Murine Model Of Heart Failure Research Articles

Sex-dependent benefit of B vitamins on cardiac function and lipid metabolism in a murine model of heart failure with reduced ejection fraction

Background: Heart failure (HF) is a global health problem requiring more effective therapeutic alternatives. While HF involves disturbances in cardiac metabolism associated with lipidomic remodeling, there is a lack of approaches to specifically target cardiac metabolism. Recently, it has been shown in murine models of HF, that the use of nicotinamide riboside (vitamin B3) alone or the combination of B9 and B12 vitamins, before the development of HF, improved cardiac metabolism and function. Our hypothesis is that the combination of B3, B9 and B12 vitamins, used when HF is declared, will improve lipid metabolism, cardiac function and survival. The aim of this study is to evaluate the curative benefit of a synthetic diet enriched with the combination of these three vitamins (VitB) in a murine model of HF with reduced ejection fraction (EF). Method: Pressure overload was induced by constriction of the transverse aorta (TAC) in male and female mice. After 4 weeks, TAC mice reaching a pressure gradient of 60 mmHg, an increased left ventricle mass >30% and a reduced EF >10%, were randomized to a VitB-enriched diet or not. Results: In females, VitB improved survival and cardiac hypertrophy (-24%, p<0.05) as well as EF (+22%, p<0.01). We also observed a reduction of cardiac fibrosis emphasized by a significant decrease expression of collagen 1a and 3 (-30% and -19% respectively) and hydroxyproline concentration (-32%, p<0.01). No benefit was observed in males in terms of survival, cardiac function and fibrosis. Untargeted mass spectrometry (MS)-based lipidomics on plasmas at 8 weeks of treatment showed a decrease in several individual triglycerides (TG from 43 to 48 %; p<0.05) in TAC females that were normalized by VitB. In contrast, in TAC males, most of the annotated TGs were increased (from ̴35 to 210%; p<0.05), a profile exacerbated upon VitB treatment. MSMS identification of TG side chains revealed a differential profile in terms of fatty acid composition according to sex and treatment. In VitB females, longer and polyunsaturated TGs (53 to 60C; 5 to 12 double bonds) were significantly increased (1.6- to 2.1-fold) while, in VitB males, the increased TGs (1.2- to 5.2-fold) were shorter (24C to 48C) and saturated (no double bonds). Conclusion: Our study pointed out a sexual dimorphism in the response to VitB treatment in HF in favor of females and implying a lipidomic remodeling promoting cardioprotective polyunsaturated TGs. European Research Area Network on Cardiovascular Diseases, Canadian Institutes of Health Research, Fonds de recherche du Québec - Santé. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteomics couples electrical remodelling to inflammation in a murine model of heart failure with sinus node dysfunction.

In patients with heart failure (HF), concomitant sinus node dysfunction (SND) is an important predictor of mortality, yet its molecular underpinnings are poorly understood. Using proteomics, this study aimed to dissect the protein and phosphorylation remodelling within the sinus node in an animal model of HF with concurrent SND. We acquired deep sinus node proteomes and phosphoproteomes in mice with heart failure and SND and report extensive remodelling. Intersecting the measured (phospho)proteome changes with human genomics pharmacovigilance data, highlighted downregulated proteins involved in electrical activity such as the pacemaker ion channel, Hcn4. We confirmed the importance of ion channel downregulation for sinus node physiology using computer modelling. Guided by the proteomics data, we hypothesized that an inflammatory response may drive the electrophysiological remodeling underlying SND in heart failure. In support of this, experimentally induced inflammation downregulated Hcn4 and slowed pacemaking in the isolated sinus node. From the proteomics data we identified proinflammatory cytokine-like protein galectin-3 as a potential target to mitigate the effect. Indeed, in vivo suppression of galectin-3 in the animal model of heart failure prevented SND. Collectively, we outline the protein and phosphorylation remodeling of SND in heart failure, we highlight a role for inflammation in electrophysiological remodelling of the sinus node, and we present galectin-3 signalling as a target to ameliorate SND in heart failure.

A Murine Model of Hyperlipidemia-Induced Heart Failure with Preserved Ejection Fraction.

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Epigenetic modulators link mitochondrial redox homeostasis to cardiac function in a sex-dependent manner

While excessive production of reactive oxygen species (ROS) is a characteristic hallmark of numerous diseases, clinical approaches that ameliorate oxidative stress have been unsuccessful. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes, mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidative defense mechanism. Paradoxically reduced expression of IDH2 associated with ventricular eccentric hypertrophy is counterbalanced by an increase in the enzyme activity. We unveil redox-dependent sex dimorphism, and extensive mutual regulation of the antioxidative activities of IDH2 and NRF2 by a feedforward network that involves 2-oxoglutarate and L-2-hydroxyglutarate and mediated in part through unconventional hydroxy-methylation of cytosine residues present in introns. Consequently, conditional targeting of ROS in a murine model of heart failure improves cardiac function in sex- and phenotype-dependent manners. Together, these insights may explain why previous attempts to treat heart failure with antioxidants have been unsuccessful and open new approaches to personalizing and, thereby, improving such treatment.

Impairment of the adrenergic reserve associated with exercise intolerance in a murine model of heart failure with preserved ejection fraction.

Exercise intolerance is the central symptom in patients with heart failure with preserved ejection fraction. In the present study, we investigated the adrenergic reserve both invivo and in cardiomyocytes of a murine cardiometabolic HFpEF model. 12-week-old male C57BL/6J mice were fed regular chow (control) or a high-fat diet and L-NAME (HFpEF) for 15 weeks. At 27 weeks, we performed (stress) echocardiography and exercise testing and measured the adrenergic reserve and its modulation by nitric oxide and reactive oxygen species in left ventricular cardiomyocytes. HFpEF mice (preserved left ventricular ejection fraction, increased E/e', pulmonary congestion [wet lung weight/TL]) exhibited reduced exercise capacity and a reduction of stroke volume and cardiac output with adrenergic stress. In ventricular cardiomyocytes isolated from HFpEF mice, sarcomere shortening had a higher amplitude and faster relaxation compared to control animals. Increased shortening was caused by a shift of myofilament calcium sensitivity. With addition of isoproterenol, there were no differences in sarcomere function between HFpEF and control mice. This resulted in a reduced inotropic and lusitropic reserve in HFpEF cardiomyocytes. Preincubation with inhibitors of nitric oxide synthases or glutathione partially restored the adrenergic reserve in cardiomyocytes in HFpEF. In this murine HFpEF model, the cardiac output reserve on adrenergic stimulation is impaired. In ventricular cardiomyocytes, we found a congruent loss of the adrenergic inotropic and lusitropic reserve. This was caused by increased contractility and faster relaxation at rest, partially mediated by nitro-oxidative signaling.

Cardiac reverse remodeling in a mouse model with many phenotypical features of heart failure with preserved ejection fraction: effects of modifying lifestyle.

Multiple factors cause heart failure with preserved ejection fraction (HFpEF) and involve various systems. HFpEF prevalence is rapidly rising, and its prognosis remains poor after the first hospitalization. Adopting a more active lifestyle has been shown to provide beneficial clinical outcomes for patients with HFpEF. Using a two-hit HfpEF murine model, we studied cardiac reverse remodeling (RR) after stopping the causing stress and introducing voluntary exercise (VE). We checked in 2-mo-old male and female C57Bl6/J mice the heart's response to angiotensin II (ANG II; 1.5 mg/kg/day for 28 days) fed or not with a high-fat diet (HFD). Then, ANG II and/or the HFD were stopped, and VE was started for an additional 4 wk. ANG II and ANG II + HFD (metabolic-hypertensive stress, MHS) caused cardiac hypertrophy (CH) and myocardial fibrosis, left ventricular (LV) concentric remodeling, atrial enlargement, and reduced exercise capacity. HFD alone induced CH and LV concentric remodeling in female mice only. CH and LV concentric remodeling were reversed 4 wk after stopping ANG II, starting VE, and a low-fat diet. Left atrial enlargement and exercise capacity were improved but differed from controls. We performed bulk LV RNA sequencing and observed that MHS upregulated 58% of the differentially expressed genes (DEGs) compared with controls. In the RR group, compared with MHS animals, 60% of the DEGs were downregulated. In an HfpEF mouse model, we show that correcting hypertension, diet, and introducing exercise can lead to extensive cardiac reverse remodeling.NEW & NOTEWORTHY Using a two-hit murine model of heart failure with preserved ejection fraction (HfpEF), combining elevated blood pressure, obesity, and exercise intolerance in male and female animals, we showed that correction of hypertension, normalization of the diet, and introduction of voluntary exercise could help reverse the remodeling of the left ventricle and double exercise capacity. We also identify genes that escape normalization after myocardial recovery and differences between males' and females' responses to stress and recovery.

Sex differences in two-hit murine model of heart failure with preserved ejection fraction

Sex differences in two-hit murine model of heart failure with preserved ejection fraction

Quantitative investigation of the mechanisms of ventricular myocyte dysfunction in a murine model of heart failure with preserved ejection fraction

Quantitative investigation of the mechanisms of ventricular myocyte dysfunction in a murine model of heart failure with preserved ejection fraction

Chymase-independent vascular Ang-(1–12)/Ang II pathway and TXA2 generation are involved in endothelial dysfunction in the murine model of heart failure

The angiotensin (Ang)-(1–12)/Ang II pathway contributes to cardiac pathology. However, its involvement in the development of peripheral endothelial dysfunction associated with heart failure (HF) remains unknown. Therefore, this study aimed to characterise the effect of exogenous Ang-(1–12) and its conversion to Ang II on endothelial function using the murine model of HF (Tgαq*44 mice), focusing on the role of chymase and vascular-derived thromboxane A2 (TXA2).Ex vivo myographic assessments of isolated aorta showed impaired endothelium-dependent vasodilation in late-stage HF in 12-month-old Tgαq*44 mice. However, endothelium-dependent vasodilation was fully preserved in the early stage of HF in 4-month-old Tgαq*44 mice and 4- and 12-month-old FVB control mice. Ang-(1–12) impaired endothelium-dependent vasodilation in 4- and 12-month-old Tgαq*44 mice, that was associated with increased Ang II production. The chymase inhibitor chymostatin did not inhibit this response. Interestingly, TXA2 production reflected by TXB2 measurement was upregulated in response to Ang-(1–12) and Ang II in aortic rings isolated from 12-month-old Tgαq*44 mice but not from 4-month-old Tgαq*44 mice or age-matched FVB mice. Furthermore, in vivo magnetic resonance imaging showed that Ang-(1–12) impaired endothelium-dependent vasodilation in the aorta of Tgαq*44 mice and FVB mice. However, this response was inhibited by angiotensin I converting enzyme (ACE) inhibitor; perindopril, angiotensin II receptor type 1 (AT1) antagonist; losartan and TXA2 receptor (TP) antagonist-picotamide in 12-month-old-Tgαq*44 mice only.In conclusion, the chymase-independent vascular Ang-(1–12)/Ang II pathway and subsequent TXA2 overactivity contribute to systemic endothelial dysfunction in the late stage of HF in Tgαq*44 mice. Therefore, the vascular TXA2 receptor represents a pharmacotherapeutic target to improve peripheral endothelial dysfunction in chronic HF.

Cardiac radiation improves ventricular function in mice and humans with cardiomyopathy.

Rapidly dividing cells are more sensitive to radiation therapy (RT) than quiescent cells. In the failing myocardium, macrophages and fibroblasts mediate collateral tissue injury, leading to progressive myocardial remodeling, fibrosis, and pump failure. Because these cells divide more rapidly than cardiomyocytes, we hypothesized that macrophages and fibroblasts would be more susceptible to lower doses of radiation and that cardiac radiation could therefore attenuate myocardial remodeling. In three independent murine heart failure models, including models of metabolic stress, ischemia, and pressure overload, mice underwent 5 Gy cardiac radiation or sham treatment followed by echocardiography. Immunofluorescence, flow cytometry, and non-invasive PET imaging were employed to evaluate cardiac macrophages and fibroblasts. Serial cardiac magnetic resonance imaging (cMRI) from patients with cardiomyopathy treated with 25 Gy cardiac RT for ventricular tachycardia (VT) was evaluated to determine changes in cardiac function. In murine heart failure models, cardiac radiation significantly increased LV ejection fraction and reduced end-diastolic volume vs. sham. Radiation resulted in reduced mRNA abundance of B-type natriuretic peptide and fibrotic genes, and histological assessment of the LV showed reduced fibrosis. PET and flow cytometry demonstrated reductions in pro-inflammatory macrophages, and immunofluorescence demonstrated reduced proliferation of macrophages and fibroblasts with RT. In patients who were treated with RT for VT, cMRI demonstrated decreases in LV end-diastolic volume and improvements in LV ejection fraction early after treatment. These results suggest that 5 Gy cardiac radiation attenuates cardiac remodeling in mice and humans with heart failure. NIH, ASTRO, AHA, Longer Life Foundation.

PDE2 is cardioprotective in chronic heart failure

Abstract Background Patients with chronic heart failure (HF) often develop life-threatening arrhythmia due to structural and electrophysiological cardiac remodelling. The chronic activation of the sympathetic nervous system is initially able to compensate the reduced myocardial contraction. Finally, long-term activation of the β-adrenergic pathway leads to deleterious activation of cAMP-dependent kinases and dysregulation of intracellular Ca2+ handling promoting HF progression and development of arrhythmia. Interestingly, phosphodiesterase 2 (PDE2), an enzyme to hydrolyze cAMP, is upregulated in human HF. Thus, PDE2 might reduce enhanced cAMP levels and pro-arrhythmic Ca2+ releases in HF. Purpose We investigated the effects of genetic PDE2 deletion on cardiac function, cellular contraction and arrhythmia occurrence in murine HF model. Methods HF was induced via high fat (60 %) diet (HFD) and the treatment with NO-synthase inhibitor L-NAME (0.5 g/l) for 5 weeks in cardiomyocyte-specific PDE2-knockout mice (KO) and control mice (WT). We characterized cardiac function by echocardiography, arrhythmia development by ECG-telemetry, cellular contraction by video-based analysis of sarcomere-shortening and cellular Ca2+ releases using Ca2+ imaging techniques. Results After 5 weeks HFD and L-NAME, mice gained weight, displayed increased blood pressure and reduced systolic and diastolic cardiac function. Interestingly, PDE2 KO showed reduced contractility and ejection fraction compared to WT mice after 5 weeks HF. On cellular level, the contraction of isolated cardiomyocytes slightly increased in HF indicating cardiac hypertrophy. However, the β-adrenergic stimulation with isoprenaline (ISO) enhanced cellular contraction only in non-failing WT mice, but was blunted in cells from failing WT hearts reflecting a desensitization of the β-adrenergic pathway. In contrast, the ISO-induced increase in cellular contraction was preserved in failing hearts from KO mice. Interestingly, PDE2 inhibition with BAY 60-7550 increased the ISO-induced cellular contraction in HF from WT, which was not observed in cells from non-failing mice. In HF, the expression of Ca2+ cycling proteins like phospholamban (PLB) were affected. Failing PDE2 KO mice showed higher phosphorylation levels of PLB compared to WT mice with HF. In diseased hearts, pro-arrhythmic triggers like spontaneous Ca2+ waves and Ca2+ sparks were increased. Importantly, diseased PDE2 KO mice developed more arrhythmia in-vivo upon arrhythmia provocation than WT animals. Conclusion Cardiac PDE2 deletion was detrimental in HF showing reduced cardiac function and higher number of arrhythmia. PDE2 stimulation might serve as potential therapeutical target in HF limiting the consequences of chronic β-adrenergic hyperactivation.

Abstract 15000: Renal Denervation Improves Cardiac Function and Mitigates Pathophysiological Changes in a Novel Murine Model of Heart Failure With Preserved Ejection Fraction

Introduction: Heart Failure with Preserved Ejection Fraction (HFpEF) is a multifaceted disease, intricately linked with sympathetic nervous system activation. Despite advancements, therapeutic options for HFpEF remain constrained, largely due to a dearth of fitting preclinical models. Renal denervation (RDN), an emerging intervention, holds potential in attenuating sympathetic activity and ameliorating HFpEF high-risk factors such as hypertension and insulin resistance. Aims: our study is designed to establish a multifactorial HFpEF mouse model and assess the therapeutic implications of RDN. Methods: Aged 12-month-old male C57BL/6N mice were exposed to a high-fat diet (60% calories from lard) and L-NAME (0.5 g/L in drinking water) over a 5-week period to generate a multi-hit HFpEF model. The efficacy of the model was assessed through a range of methods, encompassing echocardiography, histology, exercise exhaustion tests, serum NT-proBNP measurements, and intraperitoneal glucose tolerance tests. Upon successful model establishment, mice were randomized to receive either bilateral renal denervation or a sham operation. The subsequent impact of RDN on the aforementioned parameters was then evaluated. Results: In C57BL/6N mice, a cardiometabolic HFpEF phenotype was effectively induced through a multi-hit approach encompassing a high-fat diet, L-NAME administration, and aging. This potent combination triggered obesity and glucose intolerance, culminating in the emergence of characteristic HFpEF features such as cardiac hypertrophy, fibrosis, preserved fractional shortening with impaired deformation, atrial enlargement, lung congestion, hypertension and decreased exercise endurance. Intervention with RDN mitigated this cardiometabolic dysregulation and enhanced cardiac function, as demonstrated by reductions in cardiac hypertrophy, myocardial fibrosis, atrial weight, NT-proBNP levels, lung congestion, and improved exercise endurance. Conclusions: Our study suggests that RDN effectively mitigates cardiac function and structure in a multi-hit HFpEF mouse model, indicating its potential as a promising therapeutic strategy for HFpEF.

Abstract 15504: Impaired Branched-Chain Amino Acid Catabolism is Associated With Chemotherapy-Induced Heart Failure and Rescued by Metformin

Introduction: Heart failure resulting from cancer therapies represents a major cause of morbidity in cancer survivors. Interventional strategies to prevent and treat heart dysfunction in cardio-oncology patients continue to remain a challenge. Hypothesis: We posit that Branched-Chain Amino Acid Catabolism (BCAA) catabolism dysfunction is a likely causal mechanistic pathway in the pathogenesis of CTHF. Methods: In this study, we leveraged electronic health records , human-induced pluripotent stem cell ( iPSC )-derived cardiomyocyte model-based mechanistic studies, murine models, and network medicine-basedtranscriptomics analytic methodologies, to suggest that rescuing BCAA catabolic dysfunction is a promising approach for managing chemotherapy-induced heart failure, using doxorubicin as a prototypical example. Results: In our large-scale cardio-oncology cohort, we observed a significant incidence of heart dysfunction and abnormal NT-proBNP levels among patients treated with doxorubicin. Using murine heart failure models, we identified significant correlations between the abundance of branched-chain amino acids (isoleucine, leucine, and valine) and cardiac dysfunction after doxorubicin exposure. Mechanistic observations in human-induced pluripotent stem cell (iPSC)-derived cardiomyocyte models further support impaired branched-chain amino acid (BCAA) catabolism by doxorubicin. Through metabolite-enzyme network analysis, we identified metformin as a repurposable drug capable of restoring branched-chain amino acid catabolism, thereby improving mitochondrial function. Human iPSC-based mechanistic studies revealed that metformin combined with doxorubicin resulted in improved insulin signaling, endothelial cell reprogramming, increased RARA gene expression (a key doxorubicin-induced heart failure GWAS gene) and reduced pro-fibrotic signaling. Furthermore, we demonstrate that metformin improved murine cardiac function following doxorubicin exposure. Conclusions: Our findings utilized electronic health records, iPSC-based mechanistic studies, and murine models to suggest that BCAA catabolic dysfunction may be a promising approach for managing chemotherapy-induced heart failure.

Abstract 15777: Vascular Density and Fibrosis in Heart Failure Recovery

Introduction: Chronic unloading with a left ventricular assist device (LVAD) leads to LV remodeling including increased endothelial density, reduction of fibrosis, and improved cardiac function. Specific mechanisms of such recovery have not been fully explored in humans. Hypothesis: We hypothesize that recovery from non-ischemic heart failure is accompanied by increased vascular density and reduction in fibrosis. Methods: We developed and characterized a murine model of non-ischemic heart failure (HF) and recovery (HFR) using Angiotensin II infusion pumps and L-NAME/NaCl in water for 5 weeks followed by removal of these insults to induce a recovery. Ejection fraction, LV mass, fibrosis, and vessel density were assessed at various time points. We quantified vessel density using fluorescent microsphere bead perfusion and measured fibrosis using Masson’s Trichrome staining. Statistical analysis was performed using One way ANOVA, n=5 mice/group with 2 replicate experiments. ( p <0.05 was considered significant.)Results: LV mass and interstitial fibrosis increased significantly while ejection fraction and vascular density showed significant decreases in HF vs control mice while recovering hearts showed normalized ejection fraction and LV mass with a significant reduction in fibrosis. There was a very significant increase in vascular density when compared to HF mice (Fig. 1). Together, these preliminary data support our hypothesis that recovery from heart failure involves a vascular recovery. Conclusions: Morphological recovery of cardiac function is accompanied by an increase in vascular density along with a reduction in fibrosis in a non-ischemic mouse model of heart failure recovery. This increase in vascular density may be a required component of the recovery process from heart failure and further studies are necessary to understand the specifics of such mechanisms.

Exploring the mechanisms underlying stroke volume variability reduction in a murine model of heart failure with reduced ejection fraction.

Heart failure with reduced ejection fraction (HFrEF) is accompanied by disregulation of cardiovascular function. Heart rate variability (HRV) is commonly used to assess autonomic dysfunction in HFrEF. However, analysis of stroke volume variability (SVV) may provide additional insights. We examined HRV and SVV in a mouse model of HFrEF. HFrEF mice exhibited reduced stroke volume and ejection fraction versus controls, confirming cardiac contractile dysfunction. HRV was preserved in HFrEF mice. However, SVV was markedly diminished, indicating dissociation between HRV and SVV regulation. Using a mathematical model, we propose that Frank-Starling mechanism abnormalities in HFrEF disrupt SVV independent of HRV. Assessing SVV could thus provide unique insights beyond HRV into cardiovascular control deficits in HFrEF.

Protective effects of Gαi3 deficiency in a murine heart-failure model of β1-adrenoceptor overexpression

We have shown that in murine cardiomyopathy caused by overexpression of the β1-adrenoceptor, Gαi2-deficiency is detrimental. Given the growing evidence for isoform-specific Gαi-functions, we now examined the consequences of Gαi3 deficiency in the same heart-failure model. Mice overexpressing cardiac β1-adrenoceptors with (β1-tg) or without Gαi3-expression (β1-tg/Gαi3−/−) were compared to C57BL/6 wildtypes and global Gαi3-knockouts (Gαi3−/−). The life span of β1-tg mice was significantly shortened but improved when Gαi3 was lacking (95% CI: 592–655 vs. 644–747 days). At 300 days of age, left-ventricular function and survival rate were similar in all groups. At 550 days of age, β1-tg but not β1-tg/Gαi3−/− mice displayed impaired ejection fraction (35 ± 18% vs. 52 ± 16%) compared to wildtype (59 ± 4%) and Gαi3−/− mice (60 ± 5%). Diastolic dysfunction of β1-tg mice was prevented by Gαi3 deficiency, too. The increase of ANP mRNA levels and ventricular fibrosis observed in β1-tg hearts was significantly attenuated in β1-tg/Gαi3−/− mice. Transcript levels of phospholamban, ryanodine receptor 2, and cardiac troponin I were similar in all groups. However, Western blots and phospho-proteomic analyses showed that in β1-tg, but not β1-tg/Gαi3−/− ventricles, phospholamban protein was reduced while its phosphorylation increased. Here, we show that in mice overexpressing the cardiac β1-adrenoceptor, Gαi3 deficiency slows or even prevents cardiomyopathy and increases shortened life span. Previously, we found Gαi2 deficiency to aggravate cardiac dysfunction and mortality in the same heart-failure model. Our findings indicate isoform-specific interventions into Gi-dependent signaling to be promising cardio-protective strategies.

Mechanism of lncRNA HOTAIR in attenuating cardiomyocyte pyroptosis in mice with heart failure via the miR-17-5p/RORA axis

Heart failure (HF) is a complex clinical syndrome associated with significant morbidity and mortality. Dysregulation of long non-coding RNA (lncRNA) has been implicated in the pathogenesis of HF. The present study aims to investigate the role of lncRNA HOX transcript antisense RNA (HOTAIR) in cardiomyocyte pyroptosis in a murine HF model. A murine HF model was established through transverse aortic contraction surgery, and an in vitro HF cell model was developed by treating HL-1 cells with H2O2. HOTAIR was overexpressed in TAC mice and HL-1 cells via pcDNA3.1-HOTAIR transfection. Cardiac function was assessed in TAC mice, and myocardial changes were evaluated using HE staining. The expression of NLRP3 was examined by immunohistochemistry. Myocardial injury markers and pyroptosis-related inflammatory cytokines were quantified using ELISA. Protein levels of NLRP3, cleaved-caspase-1, and GSDMD-N were analyzed by Western blot. Dual-luciferase assays and RNA immunoprecipitation were employed to confirm the binding interactions between HOTAIR and miR-17-5p, miR-17-5p and RORA. Functional rescue experiments were conducted by overexpressing miR-17-5p or silencing RORA in HL-1 cells. HOTAIR exhibited reduced expression in TAC mice and H2O2-induced cardiomyocytes. Overexpression of HOTAIR ameliorated cardiac dysfunction, reduced myocardial pathological injury, enhanced cardiomyocyte viability, and decreased myocardial injury and pyroptosis. HOTAIR interacted with miR-17-5p to repress RORA transcription. Overexpression of miR-17-5p or silencing of RORA abolished the inhibitory effect of HOTAIR overexpression on cardiomyocyte pyroptosis. In conclusion, HOTAIR competitively bound to miR-17-5p, relieving its inhibition of RORA transcription and leading to increased RORA expression and suppressed cardiomyocyte pyroptosis in HF models.

Abstract P2011: Reduced Activity Of Activin Like Kinase 1 (ALK1) Increases Mortality And Myocardial Rupture After Acute Myocardial Infarction

Introduction: Acute myocardial infarction (AMI) is a primary cause of heart failure (HF). Transforming growth factor beta (TGFβ) plays a central role in cardiac remodeling after AMI. Reduced activity of the TGFβ Type I receptor, activin like kinase (ALK)-1 promotes cardiac fibrosis in murine models of heart failure. A functional role for ALK1 in post-infarct cardiac remodeling remains poorly understood. We hypothesize that reduced ALK1 activity reduces survival and promotes cardiac fibrosis after AMI. Methods: AMI was induced by left coronary ligation in wild type (WT), and ALK+/- mice. After 14 days, LV function was assessed using conductance catheters and myocardial ALK1 signaling was quantified. Using siRNAs targeting ALK1 in in human cardiac fibroblasts (H-CFs), we further tested the role of ALK1 in cardiac fibrosis. Results: Reduced ALK1 activity significantly reduced survival compared to WT (p= 0.008 respectively) after AMI. ALK1 mice demonstrated a higher rate of cardiac rupture compared to WT after AMI (0% vs 85.7%). Both WT ad ALK1+/- mice demonstrated reduced LV ejection fraction and stroke work compared to sham controls (p=0.001 and p=0.0001 respectively). Profibrotic SMAD3 signaling was increased and antifibrotic SMAD1 signaling decreased in ALK1-/+ (pSMAD3, p=0.03, pSMAD1, p=0.003) mice 14 days after AMI. Reduced SMAD1 signaling was directly associated with increased myocardial Type 1 collagen protein levels in ALK1-/+ (R2=0.42, p=0.01) mice compared to WT. Myocardial levels of the TGFb1 co-receptor, endoglin, were increased significantly in ALK+/- as compared to WT (p=0.001) after AMI. Activity of MMP9 was increased in ALK1+/- mice, but protein levels were not affected 2 weeks after MI. Conclusion: We identify for the first time that loss of ALK1 activity increases cardiac rupture and mortality after AMI. These findings identify a novel functional role for ALK1 as a determinant of post-infarct survival and cardiac remodeling and implicate ALK1 as a potential therapeutic target to improve survival after AMI.

Inhibition of miR-25 Ameliorates Cardiac Dysfunction and Fibrosis by Restoring Krüppel-like Factor 4 Expression.

Cardiac hypertrophy is an adaptive response to various pathological insults, including hypertension. However, sustained hypertrophy can cause impaired calcium regulation, cardiac dysfunction, and remodeling, accompanied by cardiac fibrosis. Our previous study identified miR-25 as a regulator of SERCA2a, and found that the inhibition of miR-25 improved cardiac function and reduced fibrosis by restoring SERCA2a expression in a murine heart failure model. However, the precise mechanism underlying the reduction in fibrosis following miR-25 inhibition remains unclear. Therefore, we postulate that miR-25 may have additional targets that contribute to regulating cardiac fibrosis. Using in silico analysis, Krüppel-like factor 4 (KLF4) was identified as an additional target of miR-25. Further experiments confirmed that KLF4 was directly targeted by miR-25 and that its expression was reduced by long-term treatment with Angiotensin II, a major hypertrophic inducer. Subsequently, treatment with an miR-25 inhibitor alleviated the cardiac dysfunction, fibrosis, and inflammation induced by Angiotensin II (Ang II). These findings indicate that inhibiting miR-25 not only enhances calcium cycling and cardiac function via SERCA2a restoration but also reduces fibrosis by restoring KLF4 expression. Therefore, targeting miR-25 may be a promising therapeutic strategy for treating hypertensive heart diseases.

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models.

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While exercise-stressed hemodynamic tests or MRI can be used to detect diastolic dysfunction and diagnose HFpEF in humans, such modalities are limited in basic research using mouse models. A treadmill exercise test is commonly used for this purpose in mice, but its results can be influenced by body weight, skeletal muscle strength, and mental state. Here, we describe an atrial-pacing protocol to detect heart rate (HR)-dependent changes in diastolic performance and validate its usefulness in a mouse model of HFpEF. The method involves anesthetizing, intubating, and performing pressure-volume (PV) loop analysis concomitant with atrial pacing. In this work, a conductance catheter was inserted via a left ventricular apical approach, and an atrial pacing catheter was placed in the esophagus. Baseline PV loops were collected before the HR was slowed with ivabradine. PV loops were collected and analyzed at HR increments ranging from 400 bpm to 700 bpm via atrial pacing. Using this protocol, we clearly demonstrated HR-dependent diastolic impairment in a metabolically induced HFpEF model. Both the relaxation time constant (Tau) and the end-diastolic pressure-volume relationship (EDPVR) worsened as the HR increased compared to control mice. In conclusion, this atrial pacing-controlled protocol is useful for detecting HR-dependent cardiac dysfunctions. It provides a new way to study the underlying mechanisms of diastolic dysfunction in HFpEF mouse models and may help develop new treatments for this condition.