Model Of Pressure Overload Research Articles

Interleukin-18 and cardiovascular diseases: a literature review

Cardiovascular disease is a serious problem of modern healthcare, which is one of the leading causes of general mortality, reduction and loss of ability to work, as well as disability. The search and the study of new cardiovascular biological markers can allow the optimization of the diagnosis of cardiovascular diseases, development of laboratory tools for assessing the effectiveness of a treatment, and improving the prediction of possible adverse clinical outcomes. The purpose of this review was to promote the consideration of interleukin-18 (IL-18) as a diagnostic and prognostic marker in cardiovascular pathology. For the first time, K. Nakamura et al. in 1989, described IL-18 and interferon-gamma (IFN--inducing factor, IFN--inducing factor, IGIF) as a new, previously unknown factor that induces the production of IFN-. Data obtained from rodent models and clinical studies have demonstrated that IL-18 is involved in the pathogenesis of several diseases, immune-inflammatory rheumatic conditions, systemic vasculitis, cardiovascular diseases, malignancy, central nervous system pathologies, inflammatory bowel disease, psoriasis, and diseases of the kidneys and lungs. In animal models of acute myocardial infarction, pressure overload, and left ventricular dysfunction, IL-18 has been demonstrated to increase cardiomyocyte hypertrophy, induce cardiac contractile dysfunction, and extracellular matrix remodeling. It is therefore expected that further scientific and clinical studies can demonstrate the possibility of using IL-18 as an additional laboratory tool for the diagnosis, risk stratification, and prediction of cardiovascular events in patients with a cardiac profile. The effect of blockade of this cytokine on reducing morbidity and mortality in patients with cardiovascular diseases remains to be assessed for more detail, while considering reasonable economic costs and the side effects of drugs.

Abstract P2015: Metabolic-epigenetic Coupling In High Fat Diet And Heart Failure With Reduced Ejection Fraction

Given rising obesity rates, understanding how metabolic changes exacerbate other cardiovascular disease (CVD) risk factors will yield novel therapeutic targets for associated comorbidities, such as heart failure. We hypothesize that obesity-related metabolic syndrome induces transcriptional programs that engender cardiac pathology via mechanisms involving chromatin regulators, thereby perpetuating the pathology by epigenetic memory. C57BL/6J adult male mice were put on a high fat diet (HFD) for 24 weeks. After HFD, hearts were in hypertrophy, as defined by an increase in heart weight to tibia length ratio (24.67 ± 5.14 mg/mm in HFD versus 17.06 ± 0.98 mg/mm in regular chow controls, p=0.026). Examination of blood plasma revealed significant increase in LDL-C (74.37 ± 22.45 mg/dL in HFD versus 21.91 ± 5.98 mg/dL in controls, p=0.0005) and insulin (452.81 ± 224.77 uIU/mL in HFD versus 89.66 ± 45.60 uIU/mL in controls, p=0.0003), indicative of metabolic disorder. RNA-seq of isolated cardiomyocytes revealed distinct transcriptomic changes at 1 week, 6 weeks, and 24 weeks of HFD. At 1 week, HFD stimulus induced promotion of lipid metabolism ( Pdk4, Per2 ) and suppression of genes related to development and proliferation ( Sox4, Epha4 ). By 6 weeks, the cardiomyocytes underwent metabolic reprogramming (downregulation of Lpl and Irs2 ) and structural changes ( Acta2, Col8a1). At 24 weeks, we observed suppression of ketolytic machinery (downregulation of Abat, Bdh1, and Oxct1 ) in response to obesity-driven hyperglycemia, suggesting that cardiomyocytes transitioned to a pre-diabetic state. To test whether this metabolic disease state compounds with other CVD risk factors, we devised a two-hit model, where mice were fed HFD for 6 weeks and then underwent transverse aortic constriction as a model of pressure overload. Cardiomyocytes from this two-hit model exhibited an enhanced hypertrophic response ( Myh7, Nppa, Col1a1 mRNA levels) compared to pressure overload with regular chow, sham surgery with HFD, or sham surgery with chow. Thus, phenotypic and transcriptional data suggest that HFD reprograms cardiomyocytes to a pre-diabetic state characterized by insulin resistance which exacerbates the effects of other CVD risk factors like hypertrophy.

Abstract EC104: Mitochondrial Microdomain Disruption Results In Sinus Node Dysfunction In Heart Failure

Introduction: Sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not entirely understood. Mitochondria are critical to cellular processes that determine life or death of the cell. Therefore, proper communication with the mitochondria is essential for normal cellular function. We tested the hypothesis that alterations in the mitochondria-sarcoplasmic reticulum (SR) connectomics underlie SAN dysfunction in HF. Methods: We took advantage of a mouse model of pressure-overload induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR/Cas9 mediated gene silencing of mitofusin-2 ( Mfn2 ). Results: TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased SR-mitochondria distance. The expression of the SR-mitochondria tether GTPase protein Mfn-2 was significantly down-regulated and showed reduced colocalization with ryanodine receptor (RyR2) in HF SAN cells. Indeed, SAN-specific gene silencing of Mfn-2 led to alterations in the mitochondrial-SR microdomains[MFN1] and SAN dysfunction. Finally, disruption in the mitochondrial-SR microdomains resulted in abnormal mitochondrial Ca 2+ handling, alterations in localized PKA activity, and impaired mitochondrial function in HF SAN cells. Conclusion: The study provides insight on the role of mitochondrial-SR microdomains in SAN automaticity and into possible therapeutic targets for SAN dysfunction in HF patients.

The sodium-glucose co-transporter-2 inhibitor ertugliflozin modifies the signature of cardiac substrate metabolism and reduces cardiac mTOR signalling, endoplasmic reticulum stress and apoptosis.

To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model. Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg-1 chow diet) for 10 weeks. Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (β-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis. SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.

SR9009 improves heart function after pressure overload independent of cardiac REV-ERB.

The core clock component REV-ERB is essential for heart function. Previous studies show that REV-ERB agonist SR9009 ameliorates heart remodeling in the pressure overload model with transverse aortic constriction (TAC). However, it is unknown whether SR9009 indeed works through cardiac REV-ERB, given that SR9009 might target other proteins and that REV-ERB in non-cardiac tissues might regulate cardiac functions indirectly. To address this question, we generated the REV-ERBα/β cardiac-specific double knockout mice (cDKO). We found that REV-ERB cardiac deficiency leads to profound dilated cardiac myopathy after TAC compared to wild-type (WT) control mice, confirming the critical role of REV-ERB in protecting against pressure overload. Interestingly, the cardioprotective effect of SR9009 against TAC retains in cDKO mice. In addition, SR9009 administered at the time points corresponding to the peak or trough of REV-ERB expression showed similar cardioprotective effects, suggesting the REV-ERB-independent mechanisms in SR9009-mediated post-TAC cardioprotection. These findings highlight that genetic deletion of REV-ERB in cardiomyocytes accelerates adverse cardiac remodeling in response to pressure overload and demonstrated the REV-ERB-independent cardioprotective effect of SR9009 upon pressure overload.

Gpx3 and Egr1 Are Involved in Regulating the Differentiation Fate of Cardiac Fibroblasts under Pressure Overload.

Objectives Although myocardial fibrosis is a common pathophysiological process associated with many heart diseases, the molecular mechanisms regulating the development of fibrosis have not been fully determined. Recently, single cell RNA sequencing (scRNA-seq) analysis has been used to examine cellular fate and function during cellular differentiation and has contributed to elucidating the mechanisms of various diseases. The main purpose of this study was to characterize the fate of cardiac fibroblasts (CFs) and the dynamic gene expression patterns in a model of cardiac pressure overload using scRNA-seq analysis. Methods The public scRNA-seq dataset of the transverse aortic coarctation (TAC) model in mice was downloaded from the GEO database, GSE155882. First, we performed quality control, dimensionality reduction, clustering, and annotation of the data through the Seurat R package (v4.0.5). Then, we constructed the pseudotime trajectory of cell development and identified key regulatory genes using the Monocle R package (v2.22.0). Different cell fates and groups were fully characterized by Gene Set Enrichment Analysis (GSEA) analysis and Transcription factor (TF) activity analysis. Finally, we used Cytoscape (3.9.1) to extensively examine the gene regulatory network related to cell fate. Results Pseudotime analysis showed that CFs differentiated into two distinct cell fates, one of which produced activated myofibroblasts, and the other which produced protective cells that were associated with reduced fibrosis levels, increased antioxidative stress responses, and the ability to promote angiogenesis. In the TAC model, activated CFs were significantly upregulated, while protective cells were downregulated. Treatment with the bromodomain inhibitor JQ1 reversed this change and improved fibrosis. Analysis of dynamic gene expression revealed that Gpx3 was significantly upregulated during cell differentiation into protective cells. Gpx3 expression was affected by JQ1 treatment. Furthermore, Gpx3 expression levels were negatively correlated with the different levels of fibrosis observed in the various treatment groups. Finally, we found that transcription factors Jun, Fos, Atf3, and Egr1 were upregulated in protective cells, especially Egr1 was predicted to be involved in the regulation of genes related to antioxidant stress and angiogenesis, suggesting a role in promoting differentiation into this cell phenotype. Conclusions The scRNA-seq analysis was used to characterize the dynamic changes associated with fibroblast differentiation and identified Gpx3 as a factor that might be involved in the regulation of myocardial fibrosis under cardiac pressure overload. These findings will help to further understanding of the mechanism of fibrosis and provide potential intervention targets.

Changes in Cardiac Function During the Development of Uremic Cardiomyopathy and the Effect of Salvianolic Acid B Administration in a Rat Model.

BackgroundUremic cardiomyopathy (UC), the main cause of death in progressive chronic kidney disease (CKD), is characterized by diastolic dysfunction. Intraventricular pressure gradients (IVPG) derived from color m-mode echocardiography (CMME) and two-dimensional speckle tracking echocardiography (2DSTE) were established as novel echocardiographic approaches for non-invasive and repeatable assessment of cardiac function. Previously, salvianolic acid B (Sal B) showed the potential to alleviate concentric LV hypertrophy in the pressure overload model. The purpose of this study was to evaluate the changes in cardiac function in UC and assess the efficacy of Sal B therapy using IVPG and 2DSTE techniques.Materials and MethodsTwenty-four rats underwent subtotal nephrectomy to produce progressive renal failure and were allocated equally into UC (n = 12) and Sal B-UC (n = 12) groups and monitored for 8 weeks. A sham-operated group was also included in this study (n = 12). Sal B was injected from weeks 4 to 8 in the Sal B-UC group. Conventional echocardiography, 2DSTE, and CMME were performed every 2 weeks post-operation, concomitantly with an evaluation of renal function. Histopathological and immunohistochemistry analyses were carried out to confirm the echocardiography findings.ResultsRenal failure and myocardial dysfunction were confirmed in the UC group from weeks 2 through 8. Eccentric and concentric hypertrophy was observed in the UC group, while the Sal B-UC group showed only eccentric hypertrophy. IVPG analysis did not reveal any significant differences between the groups. Edema, inflammation, fibrosis, and immunohistochemical expression of CD3 infiltration were higher in the UC group compared with sham and Sal B-UC groups.Conclusion2DSTE and IVPG explored the pathophysiology during the development of UC and indicated the incidence of myocardial dysfunction before ventricular morphological changes without intracardiac flow changes. This study confirmed increased ventricular stiffness and fibrosis in UC rats which was potentially treated by Sal B via decreasing edema, inflammation, and fibrosis.

BMP7-based peptide agonists of BMPR1A protect the left ventricle against pathological remodeling induced by pressure overload

Aortic stenosis (AS) exposes the left ventricle (LV) to pressure overload leading to detrimental LV remodeling and heart failure. In animal models of cardiac injury or hemodynamic stress, bone morphogenetic protein-7 (BMP7) protects LV against remodeling by counteracting TGF-β effects. BMP receptor 1A (BMPR1A) might mediate BMP7 antifibrotic effects. Herein we evaluated BMP7-based peptides, THR123 and THR184, agonists of BMPR1A, as cardioprotective drugs in a pressure overload model. We studied patients with AS, mice subjected to four-week transverse aortic constriction (TAC) and TAC release (de-TAC). The LV of AS patients and TAC mice featured Bmpr1a downregulation. Also, pSMAD1/5/(8)9 was reduced in TAC mice. Pre-emptive treatment of mice with THR123 and THR184, during the four-week TAC period, normalized pSMAD1/5/(8)9 levels in the LV, attenuated overexpression of remodeling-related genes (Col 1α1, β-MHC, BNP), palliated structural damage (hypertrophy and fibrosis) and alleviated LV dysfunction (systolic and diastolic). THR184 administration, starting fifteen days after TAC, halted the ongoing remodeling and partially reversed LV dysfunction. The reverse remodeling after pressure overload release was facilitated by THR184. Both peptides diminished the TGF-β1-induced hypertrophic gene program in cardiomyocytes, collagen transcriptional activation in fibroblasts, and differentiation of cardiac fibroblasts to myofibroblasts. Molecular docking suggests that both peptides bind with similar binding energies to the BMP7 binding domain at the BMPR1A. The present study results provide a preclinical proof-of-concept of potential therapeutic benefits of BMP7-based small peptides, which function as agonists of BMPR1A, against the pathological LV remodeling in the context of aortic stenosis.

Bone Morphogenetic Protein-9 (BMP9) is Required for Survival and Limits Left Ventricular Matrix Metalloproteinase Activity After Acute Myocardial Infarction

<h3>Purpose</h3> Reorganization of extracellular matrix is a major determinant of short and long term wound healing after acute myocardial infarction (AMI), which includes central roles for matrix metalloproteinases (MMP) and collagen turnover. We recently identified that bone morphogenetic protein (BMP)-9 is a novel endogenous inhibitor of cardiac fibrosis in murine models of pressure overload induced heart failure. We hypothesized that BMP9 regulates MMP activity and is required for survival after AMI. <h3>Methods</h3> WT or BMP9-/- mice were subjected to left anterior descending artery ligation and animals recovered for 2 weeks. Left ventricular (LV) function was assessed using conductance catheter and LV tissue was harvested for molecular analysis in animals surviving to two weeks. To further assess a functional role for BMP9, in vitro studies were performed using human atrial fibroblasts (H-Atr-Fb). <h3>Results</h3> Compared to WT controls BMP9-/- mice had reduced survival (94.4% v 66.7%, p=0.04) and tissue necropsy showed a high rate of cardiac rupture among BMP9-/- mice (BMP9-/- 80%, WT 0%). Ejection fraction and stroke work were reduced and both LV end systolic and diastolic volumes were increased in WT and BMP9-/- mice after AMI. Compared to WT, SMAD3 signaling was increased (p=0.001) and SMAD1 signaling reduced (p=0.03) in BMP9-/- mice after AMI. Compared to WT, BMP9-/- mice had higher LV protein levels of endoglin, Type 1 collagen and periostin. We further observed significantly higher LV MMP9 levels and activity in BMP9-/- compared to WT sham (p=0.003) and WT MI (p=0.02) mice after AMI . Compared to WT, BMP9-/- mice exhibited an increased ratio of circulating markers of collagen degradation and synthesis (ICTP:PINP) after AMI (p=0.02). In H-Atr-Fb, siRNA mediated knockdown of BMP9 increased protein levels of Type I collagen (p<0.05) and MMP9 (p=0.04) and increased MMP9 activity (p=0.003). <h3>Conclusion</h3> We report that BMP9 is necessary for survival after AMI and identify a novel functional role of BMP9 as a negative regulator of MMP9 activity and collagen synthesis in the LV during AMI. These findings suggest that BMP9 may be an important novel target of therapy to improve myocardial recovery and survival after AMI.

Loss of talin in cardiac fibroblasts results in augmented ventricular cardiomyocyte hypertrophy in response to pressure overload.

Pressure overload of the heart is characterized by concentric hypertrophy and interstitial fibrosis. Cardiac fibroblasts (CFs) in the ventricular wall become activated during injury and synthesize and compact the extracellular matrix, which causes interstitial fibrosis and stiffening of the ventricular heart walls. Talin1 (Tln1) and Talin2 (Tln2) are mechanosensitive proteins that participate in focal adhesion transmission of signals from the extracellular environment to the actin cytoskeleton of CFs. The aim of the present study was to determine whether the removal of Tln1 and Tln2 from CFs would reduce interstitial fibrosis and cardiac hypertrophy. Twelve-week-old male and female Tln2-null (Tln2-/-) and Tln2-null, CF-specific Tln1 knockout (Tln2-/-;Tln1CF-/-) mice were given angiotensin-II (ANG II) (1.5 mg/kg/day) or saline through osmotic pumps for 8 wk. Cardiomyocyte area and measures of heart thickness were increased in the male ANG II-infused Tln2-/-;Tln1CF-/- mice, whereas there was no increase in interstitial fibrosis. Systolic blood pressure was increased in the female Tln2-/-;Tln1CF-/- mice after ANG II infusion compared with the Tln2-/- mice. However, there was no increase in cardiac hypertrophy in the Tln2-/-;Tln1CF-/- mice, which was seen in the Tln2-/- mice. Collectively, these data indicate that in male mice, the absence of Tln1 and Tln2 in CFs leads to cardiomyocyte hypertrophy in response to ANG II, whereas it results in a hypertrophy-resistant phenotype in female mice. These findings have important implications for the role of mechanosensitive proteins in CFs and their impact on cardiomyocyte function in the pathogenesis of hypertension and cardiac hypertrophy.NEW & NOTEWORTHY The role of talins has been previously studied in cardiomyocytes; however, these mechanotransductive proteins that are members of the focal adhesion complex have not been examined in cardiac fibroblasts previously. We hypothesized that loss of talins in cardiac fibroblasts would reduce interstitial fibrosis in the heart with a pressure overload model. However, we found that although loss of talins did not alter fibrosis, it did result in cardiomyocyte and ventricular hypertrophy.

Gene Therapy With the N-Terminus of Junctophilin-2 Improves Heart Failure in Mice.

Transcriptional remodeling is known to contribute to heart failure (HF). Targeting stress-dependent gene expression mechanisms may represent a clinically relevant gene therapy option. We recently uncovered a salutary mechanism in the heart whereby JP2 (junctophilin-2), an essential component of the excitation-contraction coupling apparatus, is site-specifically cleaved and releases an N-terminal fragment (JP2NT [N-terminal fragment of JP2]) that translocates into the nucleus and functions as a transcriptional repressor of HF-related genes. This study aims to determine whether JP2NT can be leveraged by gene therapy techniques for attenuating HF progression in a preclinical pressure overload model. We intraventricularly injected adeno-associated virus (AAV) (2/9) vectors expressing eGFP (enhanced green fluorescent protein), JP2NT, or DNA-binding deficient JP2NT (JP2NTΔbNLS/ARR) into neonatal mice and induced cardiac stress by transaortic constriction (TAC) 9 weeks later. We also treated mice with established moderate HF from TAC stress with either AAV-JP2NT or AAV-eGFP. RNA-sequencing analysis was used to reveal changes in hypertrophic and HF-related gene transcription by JP2NT gene therapy after TAC. Echocardiography, confocal imaging, and histology were performed to evaluate heart function and pathological myocardial remodeling following stress. Mice preinjected with AAV-JP2NT exhibited ameliorated cardiac remodeling following TAC. The JP2NT DNA-binding domain is required for cardioprotection as its deletion within the AAV-JP2NT vector prevented improvement in TAC-induced cardiac dysfunction. Functional and histological data suggest that JP2NT gene therapy after the onset of cardiac dysfunction is effective at slowing the progression of HF. RNA-sequencing analysis further revealed a broad reversal of hypertrophic and HF-related gene transcription by JP2NT overexpression after TAC. Our prevention- and intervention-based approaches here demonstrated that AAV-mediated delivery of JP2NT into the myocardium can attenuate stress-induced transcriptional remodeling and the development of HF when administered either before or after cardiac stress initiation. Our data indicate that JP2NT gene therapy holds great potential as a novel therapeutic for treating hypertrophy and HF.

Wnt/β-catenin antagonist pyrvinium rescues high dose isoproterenol induced cardiotoxicity in rats: Biochemical and immunohistological evidences

The up-regulation of Wnt/β-catenin pathway induces cardiac function abnormalities, hypertrophy, and fibrosis in diabetic hypertensive and pressure overload models. The present study investigates the cardioprotective effects of Wnt/β-catenin inhibition on isoproterenol (ISO) induced cardiotoxicity in rats. ISO was administered at a dose of 85 mg/kg (s.c) for 2 days. Wnt/β-catenin inhibitor pyrvinium (60 μg/kg, p.o) was given 2h prior and glibenclamide at a dose of 5 mg/kg; p.o, 2 h after ISO injection. Cardiac function parameters were assessed on isolated hearts by using automated Biopac apparatus. The β-catenin transcription and expression was detected by RT-PCR technique and immunohistochemical method. Serum and cardiac tissue biochemical changes including cardiac troponin-I, CK-MB, LDH, anti-oxidant enzyme levels, inflammatory cytokines, and membrane associated Na+/K + ATPase and Ca2+ATPase and caspase-3 activity, collagen content, fibronectin protein levels were evaluated in various study groups. Histological studies were also carried out to analyze the cardiomyocyte damage, hypertrophy, fibrosis, and necrosis, while α-SMA, TGF-β expression was checked by immunostaining. ISO administration enhanced β-catenin gene expression and transcription which promoted oxidative and nitrosative stress, inflammatory cytokine release, reduced ATP levels, induced over-expression of fibrotic proteins resulting in cardiac hypertrophy, myocardial necrosis, functional and histological changes. However, antagonism of Wnt/β-catenin pathway attenuated these ISO induced pathological manifestations. Notably, the co-treatment with ATP-sensitive K+ channel inhibitor partially, reduced the cardioprotective effects of Wnt/β-catenin blocker pyrvinium in ISO rats. Thus Wnt/β-catenin inhibition exhibits cardioprotective in ISO model by anti-oxidant, anti-inflammatory, anti-fibrotic properties and by possible involvement of ATP-sensitive potassium channel activation.

Differential mRNA Expression and Circular RNA-Based Competitive Endogenous RNA Networks in the Three Stages of Heart Failure in Transverse Aortic Constriction Mice.

BackgroundThe murine transverse aortic constriction (TAC) model is frequently used to investigate molecular mechanisms underlying heart failure. However, limited data is available regarding the expression of mRNAs and circRNAs in murine heart failure progression induced by pressure overload.MethodsTransverse aortic constriction was used to induce pressure overload for 2, 4, and 8 weeks in mice. Echocardiographic measurements in B-mode and M-mode, as well as blood flow Doppler data were collected in mice without (sham) and with (2W-, 4W-, and 8W-post-TAC) pressure load. Hearts were excised and morphology, cardiomyocyte size, and fibrosis were determined. RNA sequencing, circRNA microarray, functional mRNA enrichment analysis, hub gene identification, target miRNA interaction, and competitive endogenous RNA (ceRNA) network construction were conducted.ResultsHeart weight, cardiomyocyte hypertrophy, and fibrosis gradually increased over time in the hearts with pressure overload. The 2W-post-TAC hearts displayed concentric hypertrophy, thickened left ventricular walls, and increased EF and FS. The 4W-post-TAC hearts were characterized by preserved EF and FS, dilated atria, and increased left ventricle (LV) systolic volume. The 8W-post-TAC hearts presented with ventricular and atrial dilation, increased LV systolic and diastolic volume, reduced EF and FS, and increased ejection time (MV ET). mRNA expression analysis suggested that cardiac remodeling, immune response dysregulation, and metabolic disorder were the key cellular events in heart failure progression. Depression in chemotaxis and mitochondrial function were predicted in 4W- and 8W-post-TAC myocardia, respectively. A ceRNA network analysis demonstrated that the circRNAs targeted the expression of genes enriched in metabolism dysregulation in the 2W-post-TAC hypertrophic hearts, while they targeted genes enriched in cardiac remodeling in the 4W-post-TAC EF-preserved hearts and in the suppression of oxidative phosphorylation and cardiac contraction in the 8W-post-TAC EF-reduced hearts.ConclusionOur work empirically demonstrates that distinctive features of heart failure, including ventricular hypertrophy, heart failure with preserved EF (HFpEF), and heart failure with reduced EF (HFrEF) are present in the murine pressure overload models. The three stages of heart failure vary in terms of mRNA and circRNA expression, as well as ceRNA regulation in a manner consistent with their structural, functional, and pathological differences.

Diosmetin Protects against Cardiac Hypertrophy via p62/Keap1/Nrf2 Signaling Pathway.

An important pathophysiological consequence of pressure overload-induced cardiac hypertrophy is adverse cardiac remodeling, including structural changes in cardiomyocytes and extracellular matrix. Diosmetin (DIO), a monomethoxyflavone isolated from citrus fruits, had antioxidative stress effects in multiple organs. The purpose of this study was to examine the biological effect of diosmetin on pathological cardiac hypertrophy. In mice, diosmetin treatment reduced cardiac hypertrophy and dysfunction in an aortic banding- (AB-) induced pressure overload model and reducing myocardial oxidative stress by increasing antioxidant gene expression. In vitro, diosmetin (10 or 50 μm, 12 h or 24 h) protected PE-induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes. Mechanistically, diosmetin inhibited autophagy by activating the PI3K/Akt pathway. In particular, diosmetin induced the accumulation of p62 and its interaction with Keap1, promoted the nuclear translocation of Nrf2, and increased the expression of antioxidant stress genes in the process of cardiac hypertrophy. Furthermore, knockdown of p62 in rat primary cardiomyocytes abrogate the protective effect of diosmetin on cardiomyocyte hypertrophy. Similarly, the Nrf2 inhibitor ML385 obviously abolished the above effects by diosmetin treatment. In conclusion, our results suggest that diosmetin protects cardiac hypertrophy under pressure overload through the p62/Keap1/Nrf2 signaling pathway, suggesting the potential of diosmetin as a novel therapy for pathological cardiac hypertrophy.

Transient receptor potential vanilloid 1-expressing cardiac afferent nerves may contribute to cardiac hypertrophy in accompany with an increased expression of brain-derived neurotrophic factor within nucleus tractus solitarius in a pressure overload model

ABSTRACT Introduction Increased sympathetic output contributes to cardiac hypertrophy. Sympathoexcitation is induced by activating the cardiac sympathetic afferent nerves through transient receptor potential vanilloid 1 (TRPV1) in cardiac afferent endings. Brainstem nucleus tractus solitarius (NTS) receives the sensory cardiac afferent inputs. Brain-derived neurotrophic factor (BDNF) is released within NTS from sensory neurons in an activity-dependent manner. Additionally, BDNF in NTS tonically regulates sympathetic activity. Therefore, we hypothesized that TRPV1-expressing cardiac afferent nerves contribute to cardiac hypertrophy in accompany with an increased BDNF expression in NTS. Methods and Results Abdominal aortic banding (AB) or sham operation was conducted in wild-type C57BL/6 J (WT-AB) and TRPV1 knockout mice (TRPV1 KO-AB). At 8 weeks post-operation, echocardiographic left ventricular wall thickness and heart weight/body weight ratio were significantly greater in WT-AB than WT-Sham mice, and these hypertrophic indexes were attenuated in TRPV1 KO-AB mice. Among the groups, left ventricular fractional shortening was not different. The protein levels of TRPV1 in heart and BDNF in NTS were significantly increased in WT-AB compared to WT-Sham mice, whereas BDNF expression in NTS was not increased by AB in TRPV1-KO mice. Chemical ablation of TRPV1-expressing cardiac afferents attenuated the AB-induced cardiac hypertrophy and increase in BDNF in NTS. Sympathetic activity analyzed using heart rate variability, and sympathoexcitatory responses to the stimulation of cardiac afferents were increased in WT-AB compared to WT-Sham mice. Conclusion TRPV1-expressing cardiac afferent nerves may contribute to pressure overload-induced cardiac hypertrophy in accompany with the increased BDNF within NTS.

The Critical Roles of Proteostasis and Endoplasmic Reticulum Stress in Atrial Fibrillation.

Atrial fibrillation (AF) remains the most common arrhythmia seen clinically. The incidence of AF is increasing due to the aging population. AF is associated with a significant increase in morbidity and mortality, yet current treatment paradigms have proven largely inadequate. Therefore, there is an urgent need to develop new effective therapeutic strategies for AF. The endoplasmic reticulum (ER) in the heart plays critical roles in the regulation of excitation-contraction coupling and cardiac function. Perturbation in the ER homeostasis due to intrinsic and extrinsic factors, such as inflammation, oxidative stress, and ischemia, leads to ER stress that has been linked to multiple conditions including diabetes mellitus, neurodegeneration, cancer, heart disease, and cardiac arrhythmias. Recent studies have documented the critical roles of ER stress in the pathophysiological basis of AF. Using an animal model of chronic pressure overload, we demonstrate a significant increase in ER stress in atrial tissues. Moreover, we demonstrate that treatment with a small molecule inhibitor to inhibit the soluble epoxide hydrolase enzyme in the arachidonic acid metabolism significantly reduces ER stress as well as atrial electrical and structural remodeling. The current review article will attempt to provide a perspective on our recent understandings and current knowledge gaps on the critical roles of proteostasis and ER stress in AF progression.

Autologous Umbilical Cord Blood–Derived Mononuclear Cell Therapy Promotes Cardiac Proliferation and Adaptation in a Porcine Model of Right Ventricle Pressure Overload

Congenital heart diseases, including single ventricle circulations, are clinically challenging due to chronic pressure overload and the inability of the myocardium to compensate for lifelong physiological demands. To determine the clinical relevance of autologous umbilical cord blood–derived mononuclear cells (UCB-MNCs) as a therapy to augment cardiac adaptation following surgical management of congenital heart disease, a validated model system of right ventricular pressure overload due to pulmonary artery banding (PAB) in juvenile pigs has been employed. PAB in a juvenile porcine model and intramyocardial delivery of UCB-MNCs was evaluated in three distinct 12-week studies utilizing serial cardiac imaging and end-of-study pathology evaluations. PAB reproducibly induced pressure overload leading to chronic right ventricular remodeling including significant myocardial fibrosis and elevation of heart failure biomarkers. High-dose UCB-MNCs (3 million/kg) delivered into the right ventricular myocardium did not cause any detectable safety issues in the context of arrhythmias or abnormal cardiac physiology. In addition, this high-dose treatment compared with placebo controls demonstrated that UCB-MNCs promoted a significant increase in Ki-67-positive cardiomyocytes coupled with an increase in the number of CD31+ endothelium. Furthermore, the incorporation of BrdU-labeled cells within the myocardium confirmed the biological potency of the high-dose UCB-MNC treatment. Finally, the cell-based treatment augmented the physiological adaptation compared with controls with a trend toward increased right ventricular mass within the 12 weeks of the follow-up period. Despite these adaptations, functional changes as measured by echocardiography and magnetic resonance imaging did not demonstrate differences between cohorts in this surgical model system. Therefore, this randomized, double-blinded, placebo-controlled pre-clinical trial establishes the safety of UCB-MNCs delivered via intramyocardial injections in a dysfunctional right ventricle and validates the induction of cardiac proliferation and angiogenesis as transient paracrine mechanisms that may be important to optimize long-term outcomes for surgically repaired congenital heart diseases.

Oncofetal Protein CRIPTO Is Involved in Wound Healing and Fibrogenesis in the Regenerating Liver and Is Associated with the Initial Stages of Cardiac Fibrosis.

Oncofetal protein, CRIPTO, is silenced during homeostatic postnatal life and often re-expressed in different neoplastic processes, such as hepatocellular carcinoma. Given the reactivation of CRIPTO in pathological conditions reported in various adult tissues, the aim of this study was to explore whether CRIPTO is expressed during liver fibrogenesis and whether this is related to the disease severity and pathogenesis of fibrogenesis. Furthermore, we aimed to identify the impact of CRIPTO expression on fibrogenesis in organs with high versus low regenerative capacity, represented by murine liver fibrogenesis and adult murine heart fibrogenesis. Circulating CRIPTO levels were measured in plasma samples of patients with cirrhosis registered at the waitlist for liver transplantation (LT) and 1 year after LT. The expression of CRIPTO and fibrotic markers (αSMA, collagen type I) was determined in human liver tissues of patients with cirrhosis (on a basis of viral hepatitis or alcoholic disease), in cardiac tissue samples of patients with end-stage heart failure, and in mice with experimental liver and heart fibrosis using immuno-histochemical stainings and qPCR. Mouse models with experimental chronic liver fibrosis, induced with multiple shots of carbon tetrachloride (CCl4) and acute liver fibrosis (one shot of CCl4), were evaluated for CRIPTO expression and fibrotic markers. CRIPTO was overexpressed in vivo (Adenoviral delivery) or functionally sequestered by ALK4Fc ligand trap in the acute liver fibrosis mouse model. Murine heart tissues were evaluated for CRIPTO and fibrotic markers in three models of heart injury following myocardial infarction, pressure overload, and ex vivo induced fibrosis. Patients with end-stage liver cirrhosis showed elevated CRIPTO levels in plasma, which decreased 1 year after LT. Cripto expression was observed in fibrotic tissues of patients with end-stage liver cirrhosis and in patients with heart failure. The expression of CRIPTO in the liver was found specifically in the hepatocytes and was positively correlated with the Model for End-stage Liver Disease (MELD) score for end-stage liver disease. CRIPTO expression in the samples of cardiac fibrosis was limited and mostly observed in the interstitial cells. In the chronic and acute mouse models of liver fibrosis, CRIPTO-positive cells were observed in damaged liver areas around the central vein, which preceded the expression of αSMA-positive stellate cells, i.e., mediators of fibrosis. In the chronic mouse models, the fibrosis and CRIPTO expression were still present after 11 weeks, whereas in the acute model the liver regenerated and the fibrosis and CRIPTO expression resolved. In vivo overexpression of CRIPTO in this model led to an increase in fibrotic markers, while blockage of CRIPTO secreted function inhibited the extent of fibrotic areas and marker expression (αSMA, Collagen type I and III) and induced higher proliferation of residual healthy hepatocytes. CRIPTO expression was also upregulated in several mouse models of cardiac fibrosis. During myocardial infarction CRIPTO is upregulated initially in cardiac interstitial cells, followed by expression in αSMA-positive myofibroblasts throughout the infarct area. After the scar formation, CRIPTO expression decreased concomitantly with the αSMA expression. Temporal expression of CRIPTO in αSMA-positive myofibroblasts was also observed surrounding the coronary arteries in the pressure overload model of cardiac fibrosis. Furthermore, CRIPTO expression was upregulated in interstitial myofibroblasts in hearts cultured in an ex vivo model for cardiac fibrosis. Our results are indicative for a functional role of CRIPTO in the induction of fibrogenesis as well as a potential target in the antifibrotic treatments and stimulation of tissue regeneration.

Abstract 9073: Profiling of Metabolic Remodeling in Rat Model of Pressure Overload Induced Early Systolic Dysfunction and in Progression to Overt Systolic Heart Failure

Introduction: We have previously shown left ventricular (LV) structural and hemodynamic differences in two models of rat pressure overload induced moderate remodeling and early systolic dysfunction (MOD) and overt systolic heart failure (SHF). The MOD and SHF phenotypes develop 8 weeks post ascending aortic banding via a vascular clip, area of 2 mm 2 and 1.5 mm 2 , respectively. Hypothesis: Metabolic remodeling play significant role in the development of MOD and SHF phenotypes, but is more evident in SHF than MOD. Methods: Shotgun proteomics and phospho-(p)-proteomics and non-targeted metabolomics analysis was performed in LV myocardium tissue from Sham, MOD and SHF using liquid chromatography mass spectrometry (LC-MS); n=3 biological samples per group. Raw proteomics, p-proteomics and metabolomics MS data were uploaded into Qlucore bioinformatics software for statistical analysis and data visualization. Statistically analyzed data were then uploaded into Ingenuity Pathway Analysis bioinformatics software for data validation and assessment of highly enriched canonical pathways related to metabolism that were up or down-regulated in MOD and SHF relative to Sham. Some of the proteomics and p-proteomics data were validated by immunoblotting. Results: Canonical pathways of oxidative phosphorylation, fatty acid β-oxidation, Kreb’s cycle, branched-chain amino acid and amino acid (Glutamine and Tryptophan) degradation were downregulated in MOD vs Sham and were further downregulated in SHF vs MOD. Whilst glycogen degradation and glucose degradation were upregulated in MOD and SHF vs Sham, but more so in MOD than SHF. Metabolomics data revealed that long chain and very long chain acyl-carnitines were decreased in abundance in MOD and SHF relative to Sham, while short and medium chain acyl-carnitines were decreased in abundance in SHF only. Moreover, Isoleucine degradation and glycolysis decreased and Taurine biosynthesis increased in SHF vs MOD. p-Proteomics data showed mild decrease in protein kinase A (PKA) and AMPK signaling in MOD vs Sham, which were more profoundly decreased in SHF than MOD. Conclusion: The interplay between PKA and AMPK signaling play an important role in metabolic remodeling and transitioning from MOD to SHF phenotype.

Abstract 13218: CITED4 is Required for Exercise-Induced Cardiac Growth and Cardiomyogenesis in Mice

Introduction: CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain 4) is a transcriptional regulator which limits pathologic remodeling after ischemic and pressure overload models of cardiac stress. It is also upregulated in the heart with exercise, an effective intervention which induces physiological hypertrophy and cardiomyogenesis in adult mice. We hypothesized that CITED4 is required for exercise-induced cardiac growth and cardiomyogenesis. Methods and Results: Eight- to ten-week-old mice with cardiomyocyte-specific CITED4 knockout (C4KO) and littermate controls underwent 8 weeks of voluntary wheel running in comparison to sedentary controls. 5-Ethynyl-2′-deoxyuridine (EdU) was injected subcutaneously every two days for the first two weeks. There were no significant differences in cardiac size and function at baseline between C4KO and control mice. In control mice, running increased heart weight to tibial length ratio (HW/TL, 6.26±0.25 vs 7.37±0.75, p=0.01, n=5-9 per group), heart weight to body weight ratio (HW/BW, 4.08±0.40 vs 4.93±0.48, p=0.01), and cardiomyocyte (CM) size without affecting fractional shortening (FS), consistent with physiologic remodeling. There was also a nonsignificant trend to increased EdU+ CMs in the control exercised mice (2.96±2.34% vs 5.23±6.28%, p=0.44), suggestive of previously documented exercise-induced cardiomyogenesis. In contrast, running did not change HW/TL and CM size in C4KO mice (HW/TL, 6.44±0.48 vs 6.66±0.29, p=0.22, n= 0-13 per group) but reduced %FS (58.78±3.27 vs 53.67±4.59, p=0.03) and increased chamber size (LVIDs, 1.32±0.13 vs 1.50±0.17, p=0.032), suggesting maladaptive cardiac remodeling with exercise, confirming prior findings observed in a more extreme swim-exercise model. Rather than the expected increase, hearts from exercised C4KO mice had fewer EdU+ CMs compared to sedentary C4KO mice (3.76±2.60% vs 2.11±0.76%, p=0.05) and reduced expression of cell cycle genes Cdkn1b, Cyclin B1, Cdkn2a, and Ki-67. Conclusions: CITED4 is required for exercise-induced cardiomyogenesis, CM hypertrophy, and adaptive remodeling. CITED4 deficiency leads to maladaptive cardiac remodeling with physiologic stress.