Natriuretic Peptide Expression Research Articles

Empagliflozin attenuates hypoxia-induced heart failure of zebrafish embryos via influencing MMP13 expression

BackgroundToday, sodium glucose co-transporter 2 (SGLT2) inhibitors are more than diabetes drugs. They are also indicated in chronic heart failure (HF) treatment in both diabetic and non-diabetic patients, independently of the ejection fraction. Multiple mechanisms have been suggested behind the cardioprotective effects of SGLT2 inhibitors. However, the underlying mechanisms still remain largely unexplored. Here, we used a zebrafish embryo model to search for new potential players whereby SGLT2 inhibitors attenuate HF. MethodsHF in zebrafish embryos was caused exposing them to chemically induced hypoxia. As a SGLT2 inhibitor, we used empagliflozin. Its effect on hypoxia-induced HF of the embryos was evaluated using video microscopy and calculation of fractional shortening (FS) of embryos´ hearts. RT-qPCR of brain natriuretic peptide (bnp) expression was also used to examine empagliflozin´s effect on HF. Transcriptome analysis of total RNA of the embryos was performed to search for new potential mechanisms contributing to the beneficial effect of empagliflozin on HF. ResultsEmpagliflozin significantly attenuated hypoxia-induced HF of zebrafish embryos as shown with improved FS of the hearts and decreased bnp expression. Transcriptome analysis revealed that the improvement of HF in response to empagliflozin was accompanied with decreased matrix metalloproteinase 13a (mmp13a) expression. Treatment of hypoxia-induced embryos with MMP13 inhibitor ameliorated the impaired heart function accordingly to the effect of empagliflozin. MMP13 inhibitor was not toxic to the embryos. ConclusionsOur study shows that empagliflozin´s favorable effect on attenuating HF is mediated via MMP13. MMP13 provides a novel option when developing new therapeutics for HF treatment.

Fibroblast growth factor 20 ameliorates cardiac hypertrophy via activation ErbB2

Fibroblast growth factor 20 (FGF20) is a member of the fibroblast growth factor family and involved in embryonic development and cardiac repair. This study aimed to explore the role of FGF20 in cardiac hypertrophy and the underlying molecular mechanisms. FGF20 improved cardiac hypertrophy in vivo and in vitro. Furthermore, FGF20 increased expression of erythroblastic leukemia viral oncogene homolog 2 (ErbB2), which was negatively correlated with expression of the cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). In addition, FGF20 effectively protected cardiomyocytes against apoptosis and oxidative stress. To further investigate whether protective effect of FGF20 is mediated by ErbB2, neonatal rat cardiomyocytes (NRCMs) were treated with lapatinib, an inhibitor of ErbB2. Lapatinib largely abrogated the anti-hypertrophic effect of FGF20, accompanied by increases in cardiomyocyte apoptosis and oxidative stress. In summary, this study reveals that FGF20 prevents cardiac hypertrophy by inhibiting apoptosis and oxidative stress via activating ErbB2 and may be a promising therapeutic strategy for cardiac hypertrophy.

Imatinib‑ and ponatinib‑mediated cardiotoxicity in zebrafish embryos and H9c2 cardiomyoblasts.

Tyrosine kinase inhibitors (TKIs) offer targeted therapy for cancers but can cause severe cardiotoxicities. Determining their dose‑dependent impact on cardiac function is required to optimize therapy and minimize adverse effects. The dose‑dependent cardiotoxic effects of two TKIs, imatinib and ponatinib, were assessed in vitro using H9c2 cardiomyoblasts and in vivo using zebrafish embryos. In vitro, H9c2 cardiomyocyte viability, apoptosis, size, and surface area were evaluated to assess the impact on cellular health. In vivo, zebrafish embryos were analyzed for heart rate, blood flow velocity, and morphological malformations to determine functional and structural changes. Additionally, reverse transcription‑quantitative PCR (RT‑qPCR) was employed to measure the gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), established markers of cardiac injury. This comprehensive approach, utilizing both in vitro and in vivo models alongside functional and molecular analyses, provides a robust assessment of the potential cardiotoxic effects. TKI exposure decreased viability and surface area in H9c2 cells in a dose‑dependent manner. Similarly, zebrafish embryos exposed to TKIs exhibited dose‑dependent heart malformation. Both TKIs upregulated ANP and BNP expression, indicating heart injury. The present study demonstrated dose‑dependent cardiotoxic effects of imatinib and ponatinib in H9c2 cells and zebrafish models. These findings emphasize the importance of tailoring TKI dosage to minimize cardiac risks while maintaining therapeutic efficacy. Future research should explore the underlying mechanisms and potential mitigation strategies of TKI‑induced cardiotoxicities.

MiR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice

ObjectiveCardiac fibrosis is an important contributor to atrial fibrillation (AF). Our aim was to identify biomarkers for AF using bioinformatics methods and explore the regulatory mechanism of miR-450a-2-3p in cardiac fibrosis in mice.MethodsTwo datasets, GSE115574 and GSE79768, were obtained from the Gene Expression Omnibus (GEO) database and subsequently merged for further analysis. Differential gene expression analysis was performed to identify differentially expressed genes (DEGs) and miR-450a-2-3p-related differentially expressed genes (MRDEGs). To investigate the underlying mechanism of cardiac fibrosis, a mouse model was established by treating mice with isoproterenol (ISO) and the miR-450a-2-3p agomir.ResultsA total of 127 DEGs and 31 MRDEGs were identified and subjected to Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the functions and pathways involved in AF. In the animal model, histological analysis using HE and Masson staining, as well as quantification of the collagen volume fraction (CVF), was performed. The increased expression of α-smooth muscle actin (α-SMA), collagen type I (COL1), collagen type III (COL3), and extracellular signal-regulated kinase 1/2 (ERK(1/2)) at both the transcriptional and translational levels indicated the significant development of myocardial fibrosis in mice induced with isoproterenol (ISO). In addition, the cross-sectional area of cardiomyocytes and the expression of atrial natriuretic peptide (NPPA) and brain natriuretic peptide (NPPB) were increased in the ISO group compared with the control group. However, after overexpression of the miR-450a-2-3p agomir through caudal vein injection, there was a notable improvement in cardiac morphology in the treated group. The expression levels of α-SMA, COL1, COL3, ERK(1/2), NPPA, and NPPB were also significantly decreased.ConclusionOur study reveals the mechanistic connection between ISO-induced myocardial fibrosis and the miR-450a-2-3p/ERK(1/2) signaling pathway, highlighting its role in the development of cardiac fibrosis. Modulating miR-450a-2-3p expression and inhibiting ERK(1/2) activation are promising approaches for therapeutic intervention in patients with AF.

Restoration of C-type natriuretic peptide and glial fibrillary acidic protein expression in fear centers and intrinsic cardiac ganglia by theta frequency sound during chronic stress in mice

BackgroundC-type natriuretic peptide (CNP) can be altered during stress and has protective effects in both the heart and brain; the functions of both organs can be positively affected by CNP modulation. Low arousal sounds can modulate heart–brain communication and improve stress responses. Here, we aimed to explore the modulation of CNP and glial fibrillary acidic protein (GFAP) and neuroprotective effects of low arousal theta frequency sound (TFS). MethodsChronic stress was induced in mice (n > 4) using four different stressors on alternate days for 15 days, followed by TFS therapy on alternate days. Open field and elevated plus maze tests were administered for the behavioral analysis, and enzyme-linked immunosorbent assay was used to analyze corticosterone, dopamine, and serotonin levels. Hematoxylin and eosin and cresyl violet staining were used for the morphological analysis of brain and heart sections, and immunohistochemistry for GFAP and CNP was performed. ResultsTFS significantly increased the time spent in the open arms during the elevated plus maze (p< 0.05) and improved exploration in the open field test (p < 0.05). In both tests, decision-making times were significantly reduced by TFS. Nuclear morphology and GFAP expression demonstrated significantly reduced gliosis in fear pathways after TFS therapy x. CNP levels were restored in fear pathways but not intrinsic cardiac ganglia (responsible for heart–brain communication) in TFS-treated mice. Brain corticosterone and dopamine levels increased after TFS therapy, reflecting restored motivational behaviors. ConclusionsLow arousal TFS is a potential neuromodulator for treating stress and related complications.

UCP1-dependent brown adipose activation accelerates cardiac metabolic remodeling and reduces initial hypertrophic and fibrotic responses to pathological stress.

Brown adipose tissue (BAT) is correlated to cardiovascular health in rodents and humans, but the physiological role of BAT in the initial cardiac remodeling at the onset of stress is unknown. Activation of BAT via 48 h cold (16°C) in mice following transverse aortic constriction (TAC) reduced cardiac gene expression for LCFA uptake and oxidation in male mice and accelerated the onset of cardiac metabolic remodeling, with an early isoform shift of carnitine palmitoyltransferase 1 (CPT1) toward increased CPT1a, reduced entry of long chain fatty acid (LCFA) into oxidative metabolism (0.59 ± 0.02 vs. 0.72 ± 0.02 in RT TAC hearts, p < .05) and increased carbohydrate oxidation with altered glucose transporter content. BAT activation with TAC reduced early hypertrophic expression of β-MHC by 61% versus RT-TAC and reduced pro-fibrotic TGF-β1 and COL3α1 expression. While cardiac natriuretic peptide expression was yet to increase at only 3 days TAC, Nppa and Nppb expression were elevated in Cold TAC versus RT TAC hearts 2.7- and 2.4-fold, respectively. Eliminating BAT thermogenic activation with UCP1 KO mice eliminated differences between Cold TAC and RT TAC hearts, confirming effects of BAT activation rather than autonomous cardiac responses to cold. Female responses to BAT activation were blunted, with limited UCP1 changes with cold, partly due to already activated BAT in females at RT compared to thermoneutrality. These data reveal a previously unknown physiological mechanism of UCP1-dependent BAT activation in attenuating early cardiac hypertrophic and profibrotic signaling and accelerating remodeled metabolic activity in the heart at the onset of cardiac stress.

Endurance Exercise Prevented Diabetic Cardiomyopathy through the Inhibition of Fibrosis and Hypertrophy in Rats.

Exercise training could be essential in preventing pathological cardiac remodeling in diabetes. Therefore, the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) singly or plus metformin on diabetes-induced cardiomyopathy were investigated in this study. Forty-nine Wistar rats (male) were recruited. Seven groups of animals were treated for six weeks as control, diabetes, MICT (15 m/min, 40 min/day), HIIT (20 m/min, 40 min/day), metformin (300 mg/kg), HIIT+metformin (Met-HIIT), and MICT+metformin (Met-MICT). The metformin was orally administered with an intragastrical needle, and the exercised rats were trained (5 days/week) with a motorized treadmill. Metabolic parameters, echocardiographic indices, histopathology evaluation, and assessment of gene expression connected with cardiac fibrosis, hypertrophy, mitochondrial performance, and intracellular calcium homeostasis were investigated. Our results demonstrated that all the interventions prevented weight loss and enhanced heart weight/body weight ratio and fasting plasma glucose in diabetic rats. Both types of exercise and their metformin combinations improved diabetic animals' echocardiography indices by enhancing heart rate, fractional shortening (FS), ejection fraction (EF) and reducing end-systolic and end-diastolic diameter of left ventricular (LVESD and LVEDD). Gene expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), transforming growth factor (TGF)- , and collagen increased in the diabetes group. In contrast, the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 ), AMP-activated protein kinase (AMPK), ryanodine receptors (RyR), and ATPase pump of the sarcoplasmic reticulum (SERCA) was reduced in diabetic animals. Exercise training alone or in combination with metformin reversed these changes. Moreover, diabetes-induced cardiac fibrosis was ameliorated in treated groups. All indicators of diabetic cardiomyopathy were improved more in the Met-HIIT group than in other groups. Exercise training, notably with metformin combination, alleviated diabetes-induced cardiac complications. The beneficial effects of exercise could be related to improving pathological cardiac remodeling and enhancing cardiac function.

Angiotensin-(1-7) Treatment Early in Life Prevents Cardiac Hypertrophy in Adult Hypertensive Rats.

Angiotensin (Ang)-(1-7) is a cardioprotective peptide of the renin-angiotensin system. Prepuberty has been considered as a later susceptible window of development, and stressful factors in this life phase can induce chronic diseases in adulthood. We aimed to investigate whether the treatment with Ang-(1-7) during the prepuberty could attenuate the development of hypertension and cardiac injury in adult spontaneously hypertensive rats (SHRs). SHRs were treated with Ang-(1-7) (24 μg/kg/h) from age 4 to 7 weeks. Systolic blood pressure was measured by tail-cuff plethysmography up to 17th week. Thereafter, echocardiography was performed, and the rats were euthanized for the collection of tissues and blood. Ang-(1-7) did not change the systolic blood pressure but reduced the septal and posterior wall thickness, and cardiomyocyte hypertrophy and fibrosis in SHR. In addition, Ang-(1-7) reduced the gene expression of atrial natriuretic peptide and brain natriuretic peptide, increased the metalloproteinase 9 expression, and reduced the extracellular signal-regulated kinases 1/2 phosphorylation. Ang-(1-7) also prevented the reduction of Mas receptor but did not change the protein expression of angiotensin-converting enzyme, angiotensin-converting enzyme 2, AT1, and AT2. The treatment with Ang-(1-7) decreased the malondialdehyde (MDA) levels and increased superoxide dismutase-1 and catalase activities and protein expression of catalase. Our findings demonstrate that the treatment of SHR with Ang-(1-7) for 3 weeks early in life promotes beneficial effects in the heart later in life, even without altering blood pressure, through mechanisms involving the reduction of oxidative stress and ERK1/2 phosphorylation. In addition, this study supports the prepuberty as an important programming window.

Comparison of Protective Effect of Tri-circulator and Coenzyme Q10 on Myocardial Injury and the Mechanism Study by Zebrafish Model.

Tri-Circulator (TC) is a product comprising coenzyme Q10 (CoQ10), Salvia miltiorrhiza, and Panax notoginseng. Individually, each of these constituents has demonstrated protective effects on myocardial injury. The purpose of this study is to evaluate the protective efficacy of TC on heart function and compare the differential effects between CoQ10 and TC. Two myocardial injury models of zebrafish, the hypoxia-reoxygenation model (H/R) and the isoproterenol (ISO, a β-receptor agonist) model, were used in this experiment. The zebrafish subjects were divided into 4 groups: control, H/R, TC, and CoQ10. Heart rate, stroke volume (SV), cardiac output (CO), ejection fraction (EF), fractional area change (FAC), and pericardial height were monitored to assess changes in heart function. The gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was studied as markers of injury/stress. TC significantly suppresses elevated heart rate induced by H/R and prevents the decrease of heart rate induced by ISO. It alleviates the pericardial infusion induced by ISO, whereas CoQ10 does not possess a similar effect. Both TC and CoQ10 significantly inhibit the decline in SV, CO, EF, and FAC induced by H/R and ISO, and suppress the expression of ANP and BNP in cardiomyocytes induced by ISO. It is noteworthy that TC demonstrates a more pronounced effect on EF, FAC, ANP, and BNP gene expression compared to CoQ10. Both TC and CoQ10 have a protective effect on myocardial injury of zebrafish. However, TC exhibits a greater efficacy compared to CoQ10 alone in mitigating myocardial injury.

Resveratrol alleviates hyperglycemia-induced cardiomyocyte hypertrophy by maintaining mitochondrial homeostasis via enhancing SIRT1 expression

To investigate whether resveratrol alleviates hyperglycemia-induced cardiomyocyte hypertrophy by enhancing the expression of silent information regulation 2 homolog 1 (SIRT1) to maintain mitochondrial homeostasis. Rat cardiomyocytes H9c2 cells with or without lentivirus-mediated mRNA interference of SIRT1 were cultured in high glucose (HG) and treated with resveratrol for 72 h. The changes in superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, reactive oxygen species (ROS) level, and relative surface of the cells were examined, and the mRNA expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and protein expressions of SIRT1, mitochondrial fusion related proteins optic atrophy protein 1 (OPA1) and mitofusin 2, mitochondrial division related proteins dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1), and mitophagy-related proteins BNIP3L and LC3 were detected using RT-qPCR and Western blotting. HG exposure significantly decreased SOD activity, increased MDA content, ROS production, relative cell surface, and the mRNA expressions of ANF and BNP in the cardiomyocytes; the protein expressions of SIRT1, OPA1, mitofusin 2 and BNIP3L and LC3-Ⅱ/LC3-Ⅰ ratio were all decreased and the protein expressions of DRP1 and FIS1 increased in HG-exposed cells (P<0.01). All these changes in HG-exposed cardiomyocytes were significantly alleviated by treatment with resveratrol (P<0.05). The protective effects of resveratrol against HG exposure in the cardiomyocytes were obviously attenuated by transfection of the cells with si-SIRT1 (P<0.05). Resveratrol inhibits hyperglycemia-induced cardiomyocyte hypertrophy by reducing oxidative stress, the mechanisms of which involve enhancement of SIRT1 protein expression, regulation of mitochondrial fusion and division balance, and promoting BNIP3L-mediated mitophagy to maintain mitochondrial homeostasis in the cells.

Unveiling the Therapeutic Potential of SGLT2 inhibitors and Oral Ketone Ester in Heart Failure with Preserved Ejection Fraction: A Comprehensive Exploration of Cardiac Function, Structure, and Metabolism in a Rat Model

BackgroundThis study investigated the potential efficacy of SGLT2 inhibitors empagliflozin and oral ketone supplementation as treatments for heart failure with preserved ejection fraction (HFpEF), which is often associated with obesity and type 2 diabetes mellitus. Methods20-week-old obese ZSF-1 rats were given different treatments for 12 weeks, including vehicle, empagliflozin, ketone ester (KE), or a combination of empagliflozin and KE. Cardiac magnetic resonance imaging (CMR) and hyperpolarized carbon-13 (13C) labeled pyruvate magnetic resonance spectroscopy imaging were used to evaluate cardiac function and metabolism, while histological and molecular markers of cardiac remodeling were assessed in the left ventricle. ResultsThe untreated HFpEF rats exhibited obesity, diabetes, cardiac hypertrophy, atrial enlargement, lung congestion, and increased cardiomyocyte size and cardiac expression of atrial natriuretic peptide (ANP) and fibrosis marker col1a1 and TIMP1. The treatment groups exhibited decreased body weight, increased circulating ketone levels, and reduced blood glucose levels. All treatment regimens significantly attenuated ventricular hypertrophy, atrial enlargement, and lung congestion. Furthermore, all treatments significantly reduced cardiomyocyte size and the expression of ANP and fibrosis marker. The combination of empagliflozin and KE showed the greatest improvement in diastolic function and cardiac ATP levels. Interestingly, hyperpolarized [1-13C] pyruvate detected a decrease in lactate/pyruvate ratio in the hearts treated with empagliflozin. ConclusionObese ZSF1 rats exhibit features of clinical HFpEF with predominant background of obesity and diabetes. A combination of empagliflozin and KE has greater benefits in treating HFpEF. Targeting cardiometabolic dysregulation may be a promising approach for HFpEF treatment.

Inhibition of the STIM1/Orai1 Signaling Pathway by Glycine Betaine Mitigates Myocardial Hypertrophy in Spontaneous Hypertension Rats.

Spontaneous hypertension is a leading risk factor for cardiovascular diseases morbidity and mortality. Glycine betaine (GB) is a natural vitamin that has the potential to lower blood pressure. This work attempted to investigate the role and mechanisms of GB in spontaneous hypertension. Spontaneously hypertensive rats (SHRs) were administrated with 100, 200, or 400 mg/kg of GB by gavage or combined with by injection of lentivirus-mediated STIM1 overexpression vector. The heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart weight/body weight (HW/BW) of rats were monitored. The pathological changes in myocardium were examined by hematoxylin and eosin staining and Masson staining. The expression of genes and proteins was detected by quantitative real-time PCR, western blotting, and immunohistochemistry. GB at 200 and 400 mg/kg reduced the HR, SBP, DBP and HW/BW in SHRs. GB decreased the cross-sectional area and fibrotic area in the myocardium and downregulated the expression of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) in the myocardium of SHRs. It indicated that GB treatment effectively alleviated myocardial hypertrophy in SHRs. Additionally, GB treatment repressed the expression of stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1) in the myocardium of SHRs. STIM1 overexpression reversed GB treatment-mediated inhibition of myocardial hypertrophy in SHRs. In conclusion, GB repressed STIM1/Orai1 signaling pathway, which contributed to alleviating myocardial hypertrophy in SHRs. Thus, our study provides a theoretical basis for GB as an antihypertensive drug.

Sacubitril/Valsartan Cannot Improve Cardiac Function Compared with Valsartan in Patients Suffering Nonvalvular Atrial Fibrillation without Systolic Heart Failure.

This study investigates the effect of sacubitril/valsartan (Sac/Val) in patients diagnosed with nonvalvular atrial fibrillation (AF) without systolic heart failure (SHF).Nonvalvular AF patients without SHF admitted to the People's Hospital of Bortala Mongol Autonomous Prefecture from December 2020 to December 2021 were enrolled and randomly divided into Sac/Val treatment group (group T) and valsartan treatment group (group C, control). For subgroup analysis, patients were divided into subgroups with and without diastolic heart failure (DHF). After 1-month adaptive phase and subsequent 3-month treatment period, patients were followed up in the cardiology clinic. Plasma levels of biochemical markers and echocardiographic parameters before and after treatment were evaluated, and DHF scores were computed to assess diastolic function.Of 61 enrolled patients, 46 patients completed follow-up. Sac/Val treatment did not increase the percentage of sinus rhythm. Although N-terminal pro-B-type natriuretic peptide (NT-proBNP) expression tended to be reduced in both groups after 3 months of treatment, the differences compared with respective baseline levels and between groups were not significant. According to subgroup analysis, although NT-proBNP expression in the subgroup with DHF was lower at follow-up compared to baseline, the difference was not statistically significant. Similarly, no marked differences in echocardiographic parameters or tissue Doppler parameters related to DHF were detected between the groups (P > 0.05). Additionally, a subgroup analysis found no significant variations in the echocardiographic measures (P > 0.05).Sac/Val is not superior to valsartan for the short-term treatment of patients suffering with AF without SHF in improving NT-proBNP level and cardiac function.

Melanocortin 1 receptor deficiency protects against pathological cardiac hypertrophy

Abstract Background The melanocortin system, including adrenocorticotropic hormone, melanocyte-stimulating hormones and five melanocortin receptors (MC-R), regulates important physiological functions such as pigmentation and energy homeostasis. It has been also recently implicated in the regulation of pathological cardiac remodeling but the role of different MC-R subtypes in the heart remain unknown. Purpose Based on the abundant cardiac expression of MC1-R, we hypothesized that it is functionally active in the heart and regulates pathological cardiac remodeling. Methods Rat H9c2 cells were used to study the effects of the selective MC1-R agonist LD211 on intracellular signaling cascades, gene expression and cellular growth response by using Western blotting, quantitative RT-PCR and 3H-leucine incorporation assay, respectively. Recessive yellow mice (Mc1re/e) were used as a model of global MC1-R deficiency. Inducible cardiomyocyte-specific MC1-R knockout mouse model (Mc1r-cKO) was engineered by intercrossing MC1-R floxed mice (Mc1rfl/fl) with tamoxifen-inducible Myh6-MerCreMer transgenic mice (Myh6-MCM). Mc1re/e and Mc1r-cKO mice were subjected to transverse aortic constriction (TAC, 8 wks) or subcutaneous infusion of angiotensin II (Ang II, 4 wks) to induce pathological cardiac hypertrophy. Cardiac structure and function were measured by echocardiography and by histological examination of H&amp;E-stained heart sections. Results Treatment of H9c2 cells with LD211 increased MAP-kinase p-38 phosphorylation (+53 %, P=0.005 vs control), upregulated the expression of B-type natriuretic peptide (Nppb, +40 %, P=0.03 vs control) and increased 3H-leucine incorporation (+27 %, P=0.006 vs control). Mc1re/e mice showed reduction in ventricular weight (-29 mg, P=0.03) compared to WT mice after TAC operation, and reduced left ventricular (LV) expression of atrial natriuretic peptide (Nppa) and fibrosis-related genes (Ctgf and Mmp2). In terms of LV systolic performance, TAC-operated Mc1re/e mice showed tendency towards improved LV ejection fraction (EF, +6 %, P=0.06) compared to WT mice. To further investigate whether the phenotype of Mc1re/e mice is driven by deficient MC1-R signaling in cardiomyocytes, we characterized Mc1r-cKO mice, which recapitulated the phenotypic features of Mc1re/e mice. Specifically, TAC-operated Mc1r-cKO mice displayed reduced ventricular weight (-38 mg, P=0.007) and downregulation of LV expression of Nppa and fibrosis-related genes compared to Myh6-MCM mice. Likewise, in a model of Ang II-induced cardiac hypertrophy, Mc1r-cKO mice showed reduced ventricular weight (-10 mg, P=0.015) compared to Myh6-MCM mice, indicating that the protection against pathological cardiac hypertrophy was model-independent. Conclusion Our studies demonstrate that MC1-R activation promotes cardiomyocyte growth in vitro, while global and cardiomyocyte-specific MC1-R deficiency protects against pathological cardiac hypertrophy induced by TAC or Ang II infusion.

Inhibition of ADAM10-mediated CX3CL1 shedding promotes survival and cardiac function early after myocardial infarction

Abstract Background and Purpose The metalloprotease ADAM10 has been implicated in immune regulatory processes, is upregulated in response to specific heart pathologies e.g. ischemic heart failure, and elevated serum levels of the ADAM10 substrates CX3CL1 and CXCL16 have been reported following infarction. The causal role of ADAM10 in cardiovascular diseases, however, is not known yet. By using human tissue biopsies, a cardiomyocyte-specific ADAM10 knockout mouse model (ADAM10 KO), and pharmacological ADAM10 inhibition we here comprehensively characterize the role of ADAM10 in the heart and in cardiac disease. Methods and Results We show that ADAM10 protein levels are upregulated in patients with ischemic cardiomyopathy (2.5-fold) and ADAM10 mRNA expression correlates with expression of atrial (NPPA) and brain natriuretic peptide (NPPB) mRNA. Upon infarction, ADAM10 expression is specifically induced in cardiomyocytes, and cardiomyocyte-specific ADAM10 KO as well as pharmacological inhibition (GI254023X) for only three days significantly improves overall survival, reduces scar size, and preserves cardiac function. Mechanistically, flow cytometry and bulk RNA sequencing identify a ADAM10/CX3CL1 axis to control neutrophil bone marrow egress and heart tissue infiltration after infarction. Diminished neutrophil migration results in long-term reduction of IL-1β-driven inflammation. Using cell culture experiments with ADAM10/CX3CL1 knockdown cardiomyocytes, we show that ADAM10-mediated ectodomain shedding of CX3CL1 specifically regulates neutrophil but not macrophage chemotaxis revealing a novel conceptual insight into how acute infarction triggers chemotactic signaling via ectodomain shedding. Conclusion Pharmacological and genetic targeting of the membrane sheddase ADAM10 is highly efficient for improving post-infarction cardiac function and survival via abolished post-translational CX3CL1 processing, neutrophil infiltration and IL-1β-dependent inflammation. Thus, our data reveal a novel therapeutic concept, which may close a clinically relevant therapeutic gap early after myocardial infarction.

Spatial transcriptomic analysis identifies a border zone with localized expression of brain natriuretic peptide in the inferobasal region of the left ventricle in patients with mitral valve prolapse

Abstract Background/Introduction Mitral valve prolapse (MVP) is a common cause of mitral regurgitation (MR), adverse left ventricular (LV) remodeling and arrhythmias. MVP induces mechanical stretch to the inferobasal (IB) myocardium and papillary muscles. The underlying regional and molecular pathology of MVP-induced LV remodeling remains incompletely understood. Purpose The aim was to characterize the molecular effects of mechanical stretch by comparing the regional gene expression of the IB region of LV with the interventricular septum in patients with MVP. Methods Myocardial biopsies were obtained from the IB LV (i.e. between papillary muscles) and the interventricular septum during mitral valve repair surgery for MVP-induced primary MR. The degree of cardiac fibrosis was detected and quantified by histology. We used spatial transcriptomics (ST) that - in contrast to traditional analyses of tissue homogenates - allows regional expressional analysis. Results Automated clustering of ST results correlated with histological staining of fibrosis in the IB and septal regions (R=0.97, P&amp;lt;0.0001), demonstrating good automated identification of fibrotic regions. The extent of fibrosis was similar between the inferobasal region and the septal control in the respective patients with MVP as determined by histological analysis and ST. The localized fibrotic areas of the inferobasal region showed decreased expression of cardiac myocytegenes such as RYR2, TNNI3, TNNC1 or TPM1, and increased expression of fibrous and EndMT markers such as COL3A1, BGN, SM22 or FN1. In the IB region, we identified a transition zone between healthy myocardium and fibrous tissue. This transition zone showed increased expression of the NPPB gene encoding brain natriuretic peptide (BNP), whereas NPPB was not increased in the septal region of the same patients. The upregulation of NPPB (6.4-fold, P=0.004) in the inferobasal region compared to the septal control was confirmed by qPCR in biopsies from patients with MVP. The identified transition zones were characterized by increased expression of genes such as IGFBP2 and LTBP2, which have been described to be associated with adverse LV remodeling in heart failure. Interestingly, the serum NT-proBNP levels (mean 276.8 ± 121.6 pg/ml) of the patients correlated with NPPB levels from the inferobasal region (R=0.76, P=0.01, N=9), whereas no correlation was found with NPPB levels from the septal control regions (R=0.17, P=0.65) of the same patients. Conclusions ST analysis in MVP identifies a transition zone of the inferobasal region of LV which is exposed to high mechanical stretch. This transition zone - but not the inferobasal fibrotic area nor the LV septum - is characterized by a specific increase in NBBP expression that correlates with increased NT-ProBNP serum concentrations. These special sequencing results indicate that mechanical stretch induces highly localized myocardial pathologies with systemic consequences.

Abstract 13962: Exploring the Therapeutic Potential of SGLT2 Inhibitors and Oral Ketone Ester in HFpEF: A Comprehensive Exploration of Cardiac Function, Structure, and Metabolism in a Rat Model

Introduction: This study aimed to assess the potential efficacy of the SGLT2 inhibitor empagliflozin and oral ketone ester supplementation as treatments for heart failure with preserved ejection fraction (HFpEF), which is commonly associated with obesity and type 2 diabetes mellitus. Methods: Twenty-week-old obese ZSF-1 rats were initially fed a high-fat diet (HFD) to establish and maintain the phenotype. Subsequently, the rats were randomly assigned to different treatment groups. These groups consisted of a control group receiving only the high-fat diet (HFD), as well as groups receiving empagliflozin, ketone ester (DeltaG®, KE), or a combination of both in addition to the HFD. Cardiac magnetic resonance imaging (CMR) and hyperpolarized carbon-13 (13C) labeled pyruvate magnetic resonance spectroscopy imaging were used to evaluate cardiac function and metabolism. Histological and molecular markers of cardiac remodeling were assessed in the left ventricle. Results: Untreated HFpEF rats exhibited obesity, diabetes, cardiac hypertrophy, atrial enlargement, lung congestion, increased cardiomyocyte size and fibrosis, as well as elevated expression of atrial natriuretic peptide (ANP), col1a1 (fibrosis marker), and TIMP1. The treatment groups displayed decreased body weight, increased circulating ketone levels, and reduced blood glucose levels. All treatment regimens significantly mitigated ventricular hypertrophy, atrial enlargement, and lung congestion. Furthermore, they reduced cardiomyocyte size, ANP expression, fibrosis marker expression, and increased cardiac ATP levels. CMR revealed that the combination of empagliflozin and KE resulted in improvement in diastolic function. Hyperpolarized [1-13C] pyruvate imaging revealed a decrease in lactate/pyruvate ratio in empagliflozin-treated hearts, indicating improved oxidative metabolism. Conclusions: The findings of this study suggest that targeting cardiometabolic dysregulation holds promise as an effective approach for the treatment of HFpEF. While both empagliflozin or KE alone demonstrated improvements in cardiac energetics and remodeling, the combination of empagliflozin and KE yielded the most notable benefits in enhancing cardiac diastolic function.

Abstract 15078: Atrial and Brain Natriuretic Peptides Expression and Release in Pulmonary Hypertension

Introduction: Precapillary pulmonary hypertension (PH) is characterized by progressive overload for the right ventricle and right heart failure (RHF). N-terminal Brain Natriuretic Peptides (NTproBNP) and Atrial Natriuretic Peptides (ANP) are secreted from cardiomyocytes upon stretch. NTproBNP is used as biomarker for RHF. Aim: To study the association between ANP and NTproBNP and pressure overload in blood, right heart tissue and induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from PH-patients. Methods: We analyzed NTproBNP in 344 PH-patients, 9 controls, and in 9 PH-patients before and 6-months after pulmonary endarterectomy (PEA). We measured NPPA and NPPB gene expression on end-stage human right ventricular (RV) and right atrial (RA) samples from 8 PH-patients and 5 controls. In addition, we performed immunofluoresence staining for ANP and BNP on RA tissue (N=4) and RV tissue (N=10) in PH and controls (N=5). iPSC-CMs from 1 male and 1 female PH-patients were stretched for 24h at 1Hz and 10% on the Flexcell FX-6000 system, and NTproBNP release was measured on static and stretched iPSC-CMs. NTproBNP levels were measured through ECLIA. Results: NTproBNP serum levels were elevated in PH-patients versus controls (918 vs 25 pg/mL, Fig1A). Pressure unloading in CTEPH patients did not result in a decrease in NTproBNP (Fig1B). In RV tissue of PH-patients NPPA expression was upregulated, but NPPB was not (Fig1C). ANP and BNP content was higher in RA and RV tissue of PH-patients (Fig1D-E). Finally, ~5x higher NTproBNP was released from male vs female iPSC-CMs under static conditions and after mechanical stretch (Fig1F). Conclusions: ANP and BNP showed important differences at gene and protein levels in relation to pressure overload. To confirm current results, further analyses on atrial and ventricular iPSC-CMs will be performed, in addition to mid-range proANP measurements in patients’ blood and iPSC-CMs conditioned medium.

Underlying mechanism of atrial fibrillation–associated Nppa-I137T mutation and cardiac effect of potential drug therapy

BackgroundMore than a hundred genetic loci have been associated with atrial fibrillation (AF). But the exact mechanism remains unclear and the treatment needs to be improved. ObjectiveThis study aimed to investigate the mechanism and potential treatment of NPPA mutation–associated AF. MethodsNppa knock-in (KI, p.I137T) rats were generated, and cardiac function was evaluated. Blood pressure was recorded using a tail-cuff system. The expression levels were measured using real-time polymerase chain reaction, enzyme-linked immunosorbent assay or Western blot analysis, and RNA-sequence analysis. Programmed electrical stimulation, patch clamp, and multielectrode array were used to record the electrophysical characteristics. ResultsMutant rats displayed downregulated expression of atrial natriuretic peptide but elevated blood pressure and enlarged left atrial end-diastolic diameter. Further, gene topology analysis suggested that the majority of differently expressed genes in Nppa KI rats were related to inflammation, electrical remodeling, and structural remodeling. The expression levels of C-C chemokine ligand 5 and galectin-3 involved in remodeling were higher, while there were declined levels of Nav1.5, Cav1.2, and connexin 40. AF was more easily induced in KI rats. Electrical remodeling included abbreviated action potentials, effective refractory period, increased late sodium current, and reduced calcium current, giving rise to conduction abnormalities. These electrophysiological changes could be reversed by the late sodium current blocker ranolazine and the Nav1.8 blocker A-803467. ConclusionOur findings suggest that structural remodeling related to inflammation and fibrosis and electrical remodeling involved in late sodium current underly the major effects of the Nppa (p.I137T) variant to induce AF, which can be attenuated by the late sodium current blocker and Nav1.8 blocker.

N-terminal pro atrial natriuretic peptide as a prognostic marker of cardiac resynchronization therapy recipients

BackgroundAlthough the dynamic changes of atrial natriuretic peptide (ANP) expressions in a failing heart are well-documented, the clinical implications of detailed measurements of each ANP molecular form processed from proANP remain unclear. MethodsPatients screening was conducted on patients who were eligible for cardiac resynchronization therapy (CRT) between 2014 and 2019 in our institution. Blood samples and echocardiographic parameters were collected on the day before and six months after implantation. Total ANP, proANP, and N-terminal fragment of proANP (NT-proANP) were examined as predictive biomarkers for cardiac death, left ventricular assist device implantation, and heart failure hospitalization following CRT implantation. ResultsA total of 86 subjects (mean age 70 years, 64 males) who underwent successful CRT implantation were enrolled. Plasma levels of total ANP, proANP, and NT-proANP were not normally distributed [25.8 pM (interquartile range: 11.1–53.1), 2.2 pM (1.0–5.4), and 4.1 nM (2.4–7.1), respectively]. Over a median follow-up of 2.7 years, 31 patients (2 deaths and 29 heart failure hospitalizations) reached the endpoints. Among the different ANP forms, only NT-proANP emerged as an independent predictor of the composite outcome (adjusted odds ratio of 2.542 in those with levels above vs. below the median, 95 % confidence interval 1.151–5.615, p = 0.021). NT-proANP levels were associated with left atrial volume and left diastolic functional parameters and decreased in response to echocardiographic improvements at six months post-implantation (16 ± 44 % decrease in responders vs 18 ± 60 % increase in non-responders, p = 0.005). ConclusionPre-implantation NT-proANP levels could serve as a predictive factor for clinical outcomes in recipients of CRT.