Transverse Aortic Constriction Mice Research Articles

Alpha-calcitonin gene-related peptide prevents pressure-overload induced heart failure: role of apoptosis and oxidative stress.

Alpha‐calcitonin gene‐related peptide (α‐CGRP) is a 37‐amino acid neuropeptide that plays an important protective role in modulating cardiovascular diseases. Deletion of the α‐CGRP gene increases the vulnerability of the heart to pressure‐induced heart failure and the administration of a modified α‐CGRP agonist decreases this vulnerability. Systemic administration of α‐CGRP decreases blood pressure in normotensive and hypertensive animals and humans. Here we examined the protective effect of long‐term administration of native α‐CGRP against pressure‐overload heart failure and the likely mechanism(s) of its action. Transverse aortic constriction (TAC) was performed to induce pressure‐overload heart failure in mice. We found that TAC significantly decreased left ventricular (LV) fractional shortening, ejection fraction, and α‐CGRP content, and increased hypertrophy, dilation, and fibrosis compared to sham mice. Administration of α‐CGRP‐filled mini‐osmotic pumps (4 mg/kg bwt/day) in TAC mice preserved cardiac function and LV α‐CGRP levels, and reduced LV hypertrophy, dilation, and fibrosis to levels comparable to sham mice. Additionally, TAC pressure‐overload significantly increased LV apoptosis and oxidative stress compared to the sham mice but these increases were prevented by α‐CGRP administration. α‐CGRP administration in TAC animals decreased LV AMPK phosphorylation levels and the expression of sirt1, both of which are regulatory markers of oxidative stress and energy metabolism. These results demonstrate that native α‐CGRP is protective against pressure‐overload induced heart failure. The mechanism of this cardio‐protection is likely through the prevention of apoptosis and oxidative stress, possibly mediated by sirt1 and AMPK. Thus, α‐CGRP is a potential therapeutic agent in preventing the progression to heart failure, and the cardio‐protective action of α‐CGRP is likely the result of a direct cellular effect; however, a partial vasodilatory blood pressure‐dependent mechanism of α‐CGRP cannot be excluded.

Multimodality Imaging of Inflammation and Ventricular Remodeling in Pressure-Overload Heart Failure.

Inflammation contributes to ventricular remodeling after myocardial ischemia, but its role in nonischemic heart failure is poorly understood. Local tissue inflammation is difficult to assess serially during pathogenesis. Although 18F-FDG accumulates in inflammatory leukocytes and thus may identify inflammation in the myocardial microenvironment, it remains unclear whether this imaging technique can isolate diffuse leukocytes in pressure-overload heart failure. We aimed to evaluate whether inflammation with 18F-FDG can be serially imaged in the early stages of pressure-overload-induced heart failure and to compare the time course with functional impairment assessed by cardiac MRI. Methods: C57Bl6/N mice underwent transverse aortic constriction (TAC) (n = 22), sham surgery (n = 12), or coronary ligation as an inflammation-positive control (n = 5). MRI assessed ventricular geometry and contractile function at 2 and 8 d after TAC. Immunostaining identified the extent of inflammatory leukocyte infiltration early in pressure overload. 18F-FDG PET scans were acquired at 3 and 7 d after TAC, under ketamine-xylazine anesthesia to suppress cardiomyocyte glucose uptake. Results: Pressure overload evoked rapid left ventricular dilation compared with sham (end-systolic volume, day 2: 40.6 ± 10.2 μL vs. 23.8 ± 1.7 μL, P < 0.001). Contractile function was similarly impaired (ejection fraction, day 2: 40.9% ± 9.7% vs. 59.2% ± 4.4%, P < 0.001). The severity of contractile impairment was proportional to histology-defined myocardial macrophage density on day 8 (r = -0.669, P = 0.010). PET imaging identified significantly higher left ventricular 18F-FDG accumulation in TAC mice than in sham mice on day 3 (10.5 ± 4.1 percentage injected dose [%ID]/g vs. 3.8 ± 0.9 %ID/g, P < 0.001) and on day 7 (7.8 ± 3.7 %ID/g vs. 3.0 ± 0.8 %ID/g, P = 0.006), though the efficiency of cardiomyocyte suppression was variable among TAC mice. The 18F-FDG signal correlated with ejection fraction (r = -0.75, P = 0.01) and ventricular volume (r = 0.75, P < 0.01). Western immunoblotting demonstrated a 60% elevation of myocardial glucose transporter 4 expression in the left ventricle at 8 d after TAC, indicating altered glucose metabolism. Conclusion: TAC induces rapid changes in left ventricular geometry and contractile function, with a parallel modest infiltration of inflammatory macrophages. Metabolic remodeling overshadows inflammatory leukocyte signal using 18F-FDG PET imaging. More selective inflammatory tracers are requisite to identify the diffuse local inflammation in pressure overload.

P1614Soluble guanylate cyclase as a therapeutic target in heart failure: myocardial gene expression in response to sGC stimulation in pressure overload

Abstract Background Heart Failure (HF) is associated with endothelial dysfunction and reduced bioavailability of NO with insufficient stimulation of sGC and reduced production of cGMP. Therefore, the impairment of the NO-sGC-cGMP pathway results in vasoconstriction, platelet aggregation, inflammation, fibrosis and most importantly maladaptive cardiac hypertrophy. The restoration of the NO-sGC -cGMP pathway is an attractive pharmacological target for HF therapy. Purpose Riociguat is an NO independent stimulator of the sGC that sensitizes the sGC to endogenous NO and directly stimulates sGC to produce cGMP. We therefore hypothesized that Riociguat prevents pathological effects occurring during HF. Methods Pressure overload was induced by transverse aortic constriction (TAC) in 8 weeks old male C57Bl6/N mice. Three weeks after TAC when cardiac hypertrophy has developed either Riociguat (RIO; 3 mg/kg) or a Solvent was administered daily for 5 more weeks (n=12 per group). Animals with sham surgery and same drug regime served as controls. The heart function in all groups was evaluated weekly by small animal echocardiography. Eight weeks after surgery, the transcriptome of the left ventricles (LV) of sham and TAC mice were analysed by RNA Sequencing. Differentially expressed genes (DEG) were categorised using Ingenuity Pathway Analysis (IPA). Results TAC resulted in a steady decrease of left ventricular fractional shortening (FS) in the mice until week 3. When Riociguat treatment commenced, the systolic LV function of the TAC+Rio group recovered significantly whereas the solvent group showed a further decline until week 8 (FS 21.4±3.4% vs. 9.5±2%, p&lt;0.001). Both sham groups (Sham+Sol and Sham+Rio) showed no changes in the heart function over timer. Regarding the hypertrophic response to LV pressure overload, Riociguat treatment attenuated significantly the increase of the left ventricular mass (LVM 208.3±15.8mg vs. 148.9±11.8mg, p&lt;0.001) after TAC. In line with the reduced LVM, histological staining showed a significantly reduced fibrosis and myocyte cross sectional area in the TAC+Rio group compared to TAC+Sol group. Regarding the myocardial transcriptome, the treatment with Riociguat resulted in less changes of gene expression pattern after TAC (TAC+Sol vs. Sham+Sol 3160 DEG; TAC+Rio vs. Sham+Rio 2237 DEG). The expression of heart failure marker genes like ANP (Nppa), BNP (Nppb), β-Myosin Heavy Chain (Myh7) and the Collagens 1 and 3 (Col1a1, Col1a2, Col3a1) were significantly decreased in TAC+Rio, when compared to TAC+Sol. IPA analysis revealed that the activation of biological pathways in response to TAC, like actin cytoskeleton- and Integrin signalling, renin-angiotensin or cardiac hypertrophy signalling was attenuated when Riociguat was administered. Conclusion Riociguat attenuates pressure overload induced LV remodelling resulting in less hypertrophy, improved heart function and less alteration of gene expression pattern.

P1626MicroRNA-21 deteriorates left ventricular reverse remodeling by promoting cardiac fibrosis in non-ischemic cardiomyopathy

Abstract Background Left ventricular reverse remodeling (LVRR) contributes to better outcomes in patients with non-ischemic cardiomyopathy (NICM). It is reported that LVRR is associated with progression of cardiac fibrosis. MicroRNAs (miRs) have emerged as powerful regulators of post-transcriptional gene expression. We focused on miR-21, which plays a key role in pathogenesis of fibrosis in multiple organs. The aim of this study was to clarify the effect of miR-21 on cardiac fibrosis and LVRR in patients with NICM. Methods We measured plasma miR-21 levels in 16 patients with NICM. LVRR was defined as increased LVEF by ≥10% and decreased LV end-diastolic diameter index by ≥10% from baseline data after optimal medication treatment at 1-year of follow-up. Further, we examined miR-21 expression and its potential role in cardiac fibrosis induced by transverse aortic constriction (TAC) in mice and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes (NRCMs). Results There were 12 patients without LVRR and 4 patients with LVRR. Plasma miR-21 levels were significantly higher in patients without LVRR compared with those with LVRR. In TAC mice heart, miR-21 levels were significantly increased and programmed cell death 4 (PDCD4), a main target of miR-21, was decreased. In vitro, miR-21 levels were significantly increased and its upstream transcriptional factor, activator protein 1 (AP-1), was activated by Ang II stimulation in NRCMs. After transfection of miR-21 specific inhibitor, PDCD4 levels were upregulated. Furthermore, AP-1 activity, expression of collagen type I, and α-smooth muscle actin levels were significantly decreased after miR-21 inhibition. Conclusions These findings suggested that miR-21/PDCD4/AP-1 feedback loop pathway was involved in LVRR in patients with NICM by promoting cardiac fibrosis. MiR-21 can be the therapeutic target in NICM.

P.79Malignant cardiac phenotype after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous R349P desmin knock-in mice

Which etiological factors significantly aggravate the disease progression in genetically determined forms of cardiomyopathies? In the present study, we investigated the influence of pressure overload on cardiac function in our R349P desmin knock-in mouse model for human desminopathies. We performed transverse aortic constriction (TAC) operations in heterozygous R349P desmin knock-in mice and wild-type siblings. Left heart catheterization demonstrated effective TAC procedures by marked increases in systolic carotid pressure and end-systolic and end-diastolic left ventricular pressure values. Echocardiography demonstrated a significant reduction of the left ventricular ejection fraction only in TAC R349P desmin knock-in mice as compared to controls. Electrophysiological investigation revealed an increase in spontaneous AV blocks and the longest AV block duration in the mutant TAC mice. Furthermore, the probability of induction of ventricular tachycardias was also significantly increased in these mice. Mitochondrial analyses demonstrated marked reductions of the activities of respiratory chain complexes I and IV as well as significantly reduced mtDNA copy numbers in the TAC-operated desminopathy mice. While young, 3-month-old, non-operated heterozygous R349P desmin knock-in mice do not show obvious signs of cardiomyopathy, they develop marked signs of cardiomyopathy with accompanying arrhythmias and AV blocks when subjected to pressure overload by TAC operation. The cardiac disease manifestations are associated with reductions in respiratory chain complex activities and mtDNA copy numbers. Preventive avoidance of pressure overload, i.e. arterial hypertension, to the heart might represent a crucial therapeutic measure.

Cholesterol lowering attenuates pressure overload-induced heart failure in mice with mild hypercholesterolemia.

Epidemiological studies support a strong association between non-high-density lipoprotein cholesterol levels and heart failure incidence. The objective of the current study was to evaluate the effect of selective cholesterol lowering adeno-associated viral serotype 8 (AAV8)-mediated low-density lipoprotein receptor (LDLr) gene transfer on cardiac remodelling and myocardial oxidative stress following transverse aortic constriction (TAC) in female C57BL/6 LDLr-/- mice with mild hypercholesterolemia. Cholesterol lowering gene transfer resulted in a 65.9% (p<0.0001) reduction of plasma cholesterol levels (51.2 ± 2.2 mg/dl) compared to controls (150 ± 7 mg/dl). Left ventricular wall area was 11.2% (p<0.05) lower in AAV8-LDLr TAC mice than in control TAC mice. In agreement, pro-hypertrophic myocardial proteins were potently decreased in AAV8-LDLr TAC mice. The degree of interstitial fibrosis and perivascular fibrosis was 31.0% (p<0.001) and 29.8% (p<0.001) lower, respectively, in AAV8-LDLr TAC mice compared to control TAC mice. These structural differences were associated with improved systolic and diastolic function and decreased lung congestion in AAV8-LDLr TAC mice compared to control TAC mice. Cholesterol lowering gene therapy counteracted myocardial oxidative stress and preserved the potential for myocardial fatty acid oxidation in TAC mice. In conclusion, cholesterol lowering gene therapy attenuates pressure overload-induced heart failure in mice with mild hypercholesterolemia.

Abstract P121: A Novel Delivery of Alpha-Calcitonin Gene Related Peptide (α-CGRP) Using Alginate Microcapsules Protects Against Pressure-Overload Induced Heart Failure

The cardioprotective role of α-calcitonin gene related peptide (α-CGRP), a 37-amino acid neuropeptide and potent vasodilator, in cardiac diseases has been established by our laboratory and others. Systemic delivery of α-CGRP decreases blood pressure in hypertensive humans, and improves hemodynamic variables in congestive heart failure (CHF) patients. However, the short half-life of the peptide (t 1/2 = ~5.5 min in serum) limits α-CGRP use in any long-term treatment regime. The present study utilized alginate-α-CGRP microcapsules for long-term continuous delivery of α-CGRP in a mouse CHF model. We used an electrospray method to prepare α-CGRP encapsulated alginate microcapsules approximately 200μm in size. The α-CGRP encapsulated microcapsules showed no cytotoxicity when incubated with the cardiac cell lines, HL-1 and H9c2 cells, for 7 days. Subcutaneous administration of microcapsules containing 150, 250, or 500μg α-CGRP lowered the systolic pressure up to 18 h, 3 days, and 7 days, respectively, in wild-type mice (measured by tail-cuff method), indicating that released α-CGRP remains biologically active. The cardioprotective effect of microcapsules released α-CGRP was examined using a transverse aortic constriction (TAC) induced pressure-overload mouse model of CHF. After two days of TAC, 500μl of alginate-α-CGRP microcapsules (containing 150μg α-CGRP) was administered in nine weeks old C57/BL6 mice on alternate days up to 28 days. Echocardiography data showed that microcapsule treatment of α-CGRP significantly preserved left ventricular fraction shortening (FS) and ejection fraction in the TAC mice (FS±SD: sham 47.2±1.5%, sham-alginate-α-CGRP 48.4±1.8%, TAC 29.5±1.4%, and TAC-alginate-α-CGRP 43.2±1.2%). α-CGRP delivery attenuated cardiac hypertrophy in TAC mice when reported as heart wt/tibia length (mg/mm±SD): sham 6.3±0.1, sham-alginate-α-CGRP 6.3±0.2, TAC 8.6±0.3, and TAC-alginate-α-CGRP 6.6±0.2. Alginate-only microcapsules did not affect these cardiac parameters. Our results show that α-CGRP delivery through alginate microcapsules protect hearts from cardiac failure. As alginate is immunologically inactive, the alginate microcapsules offer a new potential delivery paradigm for patients with heart failure.

Miro2 Regulates Inter-Mitochondrial Communication in the Heart and Protects Against TAC-Induced Cardiac Dysfunction.

Rationale: The constrained mitochondria in cardiomyocytes communicate with each other, through mitochondrial kissing or nanotunneling, forming a dynamically continuous network to share content and transfer signals. However, the molecular mechanism of cardiac inter-mitochondrial communication is unclear. Objective: To determine the molecular mechanism underlying the robust inter-mitochondrial communication and its pathophysiological relevance in the heart. Methods and Results: By mitochondria-targeted expressing the photoactivatable green fluorescent protein, we revealed that most mitochondrial nanotubes bridge communicating mitochondrial pairs were associated with microtubules. Miro2 (mitochondrial Rho GTPase), the outer mitochondrial membrane protein which usually mediates mitochondrial transport within cells, accompanied with mitochondrial nanotubes along microtubules in adult cardiomyocytes. Adenovirus mediated expression of Miro2 in cardiomyocytes accelerated inter-mitochondrial communication through increasing mitochondrial nanotunneling and mitochondrial kissing between adjacent mitochondrial pairs. In transverse aortic constriction-induced hypertrophic mouse hearts Miro2 protein was declined, accompanied with decreased inter-mitochondrial communication. Miro2 transgenic mice showed ameliorated cardiac function, increased mitochondrial nanotube formation and inter-mitochondrial communication, and improved mitochondrial function after transverse aortic constriction. E3 ubiquitin ligase Parkin was increased in transverse aortic constriction mouse hearts and phenylephrine stimulation-induced hypertrophic cardiomyocytes. Inhibition of proteasome blocked phenylephrine-induced decrease of Miro2, and Parkin overexpression led to the decrease of Miro2. Conclusions: Mitochondrial Miro2 expression levels regulate inter-mitochondrial communication along microtubules in adult cardiomyocytes, and degradation of Miro2 through Parkin-mediated ubiquitination contributes to impaired inter-mitochondrial communication and cardiac dysfunction during hypertrophic heart diseases.Visual Overview: An online visual overview is available for this article.

Long Noncoding RNA-DACH1 (Dachshund Homolog 1) Regulates Cardiac Function by Inhibiting SERCA2a (Sarcoplasmic Reticulum Calcium ATPase 2a).

Heart failure (HF) is a major cause of morbidity and mortality in patients with various cardiovascular diseases. Restoration of cardiac function is critical in improving the clinical outcomes of patients with HF. Long noncoding RNAs are widely involved in the development of multiple cardiac diseases, whereas their role in regulating cardiac function remains unclear. In this study, we found that the expression of long noncoding RNA-DACH1 (dachshund homolog 1) was upregulated in the failing hearts of mice and human. We tested the hypothesis that the intronic long noncoding RNA of DACH1 (LncDACH1) can participate in the regulation of cardiac function and HF. Transgenic overexpression of LncDACH1 in the cardiac myocytes of mice led to impaired cardiac function, reduced calcium transient and cell shortening, and decreased SERCA2a (sarcoplasmic reticulum calcium ATPase 2a) protein expression. In contrast, conditional knockout of LncDACH1 in cardiac myocytes resulted in increased calcium transient, cell shortening, SERCA2a protein expression, and improved cardiac function of transverse aortic constriction induced HF mice. The same qualitative data were obtained by overexpression or knockdown of LncDACH1 with adenovirus carrying LncDACH1 or its siRNA. Moreover, therapeutic administration of adenovirus carrying LncDACH1 siRNA to transverse aortic constriction mice abolished the development of HF. Mechanistically, LncDACH1 directly binds to SERCA2a. Overexpression of LncDACH1 augments the ubiquitination of SERCA2a. LncDACH1 upregulation impairs cardiac function by promoting ubiquitination-related degradation of SERCA2a.

Abstract 847: Adipocyte-specific Pharmacological Inhibition of Adipose Triglyceride Lipase (ATGL) Ameliorates Cardiac Fibrosis in Heart Failure

Objectives: ATGL is a key enzyme that regulates triglyceride lipolysis in most cells, including the adipocyte and the cardiomyocyte. Recent studies indicate that accelerated adipose tissue lipolysis contributes to worsening heart failure (HF), suggesting that ATGL inhibition specifically in the adipocyte may prevent worsening HF. Indeed, we have shown that the ATGL inhibitor, Atglistatin, which appears to be largely adipocyte-specific, ameliorates cardiac dysfunction and hypertrophy in HF induced by transverse aortic constriction (TAC). However, the mechanisms by which Atglistatin treatment prevents worsening HF in mice remains unknown. Methods: Eight week-old C57BL/6N mice underwent TAC or sham surgery. After 1 week, mice with HF were assigned into 4 groups: 1) vehicle (standard chow)-sham, 2) Atglistain-sham, 3) vehicle-TAC and 4) Atglistatin-TAC. Atglistain was administered in food pellets containing 2mmol Atglistatin/kg for 2 weeks. Thereafter, mice were euthanized and hearts were used for subsequent histological and quantitative PCR analysis. Results: Picro-sirius red staining demonstrated that hearts from the Atglistatin TAC mice had significantly less cardiac fibrosis than the vehicle TAC mice (1.6±0.4 vs 5.3±1.1%, in Atglistatin TAC vs vehicle TAC; p&lt;0.01). Consistent with this finding, Atglistatin treatment of the TAC group reduced transcript levels of fibrosis-related genes, including Col1a1 (-58.7%, p=0.03) and Postn (-54.7%, p&lt;0.01), compared to vehicle-treated hearts. Furthermore, Atglistatin decreased the transcript levels of the macrophage polarization-related genes, including Lgals3 (-55.3%, p&lt;0.01) and Spp1 (-38.7%, p=0.03) , in the hearts from TAC mice compared to controls. Moreover, immunofluorescent analysis showed that Atglistatin reduced infiltration of Galectin-3 positive macrophages in the heart compared to vehicle-TAC mice, suggesting reduced macrophage infiltration and activation and less cardiac fibrosis. Conclusion: Our data indicate that reducing triglyceride lipolysis in the adipose tissue via ATGL inhibition may ameliorate HF via modulation of HF-induced macrophage and fibroblast activation in the heart.

Abstract 518: Pharmacological Inhibition of Human Antigen R (HuR) Blunts fibroblast Activation and Cardiac Fibrosis

Myocardial fibrosis is associated with many cardiac diseases and often leads to a worsened prognosis. In a healthy heart, cardiac fibroblasts are responsible for maintaining the extracellular matrix (ECM). In response to stress or injury, however, the fibroblasts activate to myofibroblasts and begin proliferating, migrating, and depositing excess ECM. This leads to decreased compliance and contractility of the myocardium, furthering the progression to heart failure. Dampening the chronic activation of fibroblasts, therefore, is an attractive target for future cardiac therapeutics. Human Antigen R (HuR) is an RNA binding protein known to stabilize mRNA through binding to AU rich regions in the 3’UTR. Our lab has previously utilized a myocyte-specific knockout of HuR to show that HuR plays an important role in cardiac fibrosis through mediating TGFβ gene expression in the myocytes. Additionally, pharmacological inhibition of HuR markedly reduced interstitial cardiac fibrosis in an 11-week transverse aortic constriction (TAC) mouse model of heart failure, quantifying the fibrosis with Masson’s Trichrome Stains, western blots, and qPCR of profibrotic markers. The goal of this work is to identify the functional role of HuR in mediating pro-fibrotic gene expression and ECM remodeling activity in cardiac fibroblasts. Our results demonstrate that HuR inhibition blunts the activation and wound healing activity of primary adult cardiac fibroblasts. Specifically, utilizing an in vitro scratch/wound healing assay we show that HuR inhibition significantly prolongs wound closure time and blunts the expression of profibrotic genes (e.g. collagen1a1, periostin, TGFb, fibronectin). Ongoing studies will determine the effect of HuR inhibition in a two-week isoproterenol pump (30mg/kg/day) model. Quantification of fibrosis and upstream signaling of fibroblast activation, such as TGFβ-dependent phosphorylation of SMAD2/3, will be assessed to determine the role of HuR in the initiation of fibrotic signaling.

Distinct Phenotypes Induced by Three Degrees of Transverse Aortic Constriction in Mice

Introduction Experimental models of left ventricular (LV) remodeling provide important preclinical methods for identifying novel therapeutic targets and evaluating experimental therapies for heart failure. Transverse aortic constriction (TAC) is a well-established model of pressure overload-induced cardiac hypertrophy and failure in mice. The degree of constriction “tightness” dictates the TAC severity and is determined by the gauge (G) of needle used. Though many reports use the TAC model, few studies have directly compared the range of resulting phenotypes. Hypothesis We hypothesize that different gauges of TAC will induce distinct phenotypes, which may be suitable for use in different preclinical studies. Methods Mice were randomized to receive TAC surgery with varying degrees of tightness: mild (25G), moderate (26G) or severe (27G) for 4 weeks, alongside sham-operated controls. Weekly echocardiography and terminal hemodynamic analysis determined cardiac remodeling and functional changes. Results All TAC models induced significant, severity-dependent trans-TAC pressure gradients, LV hypertrophy and diastolic dysfunction compared to sham mice. Mice subjected to 26G TAC additionally exhibited cardiac fibrosis and mild systolic dysfunction, in terms of left ventricular strain rate and preload-recruitable stroke work, whereas mice in the 27G TAC group had even more severe cardiac fibrosis, global systolic and diastolic dysfunction in terms of ejection fraction and LV filling indices, and were more likely to display features of heart failure, such as elevated plasma BNP (903.6±59.2 pmol/mL in 27G TAC vs. 676.0±33.8 pmol/mL in 26G TAC; P Conclusions 25G, 26G and 27G TAC produced different responses in terms of cardiac structure and function. 25G TAC led to a mild hypertrophic phenotype characterized by diastolic dysfunction, similar to hypertensive heart disease. 26G TAC led to a moderate hypertrophic phenotype with mixed systolic and diastolic dysfunction, comparable to early decompensation. Lastly, 27G TAC led to a severe hypertrophic phenotype with severely reduced systolic function, similar to clinical heart failure with reduced ejection fraction. These distinct phenotypes may be applicable to specific preclinical settings.

ADAMTS16 activates latent TGF-β, accentuating fibrosis and dysfunction of the pressure-overloaded heart.

Cardiac fibrosis is a major cause of heart failure (HF), and mediated by the differentiation of cardiac fibroblasts into myofibroblasts. However, limited tools are available to block cardiac fibrosis. ADAMTS16 is a member of the ADAMTS superfamily of extracellular protease enzymes involved in extracellular matrix (ECM) degradation and remodelling. In this study, we aimed to establish ADAMTS16 as a key regulator of cardiac fibrosis. Western blot and qRT-PCR analyses demonstrated that ADAMTS16 was significantly up-regulated in mice with transverse aortic constriction (TAC) associated with left ventricular hypertrophy and HF, which was correlated with increased expression of Mmp2, Mmp9, Col1a1, and Col3a1. Overexpression of ADAMTS16 accelerated the AngII-induced activation of cardiac fibroblasts into myofibroblasts. Protein structural analysis and co-immunoprecipitation revealed that ADAMTS16 interacted with the latency-associated peptide (LAP)-transforming growth factor (TGF)-β via a RRFR motif. Overexpression of ADAMTS16 induced the activation of TGF-β in cardiac fibroblasts; however, the effects were blocked by a mutation of the RRFR motif to IIFI, knockdown of Adamts16 expression, or a TGF-β-neutralizing antibody (ΝAb). The RRFR tetrapeptide, but not control IIFI peptide, blocked the interaction between ADAMTS16 and LAP-TGF-β, and accelerated the activation of TGF-β in cardiac fibroblasts. In TAC mice, the RRFR tetrapeptide aggravated cardiac fibrosis and hypertrophy by up-regulation of ECM proteins, activation of TGF-β, and increased SMAD2/SMAD3 signalling, however, the effects were blocked by TGF-β-NAb. ADAMTS16 promotes cardiac fibrosis, cardiac hypertrophy, and HF by facilitating cardiac fibroblasts activation via interacting with and activating LAP-TGF-β signalling. The RRFR motif of ADAMTS16 disrupts the interaction between ADAMTS16 and LAP-TGF-β, activates TGF-β, and aggravated cardiac fibrosis and hypertrophy. This study identifies a novel regulator of TGF-β signalling and cardiac fibrosis, and provides a new target for the development of therapeutic treatment of cardiac fibrosis and HF.

Impaired branched chain amino acid oxidation contributes to cardiac insulin resistance in heart failure

BackgroundBranched chain amino acids (BCAA) can impair insulin signaling, and cardiac insulin resistance can occur in the failing heart. We, therefore, determined if cardiac BCAA accumulation occurs in patients with dilated cardiomyopathy (DCM), due to an impaired catabolism of BCAA, and if stimulating cardiac BCAA oxidation can improve cardiac function in mice with heart failure.MethodFor human cohorts of DCM and control, both male and female patients of ages between 22 and 66 years were recruited with informed consent from University of Alberta hospital. Left ventricular biopsies were obtained at the time of transplantation. Control biopsies were obtained from non-transplanted donor hearts without heart disease history. To determine if stimulating BCAA catabolism could lessen the severity of heart failure, C57BL/6J mice subjected to a transverse aortic constriction (TAC) were treated between 1 to 4-week post-surgery with either vehicle or a stimulator of BCAA oxidation (BT2, 40 mg/kg/day).ResultEchocardiographic data showed a reduction in ejection fraction (54.3 ± 2.3 to 22.3 ± 2.2%) and an enhanced formation of cardiac fibrosis in DCM patients when compared to the control patients. Cardiac BCAA levels were dramatically elevated in left ventricular samples of patients with DCM. Hearts from DCM patients showed a blunted insulin signalling pathway, as indicated by an increase in P-IRS1ser636/639 and its upstream modulator P-p70S6K, but a decrease in its downstream modulators P-AKT ser473 and in P-GSK3β ser9. Cardiac BCAA oxidation in isolated working hearts was significantly enhanced by BT2, compared to vehicle, following either acute or chronic treatment. Treatment of TAC mice with BT2 significantly improved cardiac function in both sham and TAC mice (63.0 ± 1.8 and 56.9 ± 3.8% ejection fraction respectively). Furthermore, P-BCKDH and BCKDK expression was significantly decreased in the BT2 treated groups.ConclusionWe conclude that impaired cardiac BCAA catabolism and insulin signaling occur in human heart failure, while enhancing BCAA oxidation can improve cardiac function in the failing mouse heart.

Tamarixetin protects against cardiac hypertrophy via inhibiting NFAT and AKT pathway.

Cardiac hypertrophy is a compensatory response in reaction to mechanical load that reduces wall stress by increasing wall thickness. Chronic hypertrophic remodeling involves cardiac dysfunction that will lead to heart failure and ultimately death. Studies have been carried out on cardiac hypertrophy for years, whereas the mechanisms have not been well defined. Tamarixetin (TAM), a natural flavonoid derivative of quercetin, have been demonstrated possessing anti-oxidative and anti-inflammatory effects on multiple diseases. However, little is known about the function of TAM on the development of cardiac hypertrophy. Here, we found TAM could alleviate pressure-overload-induced cardiac hypertrophy in transverse aortic constriction (TAC) mouse model, assessed by ventricular weight/body weight, lung weight/body weight, echocardiographic parameters, as well as myocyte cross-sectional area and the expression of ANP, BNP and Myh7. In vitro, TAM showed a dose dependent inhibitory effect on phenylephrine-induced hypertrophy in H9c2 cardiomyocytes. Furthermore, TAM reversed cardiac remodeling of stress overloaded heart by suppressing apoptosis and the expression of fibrotic-related genes, reduced oxidative stress and ROS production both in vivo and in vitro. In addition, TAM could negatively modulate TAC-induced nuclear translocation of NFAT and the activation of PI3K/AKT signaling pathways. Therefore, these data indicate for the first time that TAM has a protective effect on experimental cardiac hypertrophy and might be a novel candidate for the treatment of cardiac hypertrophy in clinic.

Distinct Phenotypes Induced by Three Degrees of Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a well-established model of pressure overload-induced cardiac hypertrophy and failure in mice. The degree of constriction “tightness” dictates the TAC severity and is determined by the gauge (G) of needle used. Though many reports use the TAC model, few studies have directly compared the range of resulting phenotypes. In this study adult male mice were randomized to receive TAC surgery with varying degrees of tightness: mild (25G), moderate (26G) or severe (27G) for 4 weeks, alongside sham-operated controls. Weekly echocardiography and terminal haemodynamic measurements determined cardiac remodelling and function. All TAC models induced significant, severity-dependent left ventricular hypertrophy and diastolic dysfunction compared to sham mice. Mice subjected to 26G TAC additionally exhibited mild systolic dysfunction and cardiac fibrosis, whereas mice in the 27G TAC group had more severe systolic and diastolic dysfunction, severe cardiac fibrosis, and were more likely to display features of heart failure, such as elevated plasma BNP. We also observed renal atrophy in 27G TAC mice, in the absence of renal structural, functional or gene expression changes. 25G, 26G and 27G TAC produced different responses in terms of cardiac structure and function. These distinct phenotypes may be useful in different preclinical settings.

Captopril attenuates TAC-induced heart failure via inhibiting Wnt3a/β-catenin and Jak2/Stat3 pathways

Captopril (Cap) as angiotensin-converting enzyme inhibitor (ACEi) is commonly used to treat hypertension and some types of congestive heart failure. However, few studies reported on whether Cap exerts a protective effect on myocardial apoptosis induced by transverse aortic constriction (TAC). This study aimed at investigating the possible mechanism of Cap on myocardial apoptosis induced by pressure overload. Results showed that Cap significantly decreased heart-to-body weight ratios (HBWR). Cap markedly improved cardiac function, and reduced inner diameter of ascending aorta (Asc Ao) in TAC mice as shown by echocardiography. Enzyme-linked immunosorbent assay (ELISA) results demonstrated that Cap treatment also markedly decreased the level of N-terminal pro-B-type natriuretic peptide (NT-proBNP), atrial natriuretic peptide (ANP), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cardiac pathological changes and fibrosis have been improved after Cap treatment as shown by hematoxylin-eosin (H&E) staining and Masson’s trichrome staining. Moreover, Terminal deoxynucleotidyl transferase-mediated dexoxyuridine triphosphate nick-end labeling (TUNEL) staining result indicated Cap treatment also significantly inhibited cardiac apoptosis. Western Blot results showed that Cap obviously decreased the expression of cleaved capase-3, Bax, phosphorylated Jak2 (p-Jak2), phosphorylated Stat3 (p-Stat3), Wnt3a and β-catenin proteins, as well as increased Bcl-2 expression. In conclusion, Cap showed a protective effect on TAC-induced cardiac apoptosis, which could be attributed to the inhibition of Wnt3a/β-catenin signaling pathway. Cap also attenuated myocardial hypertrophy induced by TAC via suppression of Jak2/Stat3 pathway.

Alamandine attenuates arterial remodelling induced by transverse aortic constriction in mice

Aims: The renin-angiotensin system (RAS) plays an important role in the pathophysiology of vascular diseases, especially as a mediator of inflammation and tissue remodelling. Alamandine (Ala1-angiotensin-(1-7)) is a new biologically active peptide from the RAS, interacting with Mas-related G-protein-coupled receptor member D. Although a growing number of studies reveal the cardioprotective effects of alamandine, there is a paucity of data on its participation in vascular remodelling associated events. In the present study, we investigated the effects of alamandine on ascending aorta remodelling after transverse aortic constriction (TAC) in mice. Methods and results: C57BL/6J male mice were divided into the following groups: Sham (sham-operated), TAC (operated) and TAC+ALA (operated and treated with alamandine-HPβCD (2-Hydroxypropyl-β-cyclodextrin), 30 μg/kg/day, by gavage). Oral administration of alamandine for 14 days attenuated arterial remodelling by decreasing ascending aorta media layer thickness and the cells density in the adventitia induced by TAC. Alamandine administration attenuated ascending aorta fibrosis induced by TAC, through a reduction in the following parameters; total collagen deposition, expression collagen III and transforming growth factor-β (TGF-β) transcripts, matrix metalloproteinases (MMPs) activity and vascular expression of MMP-2. Importantly, alamandine decreased vascular expression of proinflammatory genes as CCL2, tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β), and was able to increase expression of MRC1 and FIZZ1, pro-resolution markers, after TAC surgery. Conclusion: Alamandine treatment attenuates vascular remodelling after TAC, at least in part, through anti-fibrotic and anti-inflammatory effects. Hence, this work opens new avenues for the use of this heptapeptide also as a therapeutic target for vascular disease.

Reversal of Aortic Enlargement Induced by Increased Biomechanical Forces Requires AT1R Inhibition in Conjunction With AT2R Activation.

Objective- Pharmacological inhibition of the AT1R (angiotensin II type 1 receptor) with losartan can attenuate ascending aortic remodeling induced by transverse aortic constriction (TAC). In this study, we investigated the role of the AT2R (angiotensin II type 2 receptor) and MasR (Mas receptor) in TAC-induced ascending aortic dilation and remodeling. Approach and Results- Wild-type C57BL/6J mice were subjected to sham or TAC surgeries in the presence and absence of various drugs. Aortic diameters were assessed by echocardiography, central blood pressure was measured in the ascending aorta 2 weeks post-operation, and histology and gene expression analyses completed. An angiotensin-converting enzyme inhibitor, captopril, decreased systolic blood pressure to the same level as losartan but did not attenuate aortic dilation, adventitial inflammation, medial collagen deposition, elastin breakage, or Mmp9 (matrix metalloproteinase-9) expression when compared with TAC mice. In contrast, co-administration of captopril with an AT2R agonist, compound 21, attenuated aortic dilation, medial collagen content, elastin breaks, and Mmp9 expression, whereas co-administration of captopril with a MasR agonist (AVE0991) did not reverse aortic dilation and led to aberrant aortic remodeling. An AT2R antagonist, PD123319, reversed the protective effects of losartan in TAC mice. Treatment with compound 21 alone showed no effect on TAC-induced aortic enlargement, blood pressure, elastin breakage, or Mmp9 expression. Conclusions- Our data indicate that when AT1R signaling is blocked, AT2R activation is a key modulator to prevent aortic dilation that occurs with TAC. These data suggest that angiotensin-converting enzyme inhibitor may not be as effective as losartan for slowing aneurysm growth because losartan requires intact AT2R signaling to prevent aortic enlargement.

Increased ketone body oxidation provides additional energy for the failing heart without improving cardiac efficiency.

The failing heart is energy-starved and inefficient due to perturbations in energy metabolism. Although ketone oxidation has been shown recently to increase in the failing heart, it remains unknown whether this improves cardiac energy production or efficiency. We therefore assessed cardiac metabolism in failing hearts and determined whether increasing ketone oxidation improves cardiac energy production and efficiency. C57BL/6J mice underwent sham or transverse aortic constriction (TAC) surgery to induce pressure overload hypertrophy over 4-weeks. Isolated working hearts from these mice were perfused with radiolabelled β-hydroxybutyrate (βOHB), glucose, or palmitate to assess cardiac metabolism. Ejection fraction decreased by 45% in TAC mice. Failing hearts had decreased glucose oxidation while palmitate oxidation remained unchanged, resulting in a 35% decrease in energy production. Increasing βOHB levels from 0.2 to 0.6 mM increased ketone oxidation rates from 251 ± 24 to 834 ± 116 nmol·g dry wt-1 · min-1 in TAC hearts, rates which were significantly increased compared to sham hearts and occurred without decreasing glycolysis, glucose, or palmitate oxidation rates. Therefore, the contribution of ketones to energy production in TAC hearts increased to 18% and total energy production increased by 23%. Interestingly, glucose oxidation, in parallel with total ATP production, was also significantly upregulated in hearts upon increasing βOHB levels. However, while overall energy production increased, cardiac efficiency was not improved. Increasing ketone oxidation rates in failing hearts increases overall energy production without compromising glucose or fatty acid metabolism, albeit without increasing cardiac efficiency.