Pressure Overload-induced Heart Failure Research Articles

Interleukin-12 alpha deficiency attenuates pressure overload-induced cardiac inflammation, hypertrophy, dysfunction, and heart failure progression

IL-12α plays an important role in modulating inflammatory response, fibroblast proliferation and angiogenesis through modulating macrophage polarization or T cell function, but its effect on cardiorespiratory fitness is not clear. Here, we studied the effect of IL-12α on cardiac inflammation, hypertrophy, dysfunction, and lung remodeling in IL-12α gene knockout (KO) mice in response to chronic pressure-overload produced by transverse aortic constriction (TAC). Our results showed that IL-12α KO significantly ameliorated TAC-induced left ventricular (LV) failure, as evidenced by a smaller decrease of LV ejection fraction and fractional shortening. IL-12α KO also significantly attenuated TAC-induced increase of LV weight, left atrial weight, lung weight, right ventricular weight, and their ratios to body weight or tibial length. In addition, IL-12α KO significantly attenuated TAC-induced LV leukocyte infiltration, fibrosis, cardiomyocyte hypertrophy, and lung inflammation and remodeling (such as lung fibrosis and vessel muscularization). Moreover, IL-12α KO significantly attenuated TAC-induced activation of CD4+ T cells and CD8+ T cells in the lung. Furthermore, IL-12α KO significantly suppressed accumulation and activation of pulmonary macrophages and dendritic cells. Taken together, these findings indicate that inhibition of IL-12α is effective in attenuating pressure overload-induced cardiac inflammation, heart failure development, promoting transition from LV failure to lung remodeling and right ventricular hypertrophy. This work was supported by research grants R01HL161085, R01HL139797, P20GM104357, and P01HL51971 from NIH. This is the full abstract presented at the American Physiology Summit 2023 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.

PO-01-241 ROLE OF THE TWO-PORE K+CHANNEL TREK1 IN REGULATING HEART FAILURE-INDUCED VENTRICULAR ARRHYTHMIA

Despite therapeutic advances, heart failure (HF) remains a leading cause of death in the United States and is growing in prevalence. Although disease-induced defects in ion channel excitability and expression put patients at higher risk for life-threatening ventricular arrhythmias, the exact mechanisms by which HF leads to proarrhythmic ion channel dysfunction are not completely understood. Notably, several recent studies have identified important roles for the two-pore background K+ channel TREK1 in regulating sinus rhythm, atrial arrhythmia, and ventricular excitability. In the present study, we examined a putative role for TREK1 dysfunction in promoting heart failure-induced ventricular arrhythmia. We subjected wild type (WT) mice and mice with cardiac-specific TREK1 deficiency (TREK1cKO) to transaortic constriction (TAC) surgery as a model of heart failure or to a sham procedure. Over the course of 6-12 weeks following surgery, we examined structural and electrical remodeling via echocardiography and subsurface ECGs. At the end of the time course, ventricular cells were isolated and used for patch clamp, Ca2+/contractility imaging, and immunostaining. A subset of hearts were isolated for optical voltage mapping. We observed that TREK1cKO mice experienced greater reductions in cardiac function earlier in the development of pressure overload compared to WT mice, which was maintained throughout the time course. Additionally, TREK1cKO mice exhibited proarrhythmic changes to ECG parameters over 6-12 weeks of pressure overload including action potential duration (APD) prolongation, QTc prolongation, and QRS widening. However, the TREK1cKOs also experienced faster conduction, lower APD heterogeneity, and lower ventricular arrhythmia inducibility at 6 weeks post-surgery. Overall, these results demonstrate a potentially complex role of TREK1 in regulating ventricular excitability, ion dynamics, and arrhythmia susceptibility in the context of pressure overload-induced heart failure.

Temporal Progression of Coronary Microvascular Dysfunction in Swine with Repetitive Pressure Overload-Induced Heart Failure with Preserved Ejection Fraction

Objective: We have previously shown that swine subjected to 2-weeks of intermittent repetitive pressure overload (RPO) develop a cardiac phenotype seen in many patients with heart failure with preserved ejection fraction (HFpEF), characterized by an increase in left ventricular (LV) diastolic stiffness, myocyte hypertrophy, and interstitial fibrosis without an increase in LV mass. Given emerging evidence of a potential role for coronary microvascular dysfunction (CMD) in the pathophysiology of HFpEF, we serially measured adenosine (ADO)-mediated coronary vasodilation in swine subjected to RPO to evaluate the time course of CMD relative to changes in LV stiffness in this model. Methods: Swine (n=23) were subjected to up to 2 weeks of RPO via daily infusions of phenylephrine (PE; 400 μg/min; 30-60 min/day). Regional myocardial blood flow (BF) was assessed via administration of fluorescent microspheres at rest and during submaximal ADO-mediated vasodilation (0.15 mg/kg/min) at baseline, 1 hour and 24 hours after the initial episode of pressure overload (n=7), after 3 days of RPO (n=6), and after 2 weeks of RPO (n=10). Echocardiography and invasive hemodynamic analysis were performed during PE infusion at selected timepoints to assess LV diastolic stiffness (ΔLVEDP/ΔLVEDV). Results: Impaired ADO-mediated coronary vasodilation was apparent following a single episode of transient pressure overload, as coronary flow reserve (CFR) fell from 2.1±0.3 at baseline to 1.2±0.1 one hour after the first PE infusion (p=0.03). This effect was temporary, however, as CFR normalized 24 hours later (2.3±0.4; p=0.60 vs. baseline). After 3 days of RPO, the impairment in ADO-mediated coronary vasodilation was no longer reversible, with a persistent reduction in CFR (1.4±0.1; p=0.03 vs. baseline) that was still evident after 2 weeks of RPO (1.2±0.1; p<0.01 vs. baseline). The development of persistent CMD occurred before significant changes in LV stiffness, as ΔLVEDP/ΔLVEDV rose gradually from 0.6±0.1 mmHg/mL at baseline to 0.8±0.1 mmHg/mL after 3 days of RPO (p=0.12 vs. baseline) and 2.0±0.4 mmHg/mL after 2 weeks of RPO (p<0.01 vs. baseline). Conclusions: Although transient hypertension-induced CMD is initially reversible, persistent impairments in ADO-mediated coronary vasodilation are apparent as soon as 3 days after the onset of RPO in swine. The onset of CMD precedes the increase in LV diastolic stiffness in this model, suggesting that microvascular dysfunction may be mechanistically involved in the pathogenesis of LV stiffening. Future studies are necessary to test this hypothesis, as well as determine whether impaired coronary responsiveness to ADO compromises myocardial perfusion during episodes of increased metabolic demand. Supported by funding from the National Institutes of Health (1R56HL14135901, 1RO1HL60538) and the United States Department of Veteran's Affairs (1IOBX002659). This is the full abstract presented at the American Physiology Summit 2023 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.

Atrioventricular node dysfunction in pressure overload-induced heart failure-Involvement of the immune system and transcriptomic remodelling.

Heart failure is associated with atrioventricular (AV) node dysfunction, and AV node dysfunction in the setting of heart failure is associated with an increased risk of mortality and heart failure hospitalisation. This study aims to understand the causes of AV node dysfunction in heart failure by studying changes in the whole nodal transcriptome. The mouse transverse aortic constriction model of pressure overload-induced heart failure was studied; functional changes were assessed using electrocardiography and echocardiography and the transcriptome of the AV node was quantified using RNAseq. Heart failure was associated with a significant increase in the PR interval, indicating a slowing of AV node conduction and AV node dysfunction, and significant changes in 3,077 transcripts (5.6% of the transcriptome). Many systems were affected: transcripts supporting AV node conduction were downregulated and there were changes in transcripts identified by GWAS as determinants of the PR interval. In addition, there was evidence of remodelling of the sarcomere, a shift from fatty acid to glucose metabolism, remodelling of the extracellular matrix, and remodelling of the transcription and translation machinery. There was evidence of the causes of this widespread remodelling of the AV node: evidence of dysregulation of multiple intracellular signalling pathways, dysregulation of 109 protein kinases and 148 transcription factors, and an immune response with a proliferation of neutrophils, monocytes, macrophages and B lymphocytes and a dysregulation of 40 cytokines. In conclusion, inflammation and a widespread transcriptional remodelling of the AV node underlies AV node dysfunction in heart failure.

Cadherin-11-Interleukin-6 Signaling between Cardiac Fibroblast and Cardiomyocyte Promotes Ventricular Remodeling in a Mouse Pressure Overload-Induced Heart Failure Model

Heart failure is a serious and life-threatening disease worldwide. Cadherin-11 (Cad-11) is highly expressed in the heart and closely associated with inflammation. There is currently limited understanding on how Cad-11 contributes to cardiac remodeling and its underline molecular mechanism. We found an increased expression of Cad-11 in biopsy heart samples from heart failure patients, suggesting a link between Cad-11 and heart failure. To determine the role of Cad-11 in cardiac remodeling, Cad-11-deficient mice were used in a well-established mouse transverse aortic constriction (TAC) model. Loss of Cad11 greatly improved pressure overload-induced LV structural and electrical remodeling. IL (interleukin)-6 production was increased following TAC in WT mice and this increase was inhibited in cadherin-11-/- mice. We further tested the effect of IL-6 on myocyte hypertrophy and fibrosis in a primary culture system. The addition of hCad-11-Fc to cultured cardiac fibroblasts increased IL-6 production and fibroblast cell activation, whereas neutralizing IL-6 with an IL-6 antibody resulted in alleviating the fibroblast activation induced by hCad-11-Fc. On the other hand, cardiomyocytes were promoted to cardiomyocyte hypertrophy when cultured in condition media collected from cardiac fibroblasts stimulated by hCad-11-Fc.Similarly, neutralizing IL-6 prevented cardiomyocyte hypertrophy. Finally, we found that MAPKs and CaMKII-STAT3 pathways were activated in both hCad-11-Fc stimulated fibroblasts and cardiomyocytes treated with hCad-11-Fc stimulated fibroblast condition medium. IL-6 neutralization inhibited such MAPK and CaMKII-STAT3 signaling activation. These data demonstrate that Cad-11 functions in pressure overload-induced ventricular remodeling through inducing IL-6 secretion from cardiac fibroblasts to modulate the pathophysiology of neighboring cardiomyocytes.

O-GlcNAcylation-induced GSK-3β activation deteriorates pressure overload-induced heart failure via lack of compensatory cardiac hypertrophy in mice.

O-GlcNAc transferase (OGT) modulates many functions of proteins via O-GlcNAcylation that adds O-linked β-N-acetylglucosamine (O-GlcNAc) to the serine/threonine residues of proteins. However, the role of O-GlcNAcylation in cardiac remodeling and function is not fully understood. To examine the effect of O-GlcNAcylation on pressure overload-induced cardiac hypertrophy and subsequent heart failure, transverse aortic constriction (TAC) surgery was performed in wild type (WT) and Ogt transgenic (Ogt-Tg) mice. Four weeks after TAC (TAC4W), the heart function of Ogt-Tg mice was significantly lower than that of WT mice (reduced fractional shortening and increased ANP levels). The myocardium of left ventricle (LV) in Ogt-Tg mice became much thinner than that in WT mice. Moreover, compared to the heart tissues of WT mice, O-GlcNAcylation of GSK-3β at Ser9 was increased and phosphorylation of GSK-3β at Ser9 was reduced in the heart tissues of Ogt-Tg mice, resulting in its activation and subsequent inactivation of nuclear factor of activated T cell (NFAT) activity. Finally, the thinned LV wall and reduced cardiac function induced by TAC4W in Ogt-Tg mice was reversed by the treatment of a GSK-3β inhibitor, TDZD-8. These results imply that augmented O-GlcNAcylation exacerbates pressure overload-induced heart failure due to a lack of compensatory cardiac hypertrophy via O-GlcNAcylation of GSK-3β, which deprives the phosphorylation site of GSK-3β to constantly inactivate NFAT activity to prevent cardiac hypertrophy. Our findings may provide a new therapeutic strategy for cardiac hypertrophy and subsequent heart failure.

A time-series minimally invasive transverse aortic constriction mouse model for pressure overload-induced cardiac remodeling and heart failure.

Transverse aortic constriction (TAC) is a widely-used animal model for pressure overload-induced cardiac hypertrophy and heart failure (HF). The severity of TAC-induced adverse cardiac remodeling is correlated to the degree and duration of aorta constriction. Most studies of TAC are performed with a 27-gauge needle, which is easy to cause a tremendous left ventricular overload and leads to a rapid HF, but it is accompanied by higher mortality attributed to tighter aortic arch constriction. However, a few studies are focusing on the phenotypes of TAC applied with a 25-gauge needle, which produces a mild overload to induce cardiac remodeling and has low post-operation mortality. Furthermore, the specific timeline of HF induced by TAC applied with a 25-gauge needle in C57BL/6 J mice remains unclear. In this study, C57BL/6 J mice were randomly subjected to TAC with a 25-gauge needle or sham surgery. Echocardiography, gross morphology, and histopathology were applied to evaluate time-series phenotypes in the heart after 2, 4, 6, 8, and 12 weeks. The survival rate of mice after TAC was more than 98%. All mice subjected to TAC maintained compensated cardiac remodeling during the first two weeks and began to exhibit heart failure characteristics after 4 weeks upon TAC. At 8 weeks post-TAC, the mice showed severe cardiac dysfunction, hypertrophy, and cardiac fibrosis compared to sham mice. Moreover, the mice raised a severe dilated HF at 12 weeks. This study provides an optimized method of the mild overload TAC-induced cardiac remodeling from the compensatory period to decompensatory HF in C57BL/6 J mice.

Suppression of RBFox2 by Multiple MiRNAs in Pressure Overload-Induced Heart Failure.

Heart failure is the final stage of various cardiovascular diseases and seriously threatens human health. Increasing mediators have been found to be involved in the pathogenesis of heart failure, including the RNA binding protein RBFox2. It participates in multiple aspects of the regulation of cardiac function and plays a critical role in the process of heart failure. However, how RBFox2 itself is regulated remains unclear. Here, we dissected transcriptomic signatures, including mRNAs and miRNAs, in a mouse model of heart failure after TAC surgery. A global analysis showed that an asymmetric alternation in gene expression and a large-scale upregulation of miRNAs occurred in heart failure. An association analysis revealed that the latter not only contributed to the degradation of numerous mRNA transcripts, but also suppressed the translation of key proteins such as RBFox2. With the aid of Ago2 CLIP-seq data, luciferase assays verified that RBFox2 was targeted by multiple miRNAs, including Let-7, miR-16, and miR-200b, which were significantly upregulated in heart failure. The overexpression of these miRNAs suppressed the RBFox2 protein and its downstream effects in cardiomyocytes, which was evidenced by the suppressed alternative splicing of the Enah gene and impaired E-C coupling via the repression of the Jph2 protein. The inhibition of Let-7, the most abundant miRNA family targeting RBFox2, could restore the RBFox2 protein as well as its downstream effects in dysfunctional cardiomyocytes induced by ISO treatment. In all, these findings revealed the molecular mechanism leading to RBFox2 depression in heart failure, and provided an approach to rescue RBFox2 through miRNA inhibition for the treatment of heart failure.

Evaluation of the transverse aortic constriction model in ICR and C57BL/6J mice.

Transverse aortic constriction (TAC) is a frequently used model to investigate pressure overload-induced progressive heart failure (HF); however, there is considerable phenotypic variation among different mouse strains and even sub-strains. Moreover, less is known about the TAC model in ICR mice. Therefore, to determine the suitability of the ICR strain for TAC-induced HF research, we compared the effects of TAC on ICR and C57BL/6J mice at one, two and fourweeks post-TAC via echocardiography, organ index, morphology, and histology. At the end of the study, behavior and gene expression patterns were assessed, and overall survival was monitored. Compared to the sham-operated mice, ICR and C57BL/6J mice displayed hypertrophic phenotypes with a significant increase in ventricle wall thickness, heart weight and ratio, and cross-sectional area of cardiomyocytes after a 2-week TAC exposure. In addition, ICR mice developed reduced systolic function and severe lung congestion 4weeks post-TAC, whereas C57BL/6J did not. Besides, ICR mice demonstrated comparable survival, similar gene expression alteration but severer fibrotic remodeling and poor behavioral performance compared to the C57BL/6J mice. Our data demonstrated that ICR was quite sensitive to TAC-induced heart failure and can be an ideal research tool to investigate mechanisms and drug intervention for pressure overload-induced HF.

Sirtuin 1 aggravates hypertrophic heart failure caused by pressure overload via shifting energy metabolism

Sirtuin1 (SIRT1) is involved in regulating substrate metabolism in the cardiovascular system. Metabolic homeostasis plays a critical role in hypertrophic heart failure. We hypothesize that cardiac SIRT1 can modulate substrate metabolism during pressure overload-induced heart failure. The inducible cardiomyocyte Sirt1 knockout (icSirt1−/−) and its wild type littermates (Sirt1f/f) C57BL/6J mice were subjected to transverse aortic constriction (TAC) surgery to induce pressure overload. The pressure overload induces upregulation of cardiac SIRT1 in Sirt1f/f but not icSirt1−/− mice. The cardiac contractile dysfunctions caused by TAC-induced pressure overload occurred in Sirt1f/f but not in icSirt1−/− mice. Intriguingly, Sirt1f/f heart showed a drastic reduction in systolic contractility and electric signals during post-TAC surgery, whereas icSirt1−/− heart demonstrated significant resistance to pathological stress by TAC-induced pressure overload as evidenced by no significant changes in systolic contractile functions and electric properties. The targeted proteomics showed that the pressure overload triggered downregulation of the SIRT1-associated IDH2 (isocitrate dehydrogenase 2) that resulted in increased oxidative stress in mitochondria. Moreover, metabolic alterations were observed in Sirt1f/f but not in icSirt1−/− heart in response to TAC-induced pressure overload. Thus, SIRT1 interferes with metabolic homeostasis through mitochondrial IDH2 during pressure overload. Inhibition of SIRT1 activity benefits cardiac functions under pressure overload-related pathological conditions.

Abstract 9553: Sirtuin1 Aggravates Hypertrophic Heart Failure Caused by Pressure Overload via Shifting Energy Metabolism

Introduction: Sirtuin1 (SIRT1) serves as a deacetylase to regulate cardiac substrate metabolism during physiological and pathological conditions. The alteration in metabolic homeostasis plays a critical role in hypertrophic heart failure. Hypothesis: Cardiomyocyte SIRT1 modulates substrate metabolism to regulate cardiac remodeling during pressure overload-induced heart failure. Methods: The inducible cardiomyocyte specific Sirt1 knockout (icSirt1 -/- ) and its wild type littermates (Sirt1 f/f ) C57BL/6J mice were subjected to transverse aortic constriction (TAC) surgery to induce pressure overload. The cardiac functions were evaluated with echocardiography and electrocardiography measurements. Results: The pressure overload by TAC induces upregulation of cardiac SIRT1 in Sirt1 f/f but not icSirt1 -/- mice. The cardiac contractile dysfunctions caused by TAC-induced pressure overload occurred in Sirt1 f/f but not in icSirt1 -/- mice. Intriguingly, Sirt1 f/f heart showed a drastic reduction in systolic contractility and electric signals during post-TAC surgery, whereas icSirt1 -/- heart demonstrated significant resistance to the pathological stress by TAC-induced pressure overload as evidenced by no significant changes in systolic contractile functions and electric properties. The Seahorse data revealed that TAC-induced pressure overload causes a decrease in basal respiration and ATP generation, as well as reduced OXPHOS (oxidative phosphorylation) integrity of mitochondria in Sirt1 f/f but not in icSirt1 -/- cardiomyocytes. The targeted proteomics showed that the pressure overload trigged downregulation of the SIRT1-associated IDH2 (isocitrate dehydrogenase 2) that result in an increased oxidative stress in mitochondria. Moreover, a significant alteration in substrate metabolism was observed in Sirt1 f/f but not in icSirt1 -/- heart in response to TAC-induced pressure overload stress. Conclusions: SIRT1 interferes metabolic homeostasis through regulating mitochondrial IDH2 during pressure overload. Inhibition of SIRT1 activity benefits cardiac functions under pressure overload-related pathological conditions. SIRT1 antagonism could be a pharmacological approach for hypertrophic heart failure patients.

Abstract 14533: Molecular Imaging of Fibroblast Activity in Pressure Overload Heart Failure

Introduction: Cardiac fibrosis, mediated by the activation of cardiac fibroblasts, is critical in pressure overload-induced heart failure. Fibroblast activation protein (FAP), specifically expressed by activated fibroblasts, can be visualized by radiolabeled FAP inhibitors (FAPI) PET/CT. Hypothesis: To evaluate whether 68Ga-FAPI-04 could characterize the early stages of cardiac fibrosis in pressure overload heart failure. Methods: Sprague-Dawley rats underwent abdominal aortic constriction (AAC) (n = 12) and sham surgery (n = 10). All rats were scanned with 68 Ga-FAPI-04 PET/CT at 2, 4, and 8 weeks after surgery. Cardiac function was measured by echocardiography. The expression of FAP in the myocardium was detected by immunohistochemistry. Results: Compared with the sham group, the AAC group presented with decreased ejection fraction (EF) and fractional shortening (FS) and increased left ventricular internal dimensions in diastole (LVIDd) and systole (LVIDs) at 4 and 8 weeks (all p < 0.01). The AAC group showed higher 68 Ga-FAPI-04 accumulation in the heart than the sham group at 2, 4, and 8 weeks, respectively (all p < 0.001), and FAPI increased significantly from 2 to 8 weeks. Immunohistochemistry confirmed the higher density of the FAP + area in the AAC group. The intensity of the 68 Ga-FAPI-04 PET signal correlated with the density of the FAP + area on histology (p < 0.001). The expression of the 68 Ga-FAPI-04 at 4 weeks correlated with the deterioration of cardiac function at 8 weeks (EF: R = -0.87; FS: R = -0.72; LVIDd: R = 0.77; LVIDs: R= 0.79; all p < 0.001). The AAC group also showed an increased 68 Ga-FAPI-04 signal in the liver, peaking at 4 weeks and then declining. Cardiac and liver PET signals directly correlated at 4 weeks in the AAC group (R = 0.69, p = 0.0010). A combination of the FAPI intensity in the heart and liver at 4 weeks better predicted the deterioration of cardiac function at 8 weeks. Conclusion: The activated fibroblasts in the heart and liver after pressure overload can be monitored by 68 Ga-FAPI-04 PET/CT. This imaging technique reveals an early fibrotic link in cardio-liver interactions and could better predict nonischemic heart failure prognosis.

Abstract 10615: The Effects of Maternal Hypothyroidism on Progeny Cardiac Development and Disease Susceptibility

Introduction: Maternal hypothyroidism (MH) is a common clinical condition with high prevalence. MH is thought to have adverse effects on progeny's cardiac development and disease susceptibility. Hypothesis: MH impacts progeny's postnatal cardiac development and the susceptibility to adverse cardiac disease responses as adults. Methods: MH model was induced by thyroidectomy (TX) with total thyroxine (TT4) under 1ng/dl after surgery. Offspring of mice that underwent TX or Sham surgery was termed THD (Deficient) and THN (Normal) animals. Hearts were collected from THD and THN animals to determine heart weight, cardiomyocyte (CM) size, and CM proliferation. Transverse Aortic Constriction (TAC) was performed to induce pressure overload-induced cardiac hypertrophy and/or heart failure (HF) model in adult THD and THN mice. ECHO (in-vivo) and histological (in-vitro) were performed at specific times after TAC. RNA-seq was performed at embryo day (E) 18.5 and Postnatal day 5 (P5) using heart tissue. Results: The postnatal TT4 level was not different between the THD and THN mice. The heart weight (HW) to body weight (BW) ratio was similar between the two groups on P7, P14, and P60, but the THD mice had larger CM sizes. EdU+, Ki67+, and PH3+ CMs were lower in THD progenies on P0, P5, and P13. RNA-seq data showed that the genes related to cell division and DNA replication were downregulated in THD mice on E18.5 and P5, respectively. In adult animals six weeks after TAC, the THD mice had a greater HW/BW ratio and lung weight(LW) to BW ratio, consistent with more severe cardiac hypertrophy and HF than the THN mice. After TAC, THD mice had a lower LVEF, higher absolute E/e' ratio, greater end-diastolic pressure, and more severe LV fibrosis than THN mice. Conclusions: Alterations in CM proliferation during fetal and early postnatal development results in fewer adult CMs in animals from MH mothers. These changes are associated with increased disease susceptibility in adult MH progeny.

Scleraxis and fibrosis in the pressure-overloaded heart.

In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor β. Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.

Comparative effectiveness of the new calpain inhibitor NPO-2270 versus enalapril in pressure overload-induced heart failure

Abstract Background Studies performed in multiple preclinical models support the contribution of the Ca2+-dependent cysteine proteases calpains to ventricular remodelling and heart failure (HF). However, pharmacological calpain inhibition has not yet been tested in patients with HF mainly due to the limitations of available inhibitors. Purpose To determine the effect of NPO-2270 (NPO), a new ketoamide derivative calpain inhibitor, in a mouse model of pressure-overload and compare its effectiveness with that of enalapril. Methods C57BL6 mice were subjected to transverse aortic constriction (TAC) for 4 weeks. Mice were randomised to receive orally administered NPO or enalapril at the dose of 10 mg/kg/day once a day, or vehicle, starting at day 7 after TAC surgery. The combination of both drugs and the effect of NPO starting 1 day after surgery were tested in additional TAC mice. Echocardiographic data, markers of hypertrophy, fibrosis, calpain activity and cleavage of calpain substrates were measured at different time points. Results TAC increased calpain-1 and -2 expression and activity. Administration of NPO and enalapril prevented the progression of hypertrophy and interstitial fibrosis induced by TAC with no statistically significant differences between the two treatments. However, ongoing ventricular dysfunction was less severe in the NPO group than in the enalapril group (27% of LVEF reduction in control group, 6% in NPO group and 16% in the enalapril group after TAC, P=0.024 between NPO and enalapril groups). The combined treatment or the administration of NPO from the first day after TAC surgery was not superior to NPO alone starting 7 days after TAC. These differences in LVEF correlated with better preservation of cadherin-based cell adhesion complex in mice treated with NPO-2270. No adverse effects associated with long-term NPO administration were observed in a sham group. Conclusions The new calpain inhibitor NPO-2270 prevents the development of hypertrophy and fibrosis with similar efficacy than enalapril but prevents cardiac dysfunction more effectively in a preclinical model of pressure overload when given orally at equivalent doses. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III of the Spanish Ministry of Health

Abstract P202: An Efficacy Assessment Of The Cardioprotective Effects Of Alpha-calcitonin Gene Related Peptide (αCGRP) Encapsulated Alginate Microcapsules In A Murine Model Of Heart Failure

The cardioprotective role of a neuropeptide, alpha-calcitonin gene related peptide (αCGRP), has been established in a variety of cardiovascular diseases. To increase the bioavailability of the circulating peptide, we created a peptide delivery system by encapsulating αCGRP in an alginate biopolymer and showed that subcutaneous delivery of alginate-αCGRP microcapsules (αCGRP dose= 6 mg/kg/mouse) on alternate days, up to 28 days, significantly protected hearts at pathophysiological levels in a transverse aortic constriction (TAC) pressure-overload induced heart failure murine model. The present study was performed to determine if weekly subcutaneous delivery of alginate-αCGRP microcapsules exhibited similar cardioprotective effects in TAC-mice as observed in our previous study with alternate days dose delivery scheme. This study is crucial to shed light on the efficacy of these microcapsules. An electrospray method was used to prepare alginate-αCGRP microcapsules of 200 μm diameter. Male C57BL6 mice were divided into four groups (3 mice/group): i- sham, ii- sham-alginate-CGRP, iii- TAC, and iv- TAC-alginate-CGRP. Mice from TAC and TAC-alginate-CGRP groups underwent TAC procedure. Two days post-TAC, alginate-αCGRP microcapsules (αCGRP dose= 6 mg/kg/mouse) were administered subcutaneously once a week, up to 4 weeks, in the respective alginate-CGRP groups of mice. Short-axis echocardiography was performed to evaluate cardiac functions. After 28 days of microcapsules delivery, calculated percent fractional shortening (FS) and ejection fraction (EF) in mice were (in ±SD)- sham: FS= 47.6 ± 0.9, EF= 79.8 ± 0.9; sham-alginate-CGRP: FS= 47.3 ± 1.9, EF= 79.1 ± 2; TAC: FS= 34.1 ± 4.7, EF= 63.6 ± 7.1; TAC-alginate-CGRP: FS= 32.2 ± 2.3, EF= 61.0 ± 3.4. These echo data demonstrate that TAC heart failure mice had the usual significantly reduced cardiac functions, however weekly delivery of alginate-αCGRP microcapsules showed no improvement in heart performance in the TAC-mice. Combined results from our present and previous studies suggested that alginate-αCGRP microcapsules delivery on alternate days, but not weekly, is the most efficacious choice to achieve cardioprotective effects of αCGRP against pressure-induced heart failure in mice.

Tax1 banding protein 1 exacerbates heart failure in mice by activating ITCH-P73-BNIP3-mediated cardiomyocyte apoptosis.

Tax1 banding protein 1 (Tax1bp1) was originally identified as an NF-κB regulatory protein that participated in inflammatory, antiviral and innate immune processes. Tax1bp1 also functions as an autophagy receptor that plays a role in autophagy. Our previous study shows that Tax1bp1 protects against cardiomyopathy in STZ-induced diabetic mice. In this study we investigated the role of Tax1bp1 in heart failure. Pressure overload-induced heart failure model was established in mice by aortic banding (AB) surgery, and angiotensin II (Ang II)-induced heart failure model was established by infusion of Ang II through osmotic minipump for 4 weeks. We showed that the expression levels of Tax1bp1 in the heart were markedly increased 2 and 4 weeks after AB surgery. Knockdown of Tax1bp1 in mouse hearts significantly ameliorated both AB- and Ang II infusion-induced heart failure parameters. On the contrary, AB-induced heart failure was aggravated in cardiac-specific Tax1bp1 transgenic mice. Similar results were observed in neonatal rat cardiomyocytes (NRCMs) under Ang II insult. We demonstrated that the pro-heart failure effect of Tax1bp1 resulted from its interaction with the E3 ligase ITCH to promote the transcription factor P73 ubiquitination and degradation, causing enhanced BCL2 interacting protein 3 (BNIP3)-mediated cardiomyocyte apoptosis. Knockdown ITCH or BNIP3 in NRCMs significantly reduced Ang II-induced apoptosis in vitro. Similarly, BNIP3 knockdown attenuated heart failure in cardiac-specific Tax1bp1 transgenic mice. In the left ventricles of heart failure patients, Tax1bp1 expression level was significantly increased; Tax1bp1 gene expression was negatively correlated with left ventricular ejection fraction in heart failure patients. Collectively, the Tax1bp1 increase in heart failure enhances ITCH-P73-BNIP3-mediated cardiomyocyte apoptosis and induced cardiac injury. Tax1bp1 may serve as a potent therapeutic target for the treatment of heart failure.• Cardiac Tax1bp1 transgene mice were more vulnerable to cardiac dysfunction under stress.• Cardiac Tax1bp1 transgene mice were more vulnerable to cardiac dysfunction under stress.• Knockout of Tax1bp1 in mouse hearts ameliorated heart failure induced by pressure overload.• Tax1bp1 interacts with the E3 ligase Itch to promote P73 ubiquitination and degradation, causing enhanced BNIP3-mediated apoptosis.• Tax1bp1 may become a target of new therapeutic methods for treating heart failure.

The RalGAPα1–RalA signal module protects cardiac function through regulating calcium homeostasis

Sarcoplasmic/endoplasmic reticulum calcium ATPase SERCA2 mediates calcium re-uptake from the cytosol into sarcoplasmic reticulum, and its dysfunction is a hallmark of heart failure. Multiple factors have been identified to modulate SERCA2 activity, however, its regulation is still not fully understood. Here we identify a Ral-GTPase activating protein RalGAPα1 as a critical regulator of SERCA2 in cardiomyocytes through its downstream target RalA. RalGAPα1 is induced by pressure overload, and its deficiency causes cardiac dysfunction and exacerbates pressure overload-induced heart failure. Mechanistically, RalGAPα1 regulates SERCA2 through direct interaction and its target RalA. Deletion of RalGAPα1 decreases SERCA2 activity and prolongs calcium re-uptake into sarcoplasmic reticulum. GDP-bound RalA, but not GTP-bound RalA, binds to SERCA2 and activates the pump for sarcoplasmic reticulum calcium re-uptake. Overexpression of a GDP-bound RalAS28N mutant in the heart preserves cardiac function in a mouse model of heart failure. Our findings have therapeutic implications for treatment of heart failure.

Increased Remnant Lipoproteins in Apo E Deficient Mice Induce Coronary Atherosclerosis following Transverse Aortic Constriction and Aggravate the Development of Pressure Overload-Induced Cardiac Hypertrophy and Heart Failure.

Murine coronary arteries are very resistant to the development of atherosclerosis, which may be related to their intramyocardial course. Blood pressure promotes atherosclerotic plaque formation by acting as a physical force that potentiates the migration of pro-atherogenic lipoproteins across the endothelium. C57BL/6N apolipoprotein (apo) E deficient mice have increased remnant lipoproteins that are a risk factor for coronary atherosclerosis. In this study, our aim was to quantify coronary atherosclerosis and artery remodeling following transverse aortic constriction (TAC) in C57BL/6N apo E−/− mice and to evaluate the impact of increased remnant lipoproteins on the development of pressure overload-induced cardiac hypertrophy and heart failure. Advanced atherosclerotic lesions were observed in the left coronary artery of C57BL/6N apo E−/− TAC mice but not in C57BL/6N TAC mice. Pressure overload resulted in markedly increased cardiac hypertrophy and more pronounced heart failure in C57BL/6N apo E−/− TAC mice in comparison to C57BL/6N TAC mice. Pathological hypertrophy, as evidenced by increased myocardial fibrosis and capillary rarefaction, was more prominent in C57BL/6N TAC apo E−/− than in C57BL/6N TAC mice and led to more marked cardiac dysfunction. In conclusion, TAC in apo E deficient mice induces coronary atherosclerosis and aggravates the development of pathological cardiac hypertrophy and heart failure.

Mitochondrial H2S Regulates BCAA Catabolism in Heart Failure.

Hydrogen sulfide (H2S) exerts mitochondria-specific actions that include the preservation of oxidative phosphorylation, biogenesis, and ATP synthesis, while inhibiting cell death. 3-MST (3-mercaptopyruvate sulfurtransferase) is a mitochondrial H2S-producing enzyme whose functions in the cardiovascular disease are not fully understood. In the current study, we investigated the effects of global 3-MST deficiency in the setting of pressure overload-induced heart failure. Human myocardial samples obtained from patients with heart failure undergoing cardiac surgeries were probed for 3-MST protein expression. 3-MST knockout mice and C57BL/6J wild-type mice were subjected to transverse aortic constriction to induce pressure overload heart failure with reduced ejection fraction. Cardiac structure and function, vascular reactivity, exercise performance, mitochondrial respiration, and ATP synthesis efficiency were assessed. In addition, untargeted metabolomics were utilized to identify key pathways altered by 3-MST deficiency. Myocardial 3-MST was significantly reduced in patients with heart failure compared with nonfailing controls. 3-MST KO mice exhibited increased accumulation of branched-chain amino acids in the myocardium, which was associated with reduced mitochondrial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism with 3,6-dichlorobenzo1[b]thiophene-2-carboxylic acid (BT2) and administration of a potent H2S donor JK-1 ameliorates the detrimental effects of 3-MST deficiency in heart failure with reduced ejection fraction. Our data suggest that 3-MST derived mitochondrial H2S may play a regulatory role in branched-chain amino acid catabolism and mediate critical cardiovascular protection in heart failure.