Transverse Aortic Constriction Model Research Articles

Pharmacodynamics and Mechanism of Astragali Radix and Anemarrhenae Rhizoma in Treating Chronic Heart Failure by Inhibiting Complement Activation.

Astragali radix (AR) and anemarrhenae rhizoma (AAR) are used clinically in Chinese medicine for the treatment of chronic heart failure (CHF), but the exact therapeutic mechanism is unclear. In this study, a total of 60 male C57BL/6 mice were divided into 5 groups, namely sham, model, AR, AAR, and AR-AAR. In the sham group, the chest was opened without ligation. In the other groups, the chest was opened and the transverse aorta was ligated to construct the transverse aortic constriction model. After 8 weeks of feeding, mice were given medicines by gavage for 4 weeks. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were detected by echocardiography. Heart weight index (HWI) and wheat germ agglutinin staining were used to evaluate cardiac hypertrophy. Hematoxylin-eosin staining was used to observe the pathological morphology of myocardial tissue. Masson staining was used to evaluate myocardial fibrosis. The content of serum brain natriuretic peptide (BNP) was detected by enzyme-linked immunosorbent assay kit. The content of serum immunoglobulin G (IgG) was detected by immunoturbidimetry. The mechanism of AR-AAR in the treatment of CHF was explored by proteomics. Western blot was used to detect the protein expressions of complement component 1s (C1s), complement component 9 (C9), and terminal complement complex 5b-9 (C5b-9). The results show that AR-AAR inhibits the expression of complement proteins C1s, C9, and C5b-9 by inhibiting the production of IgG antibodies from B cell activation, which further inhibits the complement activation, attenuates myocardial fibrosis, reduces HWI and cardiomyocyte cross-sectional area, improves cardiomyocyte injury, reduces serum BNP release, elevates LVEF and LVFS, improves cardiac function, and exerts myocardial protection.

Abstract 15259: The Novel Antisense Oligonucleotide CDR132L Improves Cardiac Function in Heart Failure Across Different Species

Introduction: The chronic progressive condition of heart failure (HF) is, to date, largely irreversible and continues to impose global morbidity. MicroRNAs exert substantial roles in HF progression by orchestrating pathological genes and pathways. Medications directly tackling the roots of the disease are rare, but inhibition of miR-132, a microRNA that was shown to be upregulated upon cardiac remodeling and HF, is a promising approach. Thus, CDR132L, a clinical-stage synthetic antisense oligonucleotide miR-132 inhibitor, can be an innovative therapy for patients with HF. Hypothesis: To examine the effect of CDR132L, different studies were conducted, including in mice and pigs and in a first-inhuman phase 1b safety and tolerability study that revealed preclinical and exploratory efficacy data. Results: MiR-132 transgenic mice developed reduced left-ventricular ejection fraction (LVEF) and cardiac dilatation. CDR132L treatment significantly improved LVEF and reversed cardiac dilatation. The results were confirmed in a porcine transverse aortic constriction model, where the LVEF was significantly improved in the CDR132L treatment group compared to control animals (48.9% vs. 36.1%). In post-MI HF pigs subjected to different treatment and dosing regimens, resulted in a significant and dose-dependent improvement of the cardiac function, illustrated by an up to 10.4% improvement in LVEF to the placebo group at day 56. During a monthly treatment, 87.5% of the animals treated 5x 5 mg/kg with CDR132L showed a ΔLVEF of >7% compared to the placebo group. Additionally, CDR132L improved contractility and the diastolic function by reducing cardiac stiffness. A successful translation of these preclinical results into humans was performed in a CDR132L safety and tolerability Phase 1b clinical trial in HF patients. In an exploratory analysis, the combined endpoint, consisting of patients with a decrease in NT-proBNP levels and/or an absolute increase in LVEF, was significantly met by 79% of the patients in the pharmacodynamically active CDR132L group. Conclusion: Pharmacological miR-132 inhibition by CDR132L improved cardiac function in multi-species models of HF, highlighting the therapeutic potential for clinical use in HF patients.

Biomimetic nanoparticles loaded lutein functionalized by macrophage membrane for targeted amelioration pressure overload-induced cardiac fibrosis

Lutein is a strong antioxidant with anti-inflammatory, anti-oxidative and cardioprotective effects and could be a promising candidate for the treatment of hypertensive heart disease (HHD), but is not clinically appealing because of its low oral bioavailability and main distribution in the eyes. To address this, a biomimetic drug delivery system-MMLNPs was established by coating macrophage membranes (MMs) onto lutein-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (LNPs). This study characterized the physical properties of biomimetic nanoparticles and examined the targeting capability, therapeutic effects and mechanism, and biosecurity of administering them for cardiac fibrosis therapy in the transverse aortic constriction (TAC) model and in vitro. Transmission electron microscope mapping and dynamic light scattering analysis proved that MMLNPs were spherical nanoparticles camouflaged by a layer of cell membrane and had negative zeta potential. Confocal laser scanning microscopy and flow cytometry analysis showed that MMs on the biomimetic nanoparticles hindered the phagocytosis of macrophages and facilitated the targeting of activated endothelial cells. Ex vivo fluorescence imaging experiments demonstrated the targeting of biomimetic nanoparticles to the injured heart. EdU assay indicated that MMLNPs have the same potential to inhibit angiotensin (Ang) II-induced cardiac fibroblast proliferation as free lutein. Furthermore, echocardiography showed that MMLNPs improved cardiac function and structure, and Masson staining and western blotting showed that MMLNPs ameliorated cardiac fibrosis. We found MMLNPs inhibited the interleukin (IL)-11/ERK signaling pathway which was up-regulated in the TAC model compared to the sham-operated mouse. Biochemical testing and hematoxylin and eosin staining proved that the long-term use of MMLNPs lacked biological toxicity. Collectively, MMLNPs might be a promising nanodrug delivery approach to attenuate pressure overload (PO)-induced cardiac fibrosis.

Abstract P2065: Mice Under Transverse Aortic Constriction And Treated With Sodium Oxalate May Develop Cardiac Dysfunction

Background: Experimental models are important for studying the mechanisms of cardiovascular disease. The transverse aortic constriction model (TAC) is among the most used in the literature for the study of cardiac hypertrophy. The soluble sodium oxalate (OXA) model generates renal injury but is also associated with cardiac hypertrophy through hypertensive-like lesions. There is little information in the literature on direct hemodynamic recording in both models mentioned, as well as there is no data on the changes caused by the association of these two models. Therefore, our objective is to evaluate whether TAC associated with OXA gavage could be a new model of cardiac dysfunction. Methods and Results: Male C57BL mice were randomized as follows: SHAM animals that received gavage with vehicle or OXA solution, and animals submitted to TAC and gavage with vehicle or OXA solution. Echocardiography and metabolic cage assessment were used to quantify cardiac and renal function. Direct blood pressure (BP) recording was also performed to assess baroreflex sensitivity and histological analysis. The TAC+OXA group had the highest BP values (mean BP:SHAM:100±5.0 vs. TAC+OXA:132±4.8mmHg, p<0.01), the highest pulmonary congestion index, an increase in the double product, an important reduction in autonomic modulation (AM), heart rate variability (HRV) (SD-PI: SHAM+OXA:12.5±1.8 vs. TAC+OXA:2.8±0.2ms, p<0.01) and alpha index (SHAM:3.6±1.0 vs. TAC+OXA:0.6±0.1, p=0.04). The TAC group showed the greatest weight of the ventricles and the greatest increase in blood pressure variability (DP-SBP). The SHAM+OXA group showed an increase in AM and HRV. The three groups showed a significant reduction in reflex tachycardia. Conclusion: TAC surgery provided cardiac hypertrophy, reduction in reflex tachycardia and changes in autonomic modulation. In the absence of TAC, the treatment with OXA for 15 days increased autonomic modulation. TAC surgery in conjunction with OXA gavage caused greater cardiac dysfunction.

Guanxining injection alleviates fibrosis in heart failure mice and regulates SLC7A11/GPX4 axis.

Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Lamiaceae, Danshen in Chinese) and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort., Apiaceae, Chuanxiong in Chinese) both are important traditional Chinese medicine (TCM) for activating blood and eliminating stasis. Danshen-chuanxiong herb pair has been used for more than 600 years in China. Guanxinning injection (GXN) is a Chinese clinical prescription refined from aqueous extract of Danshen and Chuanxiong at the ratio of 1:1 (w/w). GXN has been mainly used in the clinical therapy of angina, heart failure (HF) and chronic kidney disease in China for almost twenty years. This study aimed to explore the role of GXN on renal fibrosis in heart failure mice and the regulation of GXN on SLC7A11/GPX4 axis. The transverse aortic constriction model was used to mimic HF accompanied by kidney fibrosis model. GXN was administrated by tail vein injection in dose of 12.0, 6.0, 3.0mL/kg, respectively. Telmisartan (6.1mg/kg, gavage) was used as a positive control drug. Cardiac ultrasound indexes of ejection fraction (EF), cardiac output (CO), left ventricle volume (LV Vol), HF biomarker of pro-B type natriuretic peptide (Pro-BNP), kidney function index of serum creatinine (Scr), kidney fibrosis index of collagen volume fraction (CVF) and connective tissue growth factor (CTGF) were evaluated and contrasted. Metabolomic method was employed to analyze the endogenous metabolites changes in kidneys. Besides, contents of catalase (CAT), xanthine oxidase (XOD), nitricoxidesynthase (NOS), glutathione peroxidase 4 (GPX4), the x(c)(-) cysteine/glutamate antiporter (SLC7A11) and ferritin heavy chain (FTH1) in kidney were quantitatively analyzed. In addition, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the chemical composition of GXN and network pharmacology was used to predict possible mechanisms and the active ingredients of GXN. The cardiac function indexes of EF, CO and LV Vol, kidney functional indicators of Scr, the degree of kidney fibrosis indicators CVF and CTGF were all relieved to different extent for the model mice treated with GXN. 21 differential metabolites involved in redox regulation, energy metabolism, organic acid metabolism, nucleotide metabolism, etc were identified. Aspartic acid, homocysteine, glycine, and serine, methionine, purine, phenylalanine and tyrosine metabolism were found to be the core redox metabolic pathways regulated by GXN. Furthermore, GXN were found to increase CAT content, upregulate GPX4, SLC7A11 and FTH1 expression in kidney significantly. Not only that, GXN also showed good effect in down-regulating XOD and NOS contents in kidney. Besides, 35 chemical constituents were initially identified in GXN. Active ingredients of GXN-targets-related enzymes/transporters-metabolites network was established to find out that GPX4 was a core protein for GXN and the top 10 active ingredients with the most relevant to renal protective effects of GXN were rosmarinic acid, caffeic acid, ferulic acid, senkyunolide E, protocatechualdehyde, protocatechuic acid, danshensu, L-Ile, vanillic acid, salvianolic acid A. GXN could significantly maintain cardiac function and alleviate the progression of fibrosis in the kidney for HF mice, and the mechanisms of action were related to regulating redox metabolism of aspartate, glycine, serine, and cystine metabolism and SLC7A11/GPX4 axis in kidney. The cardio-renal protective effect of GXN may be attributed to multi-components like rosmarinic acid, caffeic acid, ferulic acid, senkyunolide E, protocatechualdehyde, protocatechuic acid, danshensu, L-Ile, vanillic acid, salvianolic acid A et al.

Bioinformatics exploration of potential common therapeutic targets for systemic and pulmonary arterial hypertension-induced myocardial hypertrophy.

Systemic and pulmonary arterial hypertension (PAH) can induce left and right ventricular hypertrophy, respectively, but common therapeutic targets for both left and right hypertrophy are limited. In this study, we attempt to explore potential common therapeutic targets and screen out potential target drugs for further study. Cardiac mRNA expression profiles in mice with transverse aortic constriction (TAC) and pulmonary arterial constriction (PAC) are obtained from online databases. After bioinformatics analyses, we generate TAC and PAC mouse models to validate the phenotypes of cardiac remodelling as well as the identified hub genes. Bioinformatics analyses show that there are 214 independent differentially expressed genes (DEGs) in GSE136308 (TAC related) and 2607 independent DEGs in GSE30922 (PAC related), while 547 shared DEGs are associated with the function of the extracellular matrix (ECM) or involved in the PI3K-Akt signaling pathway, cytokine-cytokine receptor interactions, and ECM-receptor interactions. We identifyd Fn1, Il6, Col1a1, Igf1, Col1a2, Timp1, Col3a1, Cd44, Ctgf and Postn as hub genes of the shared DEGs, and most of them are associated with myocardial fibrosis. Those hub genes and phenotypes of cardiac remodelling are validated in our TAC and PAC mouse models. Furthermore, we identify dehydroisoandrosterone (DHEA), iloprost and 4,5-dianilinophthalimide (DAPH) as potential therapeutic drugs targeting both left and right ventricular hypertrophy and validate the effect of DHEA. These findings suggest that DHEA could be an effective drug for pressure overload-induced left or right ventricular hypertrophy by regulating the shared hub differentially expressed genes associated with fibrosis.

Expression and role of SerpinE2 in cardiac fibrosis

Cardiac fibrosis is a common histological feature of a broad spectrum of cardiovascular diseases including myocardial infarction. Proteases and their inhibitors are known to be involved in the regulation of extracellular matrix deposition and remodeling. SerpinE2 is a serine protease inhibitor expressed within the myocardium, which targets thrombin and the proteases of the plasminergic system. This inhibitor has been shown to be involved in lung fibrosis and could be an actor of cardiac remodeling. To study SerpinE2 expression in human left ventricle biopsies, from organ donors (control) and recipient explanted patients. To evaluate SerpinE2 role in cardiac remodeling and fibrosis in the mouse TAC (transverse aortic constriction) model. LV biopsies from organ donors and recipient explanted patients were characterized by histology and classified. SerpinE2 mRNA and protein expression in human samples were assessed by RT-qPCR and Western-Blot after pulverization in liquid nitrogen. Immunofluorescence was used to characterize SerpinE2 expression within paraffin embedded biopsies sections. Wild-type (WT) and SerpinE2-deficient (KO) mice were subjected to 27G TAC model and cardiac function was followed weekly by echocardiography Doppler. After 4 weeks, mice were sacrificed, hearts were harvested and frozen in liquid nitrogen. Heart cryosections were then used for histological fibrosis analysis (Sirius red) or for RNA extraction and RT-qPCR. We found that SerpinE2 expression (mRNA and protein) was significantly increased (×2.5) in the infarcted zone of the biopsies from the LV of explanted patients, compared with non-infarcted area and control donors. SerpinE2 mRNA expression in these samples was also strongly correlated with the expression on type I and III collagens, and TGF-ß. Immunofluorescence assays showed that SerpinE2 protein was mainly detected within the fibrotic areas of the explanted donors. Echocardiography Doppler of 27G mice showed significant decrease in KO animals LV mass and thickness compared with WT counterparts. A significant 2.3 times decrease in type I collagen mRNA expression was observed in KO mice but this was not confirmed by Sirius red staining in heart sections. These data suggest that SerpinE2 could be an actor and/or a biomarker or cardiac fibrosis.

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.

OTUD1 promotes pathological cardiac remodeling and heart failure by targeting STAT3 in cardiomyocytes.

Rationale: Understanding the molecular mechanisms of deleterious cardiac remodeling is important for the development of treatments for heart failure. Recent studies have highlighted a role of deubiquitinating enzymes in cardiac pathophysiology. In the present study, we screened for alteration of deubiquitinating enzymes in experimental models of cardiac remodeling, which indicated a potential role of OTU Domain-Containing Protein 1 (OTUD1). Methods: Wide-type or OTUD1 knockout mice with chronic angiotensin II infusion and transverse aortic constriction (TAC) were utilized to develop cardiac remodeling and heart failure. We also overexpressed OTUD1 in mouse heart with AAV9 vector to validate the function of OTUD1. LC-MS/MS analysis combined with Co-IP was used to identify the interacting proteins and substrates of OTUD1. Results: We found that OTUD1 is elevated in mouse heart tissues following chronic angiotensin II administration. OTUD1 knockout mice were significantly protected against angiotensin II-induced cardiac dysfunction, hypertrophy, fibrosis and inflammatory response. Similar results were obtained in the TAC model. Mechanistically, OTUD1 bounds to the SH2 domain of STAT3 and causes deubiquitination of STAT3. Cysteine at position 320 of OTUD1 exerts K63 deubiquitination to promote STAT3 phosphorylation and nuclear translocation, thereby increasing STAT3 activity to induce inflammatory responses, fibrosis, and hypertrophy in cardiomyocytes. Finally, OTUD1 overexpression by AAV9 vector increases Ang II-induced cardiac remodeling in mice and OTUD1-regulated responses can be inhibited by blocking STAT3. Conclusion: Cardiomyocyte OTUD1 promotes pathological cardiac remodeling and dysfunction by deubiquitinating STAT3. These studies have highlighted a novel role of OTUD1 in hypertensive heart failure and identified STAT3 as a target of OTUD1 in mediating these actions.

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.

Pressure overload and volume overload-induced chronic heart failure are associated with characteristic left ventricular myocardial proteomic alterations

Abstract Introduction Hemodynamic overload induces pathological remodeling of the left ventricle (LV) and eventually heart failure (HF). The two types of chronic hemodynamic stress, namely pressure overload (PO) and volume overload (VO) evoke characteristically different functional and structural alterations in the myocardium. Nevertheless, whether PO- and VO-induced HF are also associated with distinct LV proteomic alterations has not been investigated yet. Aim Hence, we thought to perform a proteomic analysis on LV myocardial samples from rat models of PO- and VO-induced HF. Methods PO–induced HF was evoked by transverse aortic constriction (TAC). VO–induced HF was established by creating an aortocaval fistula (ACF). Age-matched sham-operated animals served as controls for TAC (ShamT) and ACF (ShamA), respectively. Pressure-volume (P-V) analysis, echocardiography, histology and quantitative real-time PCR were carried out to provide a detailed characterization of the two HF models. Peptides obtained via the digestion of myocardial proteins with trypsin and LysC were labeled with isobaric tags (TMT16) and measured with LC-MS/MS in a bottom-up explorative proteomic approach. Differential expression and gene ontology enrichment analysis (GO:BP) was carried out on summarized protein reporter ion intensities. Results In both the TAC and ACF groups, presence of typical signs and symptoms of HF (dyspnea at rest, fatigue, ascites) increased lung-to-tibial length ratio and elevated LV natriuretic peptide mRNA expression levels confirmed the development of advanced HF. Furthermore, the TAC model was associated with massive wall thickening, concentric LV hypertrophy (LVH), marked interstitial fibrosis and substantially impaired active relaxation and passive filling (slope of end-diastolic P-V relationship: 0.103±0.015 vs. 0.023±0.003mmHg/μl, TAC vs. ShamT, P<0.001). In contrast, the ACF model was predominantly characterized by LV dilatation, eccentric LVH, moderate fibrosis and severely reduced LV contractility (slope of end-systolic P-V relationship: 0.5±0.1 vs. 2.3±0.3mmHg/μl, ACF vs. ShamA, P<0.001). Proteomic analysis revealed that out of the 4691 identified and quantified proteins, 1404 and 913 have shown upregulation, while 1359 and 886 downregulation in the TAC and ACF groups respectively compared to their corresponding sham groups. GO:BP analysis has indicated that the downregulation of mitochondrion organization, ATP metabolic processes and oxidative phosphorylation and the upregulation of actin cytoskeleton organization were the most profound alterations in the TAC model. In contrast, the ACF model was associated with robust downregulation of fatty acid oxidation and upregulation of endocytosis, defense and immune response on the proteomic level. Conclusions PO and VO-induced advanced HF are not only associated with characteristically different functional and structural remodeling but also with distinct LV proteomic alterations. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office (NKFIH) of Hungary

Pyrroloquinoline quinone modulates YAP-related anti-ferroptotic activity to protect against myocardial hypertrophy.

Background: Pyrroloquinoline quinone (PQQ) has been reported to exhibit cardioprotective and antioxidant activities. Accordingly, this study was developed to explore the effects of PQQ treatment on myocardial hypertrophy and the underlying mechanism of action governing any observed beneficial effects. Methods: A transverse aortic constriction (TAC) model of myocardial hypertrophy was established in vivo using C57BL/6 mice, while neonatal murine cardiomyocytes were stimulated with phenylephrine (PE) as an in vitro validation model system. Results: Treatment of TAC model mice with PQQ significantly suppressed myocardial hypertrophy and fibrosis, in addition to inhibiting the ferroptotic death of hypertrophic myocardial cells in vivo. Subsequent in vitro analyses revealed that treatment with PQQ was sufficient to significantly alleviate PE-induced hypertrophic activity and to prevent ferroptotic induction in these primary murine cardiomyocytes. At the mechanistic level, PQQ was found to promote the upregulation of Yes-associated Protein (YAP), to suppress YAP phosphorylation, and to drive the nuclear translocation of YAP within hypertrophic cardiomyocytes. The use of a specific siRNA construct to knock down YAP expression in vitro further confirmed the ability of PQQ to protect against myocardial hypertrophy at least in part through anti-ferroptotic mechanisms. Conclusion: PQQ can regulate the pathogenesis of myocardial hypertrophy through the induction of YAP-related anti-ferroptotic activity, highlighting the potential value of PQQ as a novel therapeutic agent capable of slowing or preventing the progression of myocardial hypertrophy and thus delaying the onset of heart failure.

RNA sequencing reveals novel LncRNA/mRNAs co-expression network associated with puerarin-mediated inhibition of cardiac hypertrophy in mice.

BackgroundEvidence has demonstrated that puerarin is a potential medicine for the treatment of cardiac hypertrophy. However, the precise underlying molecular mechanisms of the protective effect of puerarin are still unclear. Here, we aimed to explore the regulatory mechanisms of lncRNAs/mRNAs co-expression network in a cardiac hypertrophy mouse model after puerarin treatment.MethodsA mouse model of cardiac hypertrophy was established by transverse aortic constriction (TAC). The echocardiography, tissue staining and western blot were used to examine the protective effect of puerarin. Then RNA sequencing (RNA-seq) was carried out to analyze systematically mRNAs and lncRNAs expression. The target lncRNA were confirmed using qRT-PCR. Moreover, a coding/non-coding gene co-expression network were established to find the interaction of lncRNA and mRNAs. The biological process, cellular component, molecular function and pathways of different expression mRNAs targeted by lncRNA were explored using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis.ResultsPuerarin exhibited an obvious inhibitory effect in cardiac hypertrophy in TAC model. RNA-seq analysis was performed to investigate the lncRNAs and mRNAs expression patterns of cardiomyocytes in sham and TAC groups treated with or without puerarin. RNA-seq identified that TAC downregulated four lncRNAs, which could be revised by puerarin treatment (|log2 Fold change| > 2 and FDR < 0.05). Among them, expression alterations of lncRNA Airn (antisense of Igf2r non-protein coding RNA) was confirmed by qRT-PCR. Pearson’s correlation coefficients of co-expression levels suggested that there was an interactive relationship between Airn and 2,387 mRNAs (r > 0.95 or r < −0.95). Those co-expressed mRNAs were enriched in some important biological processes such as translational initiation, cell proliferation, insulin-like growth factor binding and poly(A) RNA binding. KEGG analyses suggested that those Airn-interacted mRNAs were enriched in endocytosis, signaling pathways regulating pluripotency of stem cells and the Jak-STAT pathway.ConclusionPuerarin may exert beneficial effects on cardiac hypertrophy through regulating the lncRNAs/mRNAs co-expression network.

Gene expression profiling and functional analysis of ventricular tissues from murine transverse aortic constriction.

Transverse aortic constriction (TAC) model is widely used to study pressure overload-induced cardiac remodeling. However, the conserved transcriptional features of TAC model and the underlying regulatory mechanisms remain unclear. In this study, we screened out the high-quality microarray data for ventricular tissue from murine TAC model. The transcriptional changes in ventricular tissue were analyzed by identifying the common differently expressed genes (DEGs) and enriched gene sets. We also analyzed the protein-protein interaction and mRNA-mRNA association of DEGs. Furthermore, the potential regulatory elements of the DEGs were explored through comparative analysis between mouse and human. 265 common DEGs and 45 enriched canonical pathways were identified in murine TAC model. 201 DEGs had the protein-protein interaction, whereas 96 DEGs had mRNA-mRNA association. 99 transcription factor (TF)-mRNA and 2997 microRNA (miRNA)-mRNA regulatory relationships were retrieved. In pressure overload-induced cardiac remodeling, inflammation, fibrosis, metabolic remodeling and hypoxia were significant features. Approaches to intervene these phenomena may have therapeutic values. TFs and miRNAs are important regulator elements of DEGs in both mouse and human. Examination of miRNAs is a promising tool to detect the occurrence of pressure overload-induced cardiac remodeling in patients.

Mechanisms of neuro- and cardioprotection of MAS agonists in the TAC model.

Alzheimer's (AD) is a debilitating disease characterized by a loss of memory and executive function. A growing number of studies have shown a direct correlation between hypertension and increased risk of AD-like symptoms, including cognitive deficits, increased Aβ production and neuronal death. Furthermore, Renin-Angiotensin System (RAS) targeted anti-hypertensives reduce risk. This has prompted studies demonstrating that the protective arm of RAS, via activation of the MAS receptor, is reduced in AD patients, suggesting MAS agonists may protect against ROS production and exotoxicity while also increasing cognition and being cardioprotective. We hypothesize that MAS agonists can ameliorate AD-like pathology in mice using a model of heart failure - transverse aortic constriction (TAC). One week after TAC surgery, C57Bl/6J male mice (8-10wk) started treatment with saline or one of four Mas agonists. Four weeks after surgery, cardiac function was assessed by echocardiography followed by cognitive assessment (10 weeks) using novel object recognition. At 90days, brain, heart and blood were collected and analyzed for phenotypic and functional changes. TAC surgery induced marked morphological changes in cardiac tissue (cardiomyocyte area, fibrosis and apoptosis). The subsequent dysfunction manifested as decreases in standard cardiac measures of left ventricular function and by extension - cognition, which were rescued by MAS agonists. This treatment effect is partly due to a protection of the mitochondrial oxidative phosphorylation pathway rather than biogenesis. Specifically, marked ROS-induced mitochondrial dysfunction occurring within neurons and microglia were reduced with MAS agonist treatment. Further Seahorse analysis revealed a resistance to TAC-induced reductions in mitochondrial complex activity. This trend extended to the heart, improving outcomes for endothelial cells and cardiomyocytes. The Mas agonist RASRx1902 significantly decreased several measures of AD-like pathology in the TAC model with particular focus on cardioprotection and mitochondrial function, indicating that it may be beneficial in those with hypertension as a risk factor for AD development. Future work will focus on further characterizing RASRx1902 effects in affected cellular pathway changes, efficacy in a pilot study examining cerebral blood flow, blood brain barrier integrity, and effects on neuronal function.

Urinary metabolomics analysis to reveal metabolic mechanism of guanxinning injection on heart failure with renal dysfunction

Consistently, the multiple heart-kidney interactions make pharmaceutical research for cardiorenal syndrome difficult and complex. Guanxinning Injection (GXN) has been reported to provide unique advantage for treating cardiac and renal diseases compared to typical monotherapies. However, the protection mechanism of GXN is largely unknown. This study explored the acting mechanism of GXN on heart failure with renal dysfunction from a metabolic perspective. Transverse aortic constriction (TAC) surgery was performed on C57/BL/6 mice to induce heart failure with renal dysfunction. Using telmisartan as a positive control, GXN treatment was applied during the 12th to 16th week after TAC. Cardiac function and structure were examined using M-mode echocardiography, and renal function was evaluated via representative biochemical parameters and hematoxylin-eosin staining. Moreover, untargeted metabolomic analyses of urine were conducted to screen for differential substances associated with the cardiorenal protection effect of GXN. As a result, GXN provided good cardioprotective effects on left ventricular ejection fraction elevation, fractional shortening, internal diastolic, and mass maintenance. GXN also reduced TAC-induced elevation of blood urea nitrogen, and serum Cystatin C and relieved kidney pathological damage. Metabolomic analyses identified 21 differential metabolites in the TAC model group. Ten metabolites involving the metabolic pathways of carnitine synthesis, valine, leucine and isoleucine degradation, and glutamate metabolism, taurine and hypotaurine metabolism, tryptophan metabolism, arginine and proline metabolism, and purine metabolism were restored by GXN. The main cardiorenal protection mechanism of GXN was found to be related to energy metabolism and oxidative stress. Taken together, this study provides the first evidence of the metabolic protection mechanism of GXN on heart failure with renal dysfunction for the first time and provides a research basis for the application of GXN in CRS-2 pharmaceuticals.

Structural and functional alterations in heart and skeletal muscle following severe TAC in mice: impact of moderate exercise training

BackgroundHeart failure (HF) is the leading cause of death in western countries. Cardiac dysfunction is accompanied by skeletal alterations resulting in muscle weakness and fatigue. Exercise is an accepted interventional approach correcting cardiac and skeletal dysfunction, thereby improving mortality, re-hospitalization and quality of life. Animal models are used to characterize underpinning mechanisms. Transverse aortic constriction (TAC) results in cardiac pressure overload and finally HF. Whether exercise training improves cardiac remodeling and peripheral cachexia in the TAC mouse model was not analyzed yet. In this study, 2 weeks post TAC animals were randomized into two groups either performing a moderate exercise program (five times per week at 60% VO2 max for 40 min for a total of 8 weeks) or staying sedentary.ResultsIn both TAC groups HF characteristics reduced ejection fraction (− 15% compared to sham, p < 0.001), cardiac remodeling (+ 22.5% cardiomyocyte cross sectional area compared to sham; p < 0.001) and coronary artery congestion (+ 34% diameter compared to sham; p = 0.008) were observed. Unexpectedly, peripheral cachexia was not detected. Furthermore, compared to sedentary group animals from the exercise group showed aggravated HF symptoms [heart area + 9% (p = 0.026), heart circumference + 7% (p = 0.002), right ventricular wall thickness − 30% (p = 0.003)] while muscle parameters were unchanged [Musculus soleus fiber diameter (p = 0.55), Musculus extensor digitorum longus contraction force (p = 0.90)].ConclusionThe severe TAC model is inappropriate to study moderate exercise effects in HF with respect to cardiac and skeletal muscle improvements. Further, the phenotype induced by different TAC procedures should be well documented and taken into account when planning experiments.

Abstract P484: Defining Disease Specific Cardiac Regulatory Networks

The transcriptional basis of homeostasis and disease is implied by the non-coding nature of genetic variation identified by GWAS. Functional genomic approaches to unveil the gene regulatory networks (GRNs) relevant to disease are therefore a high priority. Most models for transcriptional dysregulation presume that perturbation of a wild-type gene regulatory network causes disease risk. For example, the T-box transcription factor (TF) TBX5, is essential for atrial rhythm homeostasis and directly drives a physiologically relevant GRN composed of cardiac channel genes. Alternatively, the expression of many genes pertinent to cardiac pathology are upregulated after the removal of Tbx5, implicating a disease-specific GRN absent from the wild-type atrium. We applied TF-dependent noncoding RNA (ncRNA) profiling, using differential deep ncRNA sequencing from atria of wild-type and Tbx5 mutant mice, to identify TBX5-dependent enhancers and ncRNAs that were only activated following TBX5 removal. We hypothesized that these regulatory elements would reveal disease-response enhancers, essential for coping with atrial dysfunction. To identify the cell-specific regulatory elements, we generated cell-type specific Assay from Transposase-Accessible Chromatin (ATAC) datasets for left atrial tissue, cardiac fibroblasts, and cardiomyocytes. Overlap with the Tbx5 -repressed ncRNAs defined candidate cell-type specific regulatory elements. Candidate regulatory elements were identified upstream of Sox9, a known modulator of cardiac fibrosis, along with other cardiac stress-response pathways, including mediators of TGF-β signaling. Activation of the enhancer at Sox9 was confirmed in isolated cardiac fibroblasts treated with TGF-β. We hypothesized that the disease-acquired GRN in TBX5 mutant atria may be generalizable to other cardiac insults. We therefore examined the transcriptional and genomic changes in the left atria of the heart failure Transverse Aortic Constriction (TAC) mouse model. This analysis revealed remarkable correlation between differentially expressed genes and ncRNAs between TAC and TBX5 mutant disease models. The conservation of the coding and non-coding transcriptional response between arrhythmia and heart failure models supports a paradigm of a common disease-specific GRN that mediates the physiologic consequences of distinct cardiac diseases.

Fibronectin‐Mediated Inflammatory Signaling Through Integrin α5 in Vascular Remodeling

BackgroundAdhesion of vascular endothelial cells to the underlying basement membrane potently modulates endothelial cells to cells' inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin α2β1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes endothelial cells to inflammatory stimuli through integrins α5β1and and αvβ3. A chimeric integrin in which the cytoplasmic domain of α5 is replaced with that of α2 pairs with β1 and binds fibronectin but signals like α2β1.Methods and ResultsHere, we examined mice in which integrin α5 is replaced with the α5/2 chimera, using the transverse aortic constriction and partial carotid ligation models of vessel remodeling. Following transverse aortic constriction and partial carotid ligation surgery, wild‐type mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. α5/2 mice also showed reduced artery wall hypertrophy in the transverse aortic constriction model and diminished inward remodeling in the partial carotid ligation model. Acute atherosclerosis after partial carotid ligation in hyperlipidemic ApoE−/− mice on a high fat diet was dramatically decreased in α5/2 mice.ConclusionsFibronectin and integrin α5 signaling is a key element of pathological vascular remodeling in acute models of both hypertension and disturbed flow. These results underscore the key role for integrin α5 signaling in pathological vascular remodeling associated with hypertension and atherosclerosis and support its potential as a therapeutic target.

Abnormalities in lysine degradation are involved in early cardiomyocyte hypertrophy development in pressure-overloaded rats

BackgroundCardiomyocyte metabolism changes before cardiac remodeling, but its role in early cardiac hypertrophy detection remains unclear. This study investigated early changes in plasma metabolomics in a pressure-overload cardiac hypertrophy model induced by transverse aortic constriction (TAC).MethodsThe TAC model was constructed by partly ligating the aortic arch. Twelve Sprague–Dawley rats were randomly divided into the TAC group (n = 6) and sham group (n = 6). Three weeks after surgery, cardiac echocardiography was performed to assess cardiac remodeling and function. Hematoxylin/eosin (HE), Masson, and wheat germ agglutinin (WGA) stains were used to observe pathological changes. Plasma metabolites were detected by UPLC-QTOFMS and Q-TOFMS. Specific metabolites were screened by orthogonal partial least squares discriminant analysis (OPLS-DA). Metabolic pathways were characterized by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and the predictive value of the screened metabolites was analyzed by receiver operating characteristic (ROC) curve analysis.ResultsThree weeks after surgery, the TAC and sham groups had similar left heart function and interventricular septum and diastolic left ventricular posterior wall thicknesses. However, on pathological examination, the cross-sectional area of cardiomyocytes and myocardial fibrosis severity were significantly elevated in TAC rats. OPLS-DA showed different metabolic patterns between the TAC and sham groups. Based on the criteria VIP > 1 and P < 0.05, 13 metabolites were screened out. KEGG analysis identified disrupted lysine degradation through the related metabolites 5-aminopentanoic acid, N6-acetyl-l-lysine, and l-lysine, with areas under the ROC curve (AUCs) of 0.917, 0.889, and 0.806, respectively, for predicting compensated cardiomyocyte hypertrophy.ConclusionDisruption of lysine degradation might be involved in early cardiac hypertrophy development, and related metabolites might be potential predictive and interventional targets for subclinical cardiomyocyte hypertrophy.