Murine Model Of Heart Failure Research Articles

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.

Ketone Bodies Rescue Mitochondrial Dysfunction Via Epigenetic Remodeling

Ischemic cardiac disease is a major cause of mortality worldwide. However, the exact molecular processes underlying this disorder are not fully known. This study includes a comprehensive and coordinated set of invivo and invitro experiments using human cardiac specimens from patients with postischemic heart failure (HF) and healthy control subjects, a murine model of HF, and cellular systems. These approaches identified for the first time a specific pattern of maladaptive chromatin remodeling, namely a double methylation of histone 3 at lysine 27 and a single methylation at lysine 36 (H3_K27me2K36me1) consistently induced by ischemic injury in all these settings: human HF; murine HF; and invitro models. Mechanistically, this work demonstrates that this histone modification mediates the ischemia-induced transcriptional repression of PPARG coactivator 1α (PGC1α), master regulator of mitochondrial function and biogenesis. Intriguingly, both the augmented H3_K27me2K36me1 and the mitochondrial dysfunction ensued by PGC1α down-regulation were significantly attenuated by the treatment with β-hydroxybutyrate, the most abundant ketone body in humans, revealing a novel pathway coupling metabolism to gene expression. Taken together, these findings establish maladaptive chromatin remodeling as a key mechanism in postischemic heart injury, functionally modulated by ketone bodies.

Efficacy of a growth hormone-releasing hormone agonist in a murine model of cardiometabolic heart failure with preserved ejection fraction.

Heart failure (HF) with preserved ejection fraction (HFpEF) represents a major unmet medical need owing to its diverse pathophysiology and lack of effective therapies. Potent synthetic, agonists (MR-356 and MR-409) of growth hormone-releasing hormone (GHRH) improve the phenotype of models of HF with reduced ejection fraction (HFrEF) and in cardiorenal models of HFpEF. Endogenous GHRH exhibits a broad range of regulatory influences in the cardiovascular (CV) system and aging and plays a role in several cardiometabolic conditions including obesity and diabetes. Whether agonists of GHRH can improve the phenotype of cardiometabolic HFpEF remains untested and unknown. Here we tested the hypothesis that MR-356 can mitigate/reverse the cardiometabolic HFpEF phenotype. C57BL6N mice received a high-fat diet (HFD) plus the nitric oxide synthase inhibitor (l-NAME) for 9 wk. After 5 wk of HFD + l-NAME regimen, animals were randomized to receive daily injections of MR-356 or placebo during a 4-wk period. Control animals received no HFD + l-NAME or agonist treatment. Our results showed the unique potential of MR-356 to treat several HFpEF-like features including cardiac hypertrophy, fibrosis, capillary rarefaction, and pulmonary congestion. MR-356 improved cardiac performance by improving diastolic function, global longitudinal strain (GLS), and exercise capacity. Importantly, the increased expression of cardiac pro-brain natriuretic peptide (pro-BNP), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor-A (VEGF-A) was restored to normal levels suggesting that MR-356 reduced myocardial stress associated with metabolic inflammation in HFpEF. Thus, agonists of GHRH may be an effective therapeutic strategy for the treatment of cardiometabolic HFpEF phenotype.NEW & NOTEWORTHY This randomized study used rigorous hemodynamic tools to test the efficacy of a synthetic GHRH agonist to improve cardiac performance in a cardiometabolic HFpEF. Daily injection of the GHRH agonist, MR-356, reduced the HFpEF-like effects as evidenced by improved diastolic dysfunction, reduced cardiac hypertrophy, fibrosis, and pulmonary congestion. Notably, end-diastolic pressure and end-diastolic pressure-volume relationship were reset to control levels. Moreover, treatment with MR-356 increased exercise capacity and reduced myocardial stress associated with metabolic inflammation in HFpEF.

PS-BPB09-5: BROWN ADIPOSE TISSUE DYSFUNCTION PROMOTES METABOLIC DISORDER IN A FAILING HEART.

Objective: Low body temperature predicts poor clinical outcomes in patients with heart failure, however, underlying mechanisms and pathological implications are largely unknown. Brown adipose tissue (BAT) was initially characterized as a heat generating organ, and studies suggest it has crucial roles for maintaining systemic metabolic health. The purpose of this study was to determine whether alteration of BAT function contributes to development of heart failure. Design and method: We generated two murine heart failure models, thoracic aortic constriction (TAC) and myocardial infarction (MI). We analyzed cardiac function, thermogenic response, and pathological findings in the hearts of these mice. We also performed comprehensive metabolomic analyses in the heart, BAT, and plasma samples from sham- and TAC-operated mice. Results: We found murine models with TAC or MI induced BAT dysfunction, and this led to a significant reduction in body temperature. Chronic activation of adrenergic signaling induced apoptosis in brown adipocytes, and surgical denervation of sympathetic nerves innervating BAT improved thermogenic response and cardiac systolic dysfunction. Gain of BAT function model, generated with BAT implantation into peritoneal cavity, improved thermogenesis and ameliorated cardiac dysfunction in TAC. In contrast, systolic function was reduced in genetically BAT dysfunctional model, and this associated with poor survival rate in this model. Metabolomic analyses showed that BAT dysfunction led to an increase of choline and its downstream metabolite, trimethylamine N-oxide (TMAO) that promoted metabolic dysfunction in the failing heart. TMAO administration resulted in a significant reduction in phosphocreatine or ATP level in cardiac tissues, and this was mediated by the suppression of complex IV activity in mitochondria. Genetic or pharmacological inhibition of TMAO production ameliorated cardiac dysfunction during left ventricular pressure overload. Dilated cardiomyopathy patients had lower body temperature, and this associated with an increase in circulating choline and TMAO level. Conclusions: Alteration of BAT function contributes to the development of heart failure via increased TMAO. Our findings suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

Endoplasmic reticulum stress-activated nuclear factor-kappa B signaling pathway induces the upregulation of cardiomyocyte dopamine D1 receptor in heart failure

Dopamine D1 receptor (D1R), coded by the Drd1 gene, is induced in cardiomyocytes of failing hearts, triggering heart failure-associated ventricular arrhythmia, and therefore could be a potential therapeutic target for chronic heart failure. The regulation of D1R expression, however, is not fully understood. Here, we explored the molecular mechanism by which cardiomyocyte D1R is induced in failing hearts. We performed motif analysis for the promoter region of the Drd1 gene using the transcription factor affinity prediction (TRAP) method and identified nuclear factor-kappa B (NF-κB) as a candidate transcriptional factor regulating the expression of the Drd1 gene. We next employed murine models of heart failure from chronic pressure overload by transverse aortic constriction (TAC), and assessed myocardial Drd1 expression levels and NF-κB activity, as well as endoplasmic reticulum (ER) stress, which has been implicated in the pathogenesis of heart failure. Drd1 induction in TAC hearts was dependent on the severity of heart failure, and was associated with NF-κB activation and ER stress, as assessed by p65 phosphorylation and the expression of ER stress-related genes, respectively. We further tested if Drd1 was induced by ER stress via NF-κB activation in cultured neonatal rat ventricular myocytes. Tunicamycin activated NF-κB pathway in an ER stress-dependent manner and increased Drd1 expression. Importantly, inhibition of NF-κB pathway by pretreatment with Bay11-7082 completely suppressed the tunicamycin-induced upregulation of Drd1, suggesting that NF-κB activation is essential to this regulation. Our study demonstrates the pivotal role for the ER stress-induced NF-κB activation in the induction of D1R in cardiomyocytes. Intervention of this pathway might be a potential new therapeutic strategy for heart failure-associated ventricular arrhythmia.

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

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.

Targeting the Autophagy-Lysosome Pathway in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure

The key biological "drivers" that are responsible for reverse left ventricle (LV) remodeling are not well understood. To gain an understanding of the role of the autophagy-lysosome pathway in reverse LV remodeling, we used a pathophysiologically relevant murine model of reversible heart failure, wherein pressure overload by transaortic constriction superimposed on acute coronary artery (myocardial infarction) ligation leads to a heart failure phenotype that is reversible by hemodynamic unloading. Here we show transaortic constriction+ myocardial infarction leads to decreased flux through the autophagy-lysosome pathway with the accumulation of damaged proteins and organelles in cardiac myocytes, whereas hemodynamic unloading is associated with restoration of autophagic flux to normal levels with incomplete removal of damaged proteins and organelles in myocytes and reverse LV remodeling, suggesting that restoration of flux is insufficient to completely restore myocardial proteostasis. Enhancing autophagic flux with adeno-associated virus 9-transcription factor EB resulted in more favorable reverse LV remodeling in mice that had undergone hemodynamic unloading, whereas overexpressing transcription factor EB in mice that have not undergone hemodynamic unloading leadsto increased mortality, suggesting that thetherapeutic outcomes of enhancing autophagic flux will depend on the conditions in which flux is beingstudied.

Empagliflozin improves cardiac mitochondrial function and survival through energy regulation in a murine model of heart failure.

BackgroundSodium-glucose cotransporter 2 (SGLT2) inhibitors have been demonstrated to have beneficial effects on HF in large clinical trials; however, the mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms by which empagliflozin, one of SGLT2 inhibitors, affects heart failure. Method and resultsEight-week-old male mice deficient for heart and skeletal muscle-specific manganese superoxide dismutase (MnSOD-cKO mice), a murine model of dilated cardiomyopathy, were given food mixed with or without 10 mg/kg empagliflozin for 7 weeks and evaluated. Both the survival rate and cardiac fibrosis were significantly improved in the empagliflozin group. The capacity for oxidative phosphorylation in cardiac mitochondria was significantly upregulated as measured with Oxygraph-2k respirometer, and blood lactate levels produced by anaerobic metabolism were significantly lower in the empagliflozin group. Energy expenditure was significantly improved in the empagliflozin group, measured by respiratory gas analysis, with a concomitant reduction in serum leptin concentration and increase in food intake. A moderate amount of glucose was excreted in urine in the empagliflozin group; however, the available energy substrate in the body nonetheless expanded because of the much higher caloric intake. ConclusionsWe conclude that empagliflozin improved cardiac mitochondrial function and upregulated energy metabolism even in HF in mice. These findings provide novel mechanisms for the beneficial effects of SGLT2 inhibitors on HF.

Effects of Finerenone, a Novel Nonsteroidal Mineralocorticoid Receptor Antagonist, on Cardiovascular Disease, Chronic Kidney Disease, and Blood Pressure.

Finerenone, an FDA-approved nonsteroidal mineralocorticoid receptor (MR) antagonist, has been evaluated in context of chronic kidney disease (CKD) and associated cardiovascular disease (CVD). In this review, we summarize pre-clinical and clinical studies focused on the impact of finerenone on these disease processes. Activation of the MR upregulates genes encoding for facilitators of tissue damage. Finerenone binding to a helix domain in this receptor inhibits receptor function. Studies in murine models of kidney disease, heart failure, hypertension, and vascular injury demonstrate significant protective effects of finerenone against further disease progression, as well as association with reduced oxidative stress, inflammation, and fibrosis. Phase 1-3 clinical trials with finerenone show safety and efficacy in improving renal and cardiovascular outcomes in patients with CKD. Research thus far encourages the addition of finerenone to the standard of care for certain CKD patients, especially those especially at risk for or with pre-existing cardiovascular disease. Continued study of the effect of finerenone in diverse patient populations and different disease states is needed.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Chronic treatment with serelaxin mitigates adverse remodeling in a murine model of ischemic heart failure and modulates bioactive sphingolipid signaling

Relaxin is a pleiotropic hormone demonstrated to confer cardioprotection in animal models of myocardial infarction and ischemic heart failure by modulating inflammation, fibrosis and arrhythmogenesis. Several of these pathways in the ischemic myocardium are intricately tied with the downstream signaling of bioactive sphingolipids, which play an active role during post-infarction remodeling. In this current study, we examined the effects of relaxin on sphingosine 1-phosphate (S1P), and the potential benefits of relaxin treatment on cardiac health in a rodent model of ischemic heart failure. Acute (30 min) and sub-acute (24 h) treatment of primary cardiomyocytes with serelaxin (recombinant human relaxin-2) increased the cardiomyocyte content of S1P. In the rodent model, treatment with relaxin for 28 days following myocardial ischemia by way of permanent left coronary artery occlusion improved survival and cardiac function, reduced fibrosis and apoptosis, and mitigated the expression of several pro-inflammatory and pro-fibrotic markers. The expression of beclin-1 (autophagy marker) was also reduced. The expression of S1P was significantly higher in cardiac tissue and plasma samples extracted from serelaxin-treated mice at day 28. In conclusion, our studies show a significant protection from relaxin in ischemic heart disease, and demonstrate the association between relaxin signaling and S1P generation.

H2S Therapy Improves Diastolic Dysfunction and Exercise Capacity in Two‐Hit Murine Model of Heart Failure with Preserved Ejection Fraction

BackgroundHFpEF is a complex multi‐organ disease expected to represent >60% of all clinical heart failure cases by 2030. To date, the majority of therapeutic approaches to treat HFpEF have largely failed in either preclinical or clinical trials. Given the recent reports of diminished H2S bioavailability in HFpEF combined with the robust cardioprotective actions of H2S in cardiovascular disease, we sought out to test the effects of H2S therapy in a well‐established pre‐clinical HFpEF murine model.MethodsHFpEF was induced in nine‐week‐old C57/BL6N mice (n=10 per group) by high fat diet and L‐NAME (0.5g/L/day) via drinking water for 5 weeks. Mice were treated with either vehicle or the H2S donor, JK‐1 (100 mg/kg/day B.I.D., I.P.) for a period of 5 weeks. Echocardiography and exercise capacity were performed at study weeks 0, 5, and 10 for monitoring of cardiac function and progression of exercise intolerance. At week 10, endothelium‐dependent and independent aortic vascular reactivity and left ventricular invasive hemodynamic studies were performed.ResultsTreatment with JK‐1 significantly improved cardiac function as measured by decreases in both left ventricular end diastolic pressure (LVEDP) and E/e’ after 5 weeks of treatment. Similarly, endothelium‐dependent vascular relaxation with acetylcholine exhibited significant improvement. Treadmill exercise performance expressed as total work showed improvement for the JK‐1 group, demonstrating changes in exercise capacity are irrespective of body weight.ConclusionThese results suggest that the H2S donation therapy exerts beneficial effects in the setting of severe HFpEF. Studies are currently ongoing to determine the mechanisms by which H2S therapy improves both cardiac and vascular function in this devastating cardiovascular disease.

Therapeutic inhibition of RBM20 improves diastolic function in a murine heart failure model and human engineered heart tissue.

Heart failure with preserved ejection fraction (HFpEF) is prevalent and deadly, but so far, there is no targeted therapy. A main contributor to the disease is impaired ventricular filling, which we improved with antisense oligonucleotides (ASOs) targeting the cardiac splice factor RBM20. In adult mice with increased wall stiffness, weekly application of ASOs over 2 months increased expression of compliant titin isoforms and improved cardiac function as determined by echocardiography and conductance catheter. RNA sequencing confirmed RBM20-dependent isoform changes and served as a sensitive indicator of potential side effects, largely limited to genes related to the immune response. We validated our approach in human engineered heart tissue, showing down-regulation of RBM20 to less than 50% within 3 weeks of treatment with ASOs, resulting in adapted relaxation kinetics in the absence of cardiac pathology. Our data suggest anti-RBM20 ASOs as powerful cardiac splicing regulators for the causal treatment of human HFpEF.

SnRNA sequencing defines signaling by RBC-derived extracellular vesicles in the murine heart.

Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

Temporal relationship between systemic endothelial dysfunction and alterations in erythrocyte function in a murine model of chronic heart failure.

AimsEndothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs).Methods and resultsSystemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid].ConclusionIn the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF.

Neutral Effects of Combined Treatment With GLP-1R Agonist Exenatide and MR Antagonist Potassium Canrenoate on Cardiac Function in Porcine and Murine Chronic Heart Failure Models.

Background: Ischemia-reperfusion and cardiac remodeling is associated with cardiomyocyte death, excessive fibrosis formation, and functional decline, eventually resulting in heart failure (HF). Glucagon-like peptide (GLP)-1 agonists are reported to reduce apoptosis and myocardial infarct size after ischemia-reperfusion. Moreover, mineralocorticoid receptor antagonists (MRAs) have been described to reduce reactive fibrosis and improve cardiac function. Here, we investigated whether combined treatment with GLP-1R agonist exenatide and MRA potassium canrenoate could minimize cardiac injury and limit HF progression in animal models of chronic HF.Methods and Results: Forty female Topigs Norsvin pigs were subjected to 150 min balloon occlusion of the left anterior descending artery (LAD). Prior to reperfusion, pigs were randomly assigned to placebo or combination therapy (either low dose or high dose). Treatment was applied for two consecutive days or for 8 weeks with a continued high dose via a tunneled intravenous catheter. Using 2,3,5-Triphenyltetrazolium chloride (TTC) staining we observed that combination therapy did not affect the scar size after 8 weeks. In line, left ventricular volume and function assessed by three-dimensional (3D) echocardiography (baseline, 7 days and 8 weeks), and cardiac magnetic resonance imaging (CMR, 8 weeks) did not differ between experimental groups. In addition, 36 C57Bl/6JRj mice underwent permanent LAD-occlusion and were treated with either placebo or combination therapy prior to reperfusion, for two consecutive days via intravenous injection, followed by continued treatment via placement of osmotic mini-pumps for 28 days. Global cardiac function, assessed by 3D echocardiography performed at baseline, 7, 14, and 28 days, did not differ between treatment groups. Also, no differences were observed in cardiac hypertrophy, assessed by heart weight/bodyweight and heart weight/tibia length ratio.Conclusion: In the current study, combined treatment with GLP-1R agonist exenatide and MR antagonist potassium canrenoate did not show beneficial effects on cardiac remodeling nor resulted in functional improvement in a small and large animal chronic HF model.

Multidrug resistance proteins preferentially regulate natriuretic peptide-driven cGMP signalling in the heart and vasculature.

cGMP underpins the bioactivity of NO and natriuretic peptides and is key to cardiovascular homeostasis. cGMP-driven responses are terminated primarily by PDEs, but cellular efflux via multidrug resistance proteins (MRPs) might contribute. Herein, the effect of pharmacological blockade of MRPs on cGMP signalling in the heart and vasculature was investigated in vitro and in vivo. Proliferation of human coronary artery smooth muscle cells (hCASMCs), vasorelaxation of murine aorta and reductions in mean arterial BP (MABP) in response to NO donors or natriuretic peptides were determined in the absence and presence of the MRP inhibitor MK571. The ability of MRP inhibition to reverse morphological and contractile deficits in a murine model of pressure overload-induced heart failure was also explored. MK571 attenuated hCASMC growth and enhanced the anti-proliferative effects of NO and atrial natriuretic peptide (ANP). MRP blockade caused concentration-dependent relaxations of murine aorta and augmented responses to ANP (and to a lesser extent NO). MK571 did not decrease MABP per se but enhanced the hypotensive actions of ANP and improved structural and functional indices of disease severity in experimental heart failure. These beneficial actions of MRP inhibition were associated with a greater intracellular:extracellular cGMP ratio in vitro and in vivo. MRP blockade promotes the cardiovascular functions of natriuretic peptides in vitro and in vivo, with more modest effects on NO. MRP inhibition may have therapeutic utility in cardiovascular diseases triggered by dysfunctional cGMP signalling, particularly those associated with altered natriuretic peptide bioactivity. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Resveratrol Ameliorates Cardiac Remodeling in a Murine Model of Heart Failure With Preserved Ejection Fraction

Objective: Accumulating evidence suggested that resveratrol (RES) could protect against adverse cardiac remodeling induced by several cardiovascular diseases. However, the role of RES in the setting of heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain understood. This study was to determine whether RES could ameliorate HFpEF-induced cardiac remodeling and its mechanisms. Methods: In vivo, C57BL/6 mice served as either the sham or the HFpEF model. The HFpEF mice model was induced by uninephrectomy surgery and d-aldosterone infusion. RES (10 mg/kg/day, ig) or saline was administered to the mice for four weeks. In vitro, transforming growth factor β1 (TGF-β1) was used to stimulate neonatal rat cardiac fibroblasts (CFs) and Ex-527 was used to inhibit sirtuin 1 (Sirt1) in CFs. Echocardiography, hemodynamics, western blotting, quantitative real-time PCR, histological analysis, immunofluorescence, and ELISA kits were used to evaluate cardiac remodeling induced by HFpEF. Sirt1 and Smad3 expressions were measured to explore the underlying mechanisms of RES. Results: HFpEF mice developed left ventricular hypertrophy, preserved ejection fraction, diastolic dysfunction, and pulmonary congestion. Moreover, HFpEF mice showed increased infiltration of neutrophils and macrophages into the heart, including increased interleukin (IL)-1β, IL-6, and TNF-α. We also observed elevated M1 macrophages and decreased M2 macrophages, which were exhibited by increased mRNA expression of M1 markers (iNOS, CD86, and CD80) and decreased mRNA expression of M2 markers (Arg1, CD163, and CD206) in HFpEF hearts. Moreover, HFpEF hearts showed increased levels of intracellular reactive oxygen species (ROS). Importantly, HFpEF mice depicted increased collagen-I and -III and TGF-β mRNA expressions and decreased protein expression of phosphorylated endothelial nitric-oxide synthase (p-eNOS). Results of western blot revealed that the activated TGF-β/Smad3 signaling pathway mediated HFpEF-induced cardiac remodeling. As expected, this HFpEF-induced cardiac remodeling was reversed when treated with RES. RES significantly decreased Smad3 acetylation and inhibited Smad3 transcriptional activity induced by HFpEF via activating Sirt1. Inhibited Sirt1 with Ex-527 increased Smad3 acetylation, enhanced Smad3 transcriptional activity, and offset the protective effect of RES on TGF-β–induced cardiac fibroblast–myofibroblast transformation in CFs. Conclusion: Our results suggested that RES exerts a protective action against HFpEF-induced adverse cardiac remodeling by decreasing Smad3 acetylation and transcriptional activity via activating Sirt1. RES is expected to be a novel therapy option for HFpEF patients.