± 13 vs. 83.8 ± 12 mm in WT, P<0.001) and ESV (141.8 ± 13 vs. 65.6 ± 12 mm in WT, P<0.001) as compared to WT females. EF decreased (P<0.001) in all groups post-MI, but at 4 weeks post-MI, it was significantly worse in GSNOR -/- females compared to GSNOR -/- males (14 ± 4% vs. 28 ± 3%, P<0.05). Conclusion: GSNOR -/- females exhibit significantly lower EF and greater dilation of left ventricular volumes both at diastole and systole following MI than any of the other groups suggesting that S-nitrosylation plays an important role in gender-related cardiac protection following myocardial injury. These findings suggest the importance of taking gender into account when exploring novel therapeutic treatments for myocardial injury

Progenitor/Stem Cell Delivery by Suprarenal Aorta Route in Acute Kidney Injury

Progenitor/stem cell-based kidney regenerative strategies are a key step towards the development of novel therapeutic regimens for kidney disease treatment. However, the route of cell delivery, e.g., intravenous, intra-arterial, or intra-parenchymal, may affect the efficiency for kidney repair in different models of acute and chronic injury. Here, we describe a protocol of intra-aorta progenitor/stem cell injection in rats following either acute ischemia-reperfusion injury or acute proteinuria induced by puromycin aminonucleoside (PAN) – the experimental prototype of human minimal change disease and early stages of focal and segmental glomerulosclerosis. Vascular clips were applied across both renal pedicles for 35 min, or a single dose of PAN was injected via intra-peritoneal route, respectively. Subsequently, 2 x 106 stem cells [green fluorescent protein (GFP)-labeled c-Kit+ progenitor/stem cells or GFP-mesenchymal stem cells] or saline were injected into the suprarenal aorta, above the renal arteries, after application of a vascular clip to the abdominal aorta below the renal arteries. This approach contributed to engraftment rates of ∼10% at day 8 post ischemia-reperfusion injury, when c-Kit+ progenitor/stem cells were injected, which accelerated kidney recovery. Similar rates of engraftment were found after PAN-induced podocyte damage at day 21. With practice and gentle surgical technique, 100% of the rats could be injected successfully, and, in the week following injection, ∼ 85% of the injected rats will recover completely. Given the similarities in mammals, much of the data obtained from intra-arterial delivery of progenitor/stem cells in rodents can be tested in translational research and clinical trials with endovascular catheters in humans

Mechanistic basis for rescuing hypertrophic cardiomyopathy with myosin regulatory light chain phosphorylation

Abstract We investigated the impact of the phosphomimetic (Ser15 → Asp15) myosin regulatory light chain (S15D‐RLC) on the Super‐Relaxed (SRX) state of myosin using previously characterized transgenic (Tg) S15D‐D166V rescue mice, comparing them to the Hypertrophic Cardiomyopathy (HCM) Tg‐D166V model and wild‐type (WT) RLC mice. In the Tg‐D166V model, we observed a disruption of the SRX state, resulting in a transition from SRX to DRX (Disordered Relaxed) state, which explains the hypercontractility of D166V‐mutated myosin motors. The presence of the S15D moiety in Tg‐S15D‐D166V mice restored the SRX/DRX balance to levels comparable to Tg‐WT, thus mitigating the hypercontractile behavior associated with the HCM‐D166V mutation. Additionally, we investigated the impact of delivering the S15D‐RLC molecule to the hearts of Tg‐D166V mice via adeno‐associated virus (AAV9) and compared their condition to AAV9‐empty vector‐injected or non‐injected Tg‐D166V animals. Tg‐D166V mice injected with AAV9 S15D‐RLC exhibited a significantly higher proportion of myosin heads in the SRX state compared to those injected with AAV9 empty vector or left non‐injected. No significant effect was observed in Tg‐WT hearts treated similarly. These findings suggest that AAV9‐delivered phosphomimetic S15D‐RLC modality mitigates the abnormal Tg‐D166V phenotype without impacting the normal function of Tg‐WT hearts. Global longitudinal strain analysis supported these observations, indicating that the S15D moiety can alleviate the HCM‐D166V phenotype by restoring SRX stability and the SRX ↔ DRX equilibrium

Sex‐Specific Impact of Aldosterone Receptor Antagonism on Ventricular Remodeling and Gene Expression after Myocardial Infarction

Aldosterone receptor antagonism reduces mortality and improves post‐myocardial infarction (Ml) remodeling. Because aldosterone and estrogen signaling pathways interact, we hypothesized that aldosterone blockade is sex‐specific. Therefore, we investigated the mpact of eplerenone on left ventricular (LV) remodeling and gene expression of male infarcted rats versus female infarcted rats. Ml and Sham animals were randomized to receive eplerenone (100 mg/kg/day) or placebo 3 days post‐surgery for 4 weeks and assessed by echocardiography. In the Ml placebo group, left ventricular end‐diastolic dimension (LVEDD) increased from 7.3 ± 0.4 mm to 10.2 ± 1.0 mm ( p < 0.05) and ejection fraction (EF) decreased from 82.3 + 4% to 45.5 + 11% ( p < 0.05) in both sexes ( p = NS between groups). Eplerenone attenuated LVEDD enlargement more effectively in females (8.8 ± 0.2 mm, p < 0.05 vs. placebo) than in males (9.7 ± 0.2 mm, p = NS vs. placebo) and improved EF in females (56.7 ± 3%, p < 0.05 vs. placebo) but not in males (50.6 + 3%, p = NS vs. placebo). Transcriptomic analysis using Rat_230–2.0 microarrays (Affymetrix) revealed that in females 19% of downregu‐lated genes and 44% of upregulated genes post‐MI were restored to normal by eplerenone. In contrast, eplerenone only restored 4% of overexpressed genes in males. Together, these data suggest that aldosterone blockade reduces Ml‐induced cardiac remodeling and phenotypic alterations of gene expression preferentially in females than in males. The use of transcriptomic signatures to detect greater benefit of eplerenone in females has potential implications for personalized medicine

Hydroxychloroquine Mitigates Dilated Cardiomyopathy Phenotype in Transgenic D94A Mice

In this study, we aimed to investigate whether short-term and low-dose treatment with hydroxychloroquine (HCQ), an antimalarial drug, can modulate heart function in a preclinical model of dilated cardiomyopathy (DCM) expressing the D94A mutation in cardiac myosin regulatory light chain (RLC) compared with healthy non-transgenic (NTg) littermates. Increased interest in HCQ came with the COVID-19 pandemic, but the risk of cardiotoxic side effects of HCQ raised concerns, especially in patients with an underlying heart condition, e.g., cardiomyopathy. Effects of HCQ treatment vs. placebo (H2O), administered in Tg-D94A vs. NTg mice over one month, were studied by echocardiography and muscle contractile mechanics. Global longitudinal strain analysis showed the HCQ-mediated improvement in heart performance in DCM mice. At the molecular level, HCQ promoted the switch from myosin&rsquo;s super-relaxed (SRX) to disordered relaxed (DRX) state in DCM-D94A hearts. This result indicated more myosin cross-bridges exiting a hypocontractile SRX-OFF state and assuming the DRX-ON state, thus potentially enhancing myosin motor function in DCM mice. This bottom-up investigation of the pharmacological use of HCQ at the level of myosin molecules, muscle fibers, and whole hearts provides novel insights into mechanisms by which HCQ therapy mitigates some abnormal phenotypes in DCM-D94A mice and causes no harm in healthy NTg hearts

Abstract 13371: Beneficial Effects of Growth Hormone Releasing Hormone Agonist in a Murine Model of Cardiometabolic Heart Failure With Preserved Ejection Fraction

Byline: Rosemeire M Kanashiro-Takeuchi, UNIVERSITY OF MIAMI MILLER SCHOOL OF MEDICINE, Miami, FL; Lauro M Takeuchi, Interdisciplinary Stem Cell Institute, UNIVERSITY OF MIAMI MILLER SCHOOL OF MEDICINE, Miami, FL; Raul A Dulce, Interdisciplinary Stem Cell Institute, UNIVERSITY OF MIAMI MILLER SCHOOL OF MEDICINE, Miami, FL; Katarzyna Kazmierczak, UNIVERSITY OF MIAMI MILLER SCHOOL OF MEDICINE, Miami, FL; Wayne Balkan, UNIVERSITY MIAMI MILLER SCHOOL OF MEDICINE, Miami, FL; Renzhi Cai, Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Med Cntr, Miami, FL; Andrew Schally, Endocrine, Polypeptide and Cancer Institute, Veteran Affairs Med Cntr, Miami, FL; Joshua M Hare, UNIVERSITY OF MIAMI MILLER SCHOOL OF MEDICINE, Miami, FL Introduction: Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major unmet medical need due to its complex pathophysiology and lack of effective therapies. We previously showed that growth hormone-releasing hormone (GHRH)-agonists improve the phenotype of models of HF with reduced EF (HFrEF) and cardiorenal models of HFpEF. Hypothesis: We tested the hypothesis that the GHRH-agonist, MR-356 mitigates/reverses the cardio-metabolic HFpEF phenotype. Methods: C57BL6N mice received a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (L-NAME) for 9 weeks. After 5 weeks of the HFD+L-NAME diet, animals were randomized to receive daily injections of MR-356 or placebo for 4-weeks. Control animals received no HFD+L-NAME or agonist treatment. Cardiac performance was assessed by echocardiography and hemodynamic measurements. Blood pressure, glucose tolerance, and exercise capacity, as well as cardiac hypertrophy, fibrosis, capillary density, and lung congestion were evaluated. Results: After 5 weeks of HFD+L-NAME diet, the placebo group showed impaired diastolic function (increased E/E'ratio, EDPVR, end-diastolic pressure), glucose intolerance and reduced global longitudinal strain and exercise capacity (p<0.05). MR-356 treatment improved all of these parameters (p<0.05). Cardiac hypertrophy, fibrosis, and capillary rarefaction in the placebo group (p<0.05) were completely restored by MR-356 (p<0.05). The increased expression of cardiac pro-BNP, and inducible nitric oxide synthase (iNOS) in the placebo group (p<0.05) was brought to normal levels by MR-356 (p<0.05). Hypophosphorylation of the titin isoforms in the placebo group (p<0.05) was attenuated in the MR-356 group. Conclusions: Our data show the unique potential of MR-356 to treat several HFpEF-like features including diastolic dysfunction, cardiac hypertrophy, fibrosis, and exercise intolerance, suggesting that MR-356 reduces myocardial stress associated with metabolic inflammation in HFpEF. Thus, GHRH-agonists may be an effective therapeutic strategy for the treatment of the cardiometabolic HFpEF phenotype.Professiona