Weeks Of Transverse Aortic Constriction Research Articles

Deleting the Ribosomal Prolyl Hydroxylase OGFOD1 Leads to Protection in Pressure Overload-Induced Cardiac Hypertrophy in Mice

Molecular changes occurring in heart failure have largely focused on the transcriptional landscape. However, disparities between transcript and protein abundances emphasize the need to understand proteomic changes occurring in heart failure as well the mechanisms governing these changes. Post-translational modifications (PTMs) are a well-known mechanism for regulating protein turnover and activity. A lesser-known enzyme that catalyzes the addition of PTMs on the translational machinery is 2-oxoglutarate- and Fe2+-dependent oxygenase domain-containing protein 1 (OGFOD1). Our published evidence shows OGFOD1 RNA and protein accumulate in human failing hearts, indicating a potential role for this enzyme in heart disease. Because heart failure can develop from many pathologies, including hypertrophy, we investigated the role of OGFOD1 in cardiac hypertrophy. We induced hypertrophy via pharmacological β-adrenergic stimulation by treating wildtype (WT) and OGFOD1-knockout (KO) mice with isoproterenol (ISO), where we found that KO mice were protected from ISO-induced hypertrophy. Based on our findings that OGFOD1-KO mice are protected in ISO-induced cardiac hypertrophy, we tested the hypothesis that KO mice were protected in pressure overload-induced cardiac hypertrophy mediated via transverse aortic constriction (TAC). WT and KO mice were subjected to sham or TAC surgery, and heart weight-to-tibia length (HW/TL) and heart weight-to-body weight (HW/BW) ratios were used to determine extent of hypertrophy. Additionally, hearts were subjected to histological analysis and stained with Masson’s trichrome to assess fibrosis. KO mouse hearts showed significantly less hypertrophy than WT hearts after 2 weeks of TAC according to both HW/TL and HW/BW ratios. KO mice were also protected against cardiac fibrosis. To identify translational changes mediating this protection, future studies will focus on utilizing ribosome profiling to identify actively-translated transcripts whose synthesis is regulated by OGFOD1-mediated prolyl hydroxylation. Results from these studies will provide important mechanistic insight into the therapeutic potential of OGFOD1 in human disease. NHLBI-NIH Intramural Research Program (ZO1-HL002066). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Crosstalk between endothelial cells with a non-canonical EndoMT phenotype and cardiomyocytes/fibroblasts via IGFBP5 aggravates TAC-induced cardiac dysfunction

Heart failure (HF) is a complex chronic condition characterized by structural and functional impairments. The differentiation of endothelial cells into myofibroblasts (EndoMT) in response to cardiac fibrosis is controversial, and the relative contribution of endothelial plasticity remains to be explored. Single-cell RNA sequencing was used to identify endothelial cells undergoing fibrotic differentiation within 2 weeks of transverse aortic constriction (TAC). This subset of endothelial cells transiently expressed fibrotic genes but had low expression of alpha-smooth muscle actin, indicating a non-canonical EndoMT, which we named a transient fibrotic-like phenotype (EndoFP). The role of EndoFP in pathological cardiac remodeling may be correlated with increased levels of osteopontin. Cardiomyocytes and fibroblasts co-cultured with EndoFP exhibited heightened pro-hypertrophic and pro-fibrotic effects. Mechanistically, we found that the upregulated expression of insulin-like growth factor-binding protein 5 may be a key mediator of EndoFP-induced cardiac dysfunction. Furthermore, our findings suggested that Rab5a is a novel regulatory gene involved in the EndoFP process. Our study suggests that the specific endothelial subset identified in TAC-induced pressure overload plays a critical role in the cellular interactions that lead to cardiac fibrosis and hypertrophy. Additionally, our findings provide insight into the mechanisms underlying EndoFP, making it a potential therapeutic target for early heart failure.

Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts

AimsEndothelial dysfunction plays a pivotal role in atherosclerosis, but the detailed mechanism remains incomplete understood. Nogo-B is an endoplasmic reticulum (ER)-localized protein mediating ER-mitochondrial morphology. We previously showed endothelial Nogo-B as a key regulator of endothelial function in the setting of hypertension. Here, we aim to further assess the role of Nogo-B in coronary atherosclerosis in ApoE−/− mice with pressure overload. Methods and resultsWe generated double knockout (DKO) mouse models of systemically or endothelium-specifically excising Nogo-A/B gene on an ApoE−/− background. After 7 weeks of transverse aortic constriction (TAC) surgery, compared to ApoE−/− mice DKO mice were resistant to the development of coronary atherosclerotic lesions and plaque rapture. Sustained elevation of Nogo-B and adhesion molecules (VCAM-1/ICAM-1), early markers of atherosclerosis, was identified in heart tissues and endothelial cells (ECs) isolated from TAC ApoE−/− mice, changes that were significantly repressed by Nogo-B deficiency. In cultured human umbilical vein endothelial cells (HUVECs) exposure to inflammatory cytokines (TNF-α, IL-1β), Nogo-B was upregulated and activated reactive oxide species (ROS)-p38-p65 signaling axis. Mitofusin 2 (Mfn2) is a key protein tethering ER to mitochondria in ECs, and we showed that Nogo-B expression positively correlated with Mfn2 protein level. And Nogo-B deletion in ECs or in ApoE−/− mice reduced Mfn2 protein content and increased ER-mitochondria distance, reduced ER-mitochondrial Ca2+ transport and mitochondrial ROS generation, and prevented VCAM-1/ICAM-1 upregulation and EC dysfunction, eventually restrained atherosclerotic lesions development. ConclusionOur study revealed that Nogo-B is a critical modulator in promoting endothelial dysfunction and consequent pathogenesis of coronary atherosclerosis in pressure overloaded hearts of ApoE−/− mice. Nogo-B may hold the promise to be a common therapeutic target in the setting of hypertension.

Suppression of Heart Failure With PAR1 Pepducin Technology in a Pressure Overload Model in Mice.

PAR1 (protease-activated receptor-1) contributes to acute thrombosis, but it is not clear whether the receptor is involved in deleterious inflammatory and profibrotic processes in heart failure. Here, we employ the pepducin technology to determine the effects of targeting PAR1 in a mouse heart failure with reduced ejection fraction model. After undergoing transverse aortic constriction pressure overload or sham surgery, C57BL/6J mice were randomized to daily sc PZ-128 pepducin or vehicle, and cardiac function, inflammation, fibrosis, and molecular analyses conducted at 7 weeks RESULTS: After 7 weeks of transverse aortic constriction, vehicle mice had marked increases in macrophage/monocyte infiltration and fibrosis of the left ventricle as compared with Sham mice. PZ-128 treatment significantly suppressed the inflammatory cell infiltration and cardiac fibrosis. Despite no effect on myocyte cell hypertrophy, PZ-128 afforded a significant reduction in overall left ventricle weight and completely protected against the transverse aortic constriction-induced impairments in left ventricle ejection fraction. PZ-128 significantly suppressed transverse aortic constriction-induced increases in an array of genes involved in myocardial stress, fibrosis, and inflammation. The PZ-128 pepducin is highly effective in protecting against cardiac inflammation, fibrosis, and loss of left ventricle function in a mouse model.

Genetic knockout of prestin limits cardiac hypertrophy in a murine pressure overload model.

Up until recently, Prestin (Slc26a5) was considered to be exclusively expressed in the outer hair cells of the inner ear, where it serves as a voltage-to-force convertor and mediates electromotility. Our recent studies demonstrate that prestin is expressed in the heart, where it amplifies cardiac motor function. Due to prestin's ability to modulate cardiac contractility, we hypothesize that it plays a pivotal role in pathological cardiac hypertrophy. We subjected wild-type (WT) and Slc26a5-/- mice to sham operation or chronic pressure overload induced by transverse aortic constriction (TAC) surgery. Cardiac structure and function were assessed by echocardiography. We performed single cell Ca2+ transients and cell shortening recordings on isolated cardiomyocytes. Atomic force microscopy, a high-resolution technique, was also used to quantify the Young's modulus. After 8 weeks of TAC, the expression of prestin was markedly elevated in WT TAC mice relative to WT sham mice. To test whether the increased expression is maladaptive or cardioprotective, Slc26a5-/- mice underwent the same surgeries. Slc26a5-/- sham mice had depressed cardiac function relative to WT sham mice. In contrast and unexpectedly, 4 weeks after TAC, Slc26a5-/- mice exhibited blunted cardiac structural remodeling and more robust cardiac function as compared to WT TAC mice. Moreover, we found that cardiomyocytes from Slc26a5-/- mice showed more normalized Ca2+ transients and cellular shortening relative to cardiomyocytes from WT TAC mice. Our findings demonstrate that knockout of Slc26a5 is cardioprotective in a murine model of pressure overload, highlighting its crucial role in both physiological and pathological conditions. Overexpression of prestin in cardiac hypertrophy may significantly alter the mechanical properties of cardiomyocytes, leading to worsening in hypertrophy-induced cardiac dysfunction. Our study suggests that targeting prestin may represent a viable therapeutic avenue for cardiac hypertrophy.

Abstract P2002: Mst1 Suppresses Pressure Overload-induced Cardiac Hypertrophy But Reduces Cardiac Function Through PERK Phosphorylation

Cardiac hypertrophy in response to hemodynamic overload is initially an adaptive mechanism to maintain normal cardiac function by reducing left ventricular (LV) wall stress. Conversely, insufficient hypertrophy could lead to elevated wall stress, causing increased cell death and cardiac dysfunction. The Hippo pathway plays an important role in determining organ size. Mammalian sterile 20-like kinase 1 (Mst1) is the key serine-threonine kinase in the Hippo pathway, controlling cell death, proliferation, and differentiation. Mice with cardiac-specific overexpression of Mst1 (Tg-Mst1) fail to develop compensatory hypertrophy despite LV dysfunction and dilation at baseline. To elucidate the mechanism through which Mst1 inhibits cardiac hypertrophy, pressure overload (PO) was imposed on WT and Tg-Mst1 mice by transverse aortic constriction (TAC). TAC-induced increases in LV weight/body weight were significantly suppressed in Tg-Mst1 mice (5.8 vs WT 5.1, p<0.05). LV ejection fraction (EF) after TAC was lower in Tg-Mst1 mice than in WT mice (40% vs WT 60%, p<0.01), consistent with the notion that Mst1 promotes PO-induced systolic dysfunction in part by inhibiting compensatory hypertrophy. Mst1 interacted with and induced phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). We generated knock-in mice expressing phosphorylation-resistant PERK (PERK-KI) and performed TAC on WT and PERK-KI mice. LVEF was preserved in PERK-KI mice compared to in WT mice (67% vs WT 46%, p<0.05) after 8 weeks of TAC. PO-induced increases in lung congestion were attenuated in PERK-KI mice compared to in WT mice (10 vs WT 11.5, p<0.05). Despite the improvement in cardiac function, cardiac hypertrophy was not attenuated in PERK-KI mice. These results suggest that phosphorylation of PERK by Mst1 is detrimental for the heart during PO, in part due to suppression of compensatory hypertrophy.

Cardioprotective Effects of n-3 Polyunsaturated Fatty Acids: Orchestration of mRNA Expression, Protein Phosphorylation, and Lipid Metabolism in Pressure Overload Hearts.

Background: Pressure overload can result in dilated cardiomyopathy. The beneficial effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on heart disorders have been widely recognized. However, the molecular mechanisms underlying their protective effects against cardiomyopathy remain unclear.Methods: Pressure overload in mice induced by 8 weeks of transverse aortic constriction was used to induce dilated cardiomyopathy. A transgenic fat-1 mouse model carrying the n-3 fatty acid desaturase gene fat-1 gene from Caenorhabditis elegans was used to evaluate the mechanism of n-3 PUFAs in this disease. Echocardiography, transmission electron microscopy, and histopathological analyses were used to evaluate the structural integrity and function in pressure overloaded fat-1 hearts. mRNA sequencing, label-free phosphoprotein quantification, lipidomics, Western blotting, RT-qPCR, and ATP detection were performed to examine the effects of n-3 PUFAs in the heart.Results: Compared with wild-type hearts, left ventricular ejection fraction was significantly improved (C57BL/6J [32%] vs. fat-1 [53%]), while the internal diameters of the left ventricle at systole and diastole were reduced in the fat-1 pressure overload hearts. mRNA expression, protein phosphorylation and lipid metabolism were remodeled by pressure overload in wild-type and fat-1 hearts. Specifically, elevation of endogenous n-3 PUFAs maintained the phosphorylation states of proteins in the subcellular compartments of sarcomeres, cytoplasm, membranes, sarcoplasmic reticulum, and mitochondria. Moreover, transcriptomic analysis predicted that endogenous n-3 PUFAs restored mitochondrial respiratory chain function that was lost in the dilated hearts, and this was supported by reductions in detrimental oxylipins and protection of mitochondrial structure, oxidative phosphorylation, and ATP production.Conclusions: Endogenous n-3 PUFAs prevents dilated cardiomyopathy via orchestrating gene expression, protein phosphorylation, and lipid metabolism. This is the first study provides mechanistic insights into the cardioprotective effects of n-3 PUFAs in dilated cardiomyopathy through integrated multi-omics data analysis.

Myofibroblast Deficiency of LSD1 Alleviates TAC-Induced Heart Failure.

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Old Drug, New Trick: Tilorone, a Broad-Spectrum Antiviral Drug as a Potential Anti-Fibrotic Therapeutic for the Diseased Heart.

Cardiac fibrosis is associated with most forms of cardiovascular disease. No reliable therapies targeting cardiac fibrosis are available, thus identifying novel drugs that can resolve or prevent fibrosis is needed. Tilorone, an antiviral agent, can prevent fibrosis in a mouse model of lung disease. We investigated the anti-fibrotic effects of tilorone in human cardiac fibroblasts in vitro by performing a radioisotopic assay for [3H]-proline incorporation as a proxy for collagen synthesis. Exploratory studies in human cardiac fibroblasts treated with tilorone (10 µM) showed a significant reduction in transforming growth factor-β induced collagen synthesis compared to untreated fibroblasts. To determine if this finding could be recapitulated in vivo, mice with established pathological remodelling due to four weeks of transverse aortic constriction (TAC) were administered tilorone (50 mg/kg, i.p) or saline every third day for eight weeks. Treatment with tilorone was associated with attenuation of fibrosis (assessed by Masson’s trichrome stain), a favourable cardiac gene expression profile and no further deterioration of cardiac systolic function determined by echocardiography compared to saline treated TAC mice. These data demonstrate that tilorone has anti-fibrotic actions in human cardiac fibroblasts and the adult mouse heart, and represents a potential novel therapy to treat fibrosis associated with heart failure.

Stimulation of soluble guanylyl cyclase (sGC) by riociguat attenuates heart failure and pathological cardiac remodelling

Heart failure is associated with an impaired NO-soluble guanylyl cyclase (sGC)-cGMP pathway and its augmentation is thought to be beneficial for its therapy. We hypothesized that stimulation of sGC by the sGC stimulator riociguat prevents pathological cardiac remodelling and heart failure in response to chronic pressure overload. Transverse aortic constriction or sham surgery was performed in C57BL/6N mice. After 3weeks of transverse aortic constriction when heart failure was established, animals receive either riociguat or its vehicle for 5 additional weeks. Cardiac function was evaluated weekly by echocardiography. Eight weeks after surgery, histological analyses were performed to evaluate remodelling and the transcriptome of the left ventricles (LVs) was analysed by RNA sequencing. Cell culture experiments were used for mechanistically studies. Transverse aortic constriction resulted in a continuous decrease of LV ejection fraction and an increase in LV mass until week 3. Five weeks of riociguat treatment resulted in an improved LV ejection fraction and a decrease in the ratio of left ventricular mass to total body weight (LVM/BW), myocardial fibrosis and myocyte cross-sectional area. RNA sequencing revealed that riociguat reduced the expression of myocardial stress and remodelling genes (e.g. Nppa, Nppb, Myh7 and collagen) and attenuated the activation of biological pathways associated with cardiac hypertrophy and heart failure. Riociguat reversed pathological stress response in cultivated myocytes and fibroblasts. Stimulation of the sGC reverses transverse aortic constriction-induced heart failure and remodelling, which is associated with improved myocardial gene expression. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

TMEM173 protects against pressure overload-induced cardiac hypertrophy by modulating autophagy.

TMEM173 has been reported to participate in endoplasmic reticulum stress, inflammation and immunology, all of which closely involved with cardiac hypertrophy. But its role in autophagy is not fully figured out. In our research, Tmem173 global knockout (KO) mice manifested more deteriorated hypertrophy, fibrosis, inflammatory infiltration and cardiac malfunction compared with wild type C57BL/6 mice after 6 weeks of transverse aortic constriction. And KO mice showed inhibited autophagosome degradation in myocardium observed under transmission electron microscope and in protein level. In in vitro experiments conducted in neonatal rat cardiomyocytes under phenylephrine treatment, the abundance of Tmem173 gene was negatively related to the abundance of LC3-Ⅱ and the number of red and yellow fluorescent dots, of which reflected the capacity of autophagosome degradation. These results indicated that TMEM173 might be a promoter of autophagic flux and protected against pressure overload-induced cardiac hypertrophy. It may serve as a potential therapeutic target for cardiac hypertrophy in the future.

Abstract 13114: P22 phox Protects the Heart Against Pressure Overload

Introduction: p22 phox forms a complex with NADPH oxidases, major sources of O 2 - and H 2 O 2 . However, the role of p22 phox during stress remains to be elucidated. Purpose: To investigate the role of endogenous p22 phox during pressure overload (PO). Methods and Results: The level of p22 phox protein in isolated cardiomyocytes after 4 weeks of transverse aortic constriction (TAC) was significantly higher than after sham operation (1.7-fold, p<0.05). The cardiac phenotype of cardiac-specific p22 phox knockout ( p22 phox cKO) mice was normal at baseline. However, four weeks after TAC, p22 phox cKO mice exhibited a lower left ventricular ejection fraction (32.0±10.0 vs 53.2±8.4%, p<0.05), a higher lung weight to tibial length ratio (23.0±6.0 vs 13.1±6.6, p<0.05), and more interstitial fibrosis (6.1±1.0 vs 4.4±1.1%, p<0.05) than control mice, indicating that the loss of p22 phox exacerbates TAC-induced cardiac dysfunction. The level of oxidative stress in the heart, evaluated by dityrosine immunoblot, was significantly lower in p22 phox cKO mice than in control mice (0.71±0.04 vs 1.00±0.04, p<0.01). The peak Ca 2+ amplitude in isolated cardiomyocytes was lower in p22 phox cKO mice than in control mice at baseline (2.4±0.1 vs 3.0±0.2, p<0.01). Although mRNA expression of SERCA2a did not differ, there was significantly less SERCA2a protein in p22 phox cKO mice than in control mice (0.62±0.10 vs 1.00±0.23, p<0.01) at baseline. The amount of biotinylated iodoacetamide labeled SERCA2a was significantly smaller in p22 phox cKO hearts than in control mouse hearts (0.4-fold, p<0.01), indicating that cysteine residues in SERCA2a are oxidized to a greater extent in p22 phox cKO hearts than in control mouse hearts. Since cysteine oxidation decreases the stability of SERCA2a, our results suggest that p22 phox stabilizes SERCA2a by preventing cysteine oxidation. Conclusions: Endogenous p22 phox is protective against PO, possibly by maintaining SERCA2a stability.

DPP-4 inhibitor induces FGF21 expression via sirtuin 1 signaling and improves myocardial energy metabolism

Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used incretin-based therapy for the treatment of type 2 diabetes. We investigated the cardioprotective effect of a DPP-4 inhibitor, vildagliptin (vilda), on myocardial metabolism and cardiac performance under pressure overload. Mice were treated with either vehicle or vilda, followed by transverse aortic constriction (TAC). After 3 weeks of TAC, cardiac hypertrophy and impairment of systolic function were attenuated in vilda-treated mice. Pressure–volume analysis showed that vilda treatment significantly improved left-ventricular contractile efficiency in TAC heart. Myocardial energy substrate analysis showed that vilda treatment significantly increased glucose uptake as well as fatty acid uptake. Fibroblast growth factor 21 (FGF21), a peptide involved in the regulation of energy metabolism, increased in TAC heart and was further increased by vilda treatment. FGF21 was strongly expressed in cardiac fibroblasts than in cardiomyocytes in mouse heart after TAC with vilda treatment. Vilda treatment markedly induced FGF21 expression in human cardiac fibroblasts through a sirtuin (Sirt) 1-mediated pathway, suggesting that fibroblast-mediated FGF21 expression may regulate energy metabolism and exert vilda-mediated beneficial effects in stressed heart. Vilda induced a metabolic regulator, FGF21 expression in cardiac fibroblasts via Sirt1, and increased contractile efficiency in murine pressure-overloaded heart.

Gut Dysbiosis Induced by Cardiac Pressure Overload Promotes T Cell Activation and Adverse Cardiac Remodeling Through Alterations in the Aryl Hydrocarbon Receptor Expression

Heart failure (HF), a leading cause of morbidity and mortality worldwide, has been recently associated with gut microbiota alterations (gut dysbiosis) and T cell mediated inflammation in humans, and in experimental models of HF. The gut is a major reservoir of T cells, which are in close proximity with the gut microbiota. Gut bacterial populations process nutrients and produce metabolites that signal through cellular receptors, to maintain homeostasis or induce inflammation at distal organs. The aryl hydrocarbon receptor (AhR) is one of such receptors that recognize ligands that include gut derived tryptophan metabolites to modulate immune responses in the gut and systemically. We hypothesized that cardiac pressure overload induces gut dysbiosis and contributes to adverse cardiac remodeling through T cell activation and via AhR signaling in the gut and in the heart. We performed 16S rRNA sequencing in feces from mice undergoing Sham or transverse aortic constriction (TAC) surgery to test whether cardiac pressure overload induced gut dysbiosis, and in T cell deficient (Tcra−/−) mice to evaluate the contribution of T cells in gut dysbiosis induced by TAC. In addition, we studied the role of gut microbes in T cell activation, cardiac remodeling, and AhR expression, in mice that were orally treated with an antibiotic cocktail (ABX) to deplete the microbiota, and in ABX‐treated mice that spontaneously reconstituted the microbiota upon ABX removal during the 4 weeks of TAC (TAC‐REC). TAC resulted in increased abundance of phylum Bacteroidetes, and in a decrease in genus Lactobacillus, a tryptophan derivative producer, as compared to Sham. These microbiota alterations were not observed in Tcra−/− TAC mice, which were protected from adverse cardiac remodeling and exhibited a closer gut microbiota profile to WT Sham than to WT TAC mice in principal component analysis. However, TAC did not induce leaky gut, determined by LPS levels in serum, or T cell activation in the lamina propria, but increased T cell activation in the mediastinal lymph nodes (mLNs) that drain the heart, and drastically resulted in decreased AhR expression in the gut and in the heart. Strikingly, depletion of the gut microbiota in the onset of TAC (TAC‐ABX) resulted in a reduction in the number of activated T cells in the mLNs and heart infiltrated T cells, lack of cardiac fibrosis and systolic dysfunction, and an induction of AhR expression in the gut and the heart as compared to TAC mice that had not been depleted of the microbiota. Moreover, gut microbiota reconstitution in TAC‐REC mice resulted in decreased expression of AhR in the gut and in the heart as compared to ABX treated mice, and showed similar expression levels of AhR and systolic dysfunction as TAC mice. Our data demonstrate a role of gut microbes in T cell activation and adverse cardiac remodeling induced by cardiac pressure overload that likely involves decreased AhR signaling in the gut and in the heart.Support or Funding InformationThese studies were supported by NIH HL 123658 and the American Heart Association grant 19POST34430075/2019

Rats are Protected from Chronic Pressure Overload Compared with Mice Through Angiogenesis, but not Myogenesis.

It is generally assumed that rats and mice respond to cardiovascular stress similarly. The first goal of this investigation was to compare the effects of chronic pressure overload, induced by 4 weeks of transverse aortic constriction (TAC), in Sprague Dawley rats and C57/BL/6J mice. TAC, after 1 day, induced similar left ventricular (LV) pressure gradients in both rats and mice (113 ± 5.4 vs. 103 ± 11.5 mmHg), and after 4weeks a similar amount of LV hypertrophy (LV/tibial length increase: 51 ± 6.0 vs. 51 ± 5.7). After 4 weeks of TAC, LV systolic and diastolic function were preserved in rats, whereas LV ejection fraction decreased in mice (19 ± 2%), along with increases in LV end diastolic pressure (159 ± 54%), LV systolic wall stress (90 ± 12%), and LV diastolic wall stress (102 ± 41%). The second goal was to determine whether angiogenesis or myogenesis was the mechanism protecting against chronic pressure overload. Immunofluorescent Ki67 staining of capillaries revealed greater increases in angiogenesis with TAC in rats (10 ± 2 positive cells/field) than in mice (6 ± 1 positive cells/field). Myocardial blood flow fell by 55 ± 15% in mice but was preserved in rats. Coronary reserve, the increase in blood flow to the heart with adenosine, was preserved in rats, whereas it fell by 28% in mice with TAC. The next goal was to determine if myogenesis, through generation of stem cells, mediated these differences. This was accomplished by staining the myocardium with c‐kit. More c‐kit positive cells were found in rats with TAC compared to mice with TAC (0.30 ± 0.03 vs. 0.07 ± 0.02 cells/mm2). Surprisingly, these c‐kit positive cells were located not in myocytes, but in the interstitial space and were all negative for Troponin I. However, some of these c‐kit positive cells were positive for CD31, i.e., endothelial cells, which could be involved in mediation of the angiogenesis. Thus, rats tolerate severe pressure overload better than mice, with mechanisms involving increased angiogenesis, but not myogenesis.

SUN-100 Exosome-packaged sonic hedgehog in cardiomyocyte plays a central role in type 2 cardiorenal syndrome

Cardiorenal syndrome type 2 (CRS2) is characterized by chronic abnormalities in cardiac function causing progressive chronic kidney disease (CKD). However, the mediators connecting heart and kidney remains largely unknown. In this study, we provide evidence supporting that exosomes containing Shh generated by injured cardiomyocyte plays a vital role in mediating cardiorenal connection in mouse model of transverse aortic constriction (TAC). Four weeks after TAC, mice were randomized into three groups: 1) sham control; 2) TAC mice; 3) TAC mice injected daily with cyclopamine (CPN), a specific inhibitor of hedgehog signaling. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Another set of mice were randomized into three groups after 4 weeks of TAC: 1) sham control mice injected with pcDNA3 plasmid; 2) TAC mice injected with pcDNA3 plasmid; 3) TAC mice injected with shRab27a plasmid, a small GTPases implicated in regulating exosome biogenesis and secretion. These mice were injected with pcDNA3 or shRab27a plasmids once a week for three weeks beginning at 4 weeks after TAC. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Exosomes were isolated from TAC mice serum and CRS2 patients’ serum, and incubated with normal rat kidney interstitial fibroblast (NRK-49F) cells. At 8 weeks after TAC, cardiac hypertrophy and fibrosis were prominent, as evidenced by increased expression of β-myosin heavy chain, α-actin and fibronectin. Echocardiography also revealed an impaired cardiac function in TAC mice. These cardiac lesions were accompanied by an upregulation of Shh and increased production of exosomes in cardiac tissue. Electron microscopy confirmed abundant exosomes derived from the injured cardiomyocytes in the heart of TAC mice. Blockade of Shh signaling by CPN ameliorated cardiac injury and restored heart function. Moreover, TAC mice also developed kidney injury secondary to chronic heart failure, manifested by proteinuria, kidney fibrosis, podocyte injury, and renal upregulation of Shh. CPN mitigated all these lesions in the kidneys. Notably, serum derived from TAC mice or CRS patients was able to induce renal interstitial fibroblast activation in vitro, indicating the circulating factors as mediators of cardiorenal connection. Furthermore, exosomes isolated from TAC serum was sufficient to cause fibroblast activation and matrix production. Furthermore, blockade of exosome biogenesis and secretion by knockdown of Rab27a expression not only protected against cardiac hypertrophy and fibrosis but also ameliorated renal fibrotic lesions. These studies demonstrate that heart-derived, circulating exosomes are a unique and effective vehicle to deliver Shh into the kidney after TAC. Targeting exosome generation and secretion or inhibition of Shh signaling could be novel therapeutic strategies for protecting both heart and kidney in cardiorenal syndrome.

Phosphodiesterase type 3A (PDE3A), but not type 3B (PDE3B), contributes to the adverse cardiac remodeling induced by pressure overload

Phosphodiesterase type 3 (PDE3) inhibitors block the cAMP hydrolyzing activity of both PDE3 isoforms, PDE3A and PDE3B, which have distinct roles in the heart. Although PDE3 inhibitors improve cardiac function in heart disease patients, they also increase mortality. Nevertheless, PDE3 inhibitors can provide benefit to non-ischemic heart disease patients and are used extensively to treat heart failure in dogs. Since the isoform-dependence of the complex cardiac actions of PDE3 inhibition in diseased hearts remains unknown, we assessed the effects of PDE3 inhibitors as well as gene ablation of PDE3A or PDEB in mice following the induction of non-ischemic heart disease by pressure-overload with transverse-aortic constriction (TAC). As expected, after 6 weeks of TAC, mice exhibited left ventricular contractile dysfunction, dilation, hypertrophy and interstitial fibrosis, in association with increased macrophage numbers, activation of p38 MAPK and elevated PDE3 activity. Chronic PDE3 inhibition with milrinone (MIL), at doses that did not affect either cardiac contractility or arterial blood pressure, profoundly attenuated the adverse ventricular remodeling, reduced macrophage number and diminished p38-MAPK activation induced by TAC. Surprisingly, whole-body ablation of PDE3A, but not PDE3B, provided similar protection against TAC-induced adverse ventricular remodeling, and the addition of MIL to mice lacking PDE3A provided no further protection. Our results support the conclusion that PDE3A plays an important role in adverse cardiac remodeling induced by chronic pressure overload in mice, although the underlying biochemical mechanisms remain to be fully elucidated. The implications of this conclusion on the clinical use of PDE3 inhibitors are discussed.

Gut microbe-derived metabolite trimethylamine N-oxide induces cardiac hypertrophy and fibrosis

Trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, has been linked to increased cardiovascular disease risk. It is unknown whether TMAO plays a role in the development of cardiac hypertrophy. Transverse aortic constriction (TAC) was performed to induce cardiac hypertrophy in Sprague-Dawley (SD) rats. We observed that TMAO levels were significantly elevated in SD rats after 6 weeks of TAC, suggesting the potential role of TMAO in regulating cardiac hypertrophy. In cultured cardiomyocytes, TMAO treatment stimulated cardiac hypertrophy, as indicated by increased cell area of cardiomyocytes and expression of hypertrophic markers including atrial natriuretic peptide (ANP) and beta-myosin heavy chain (β-MHC). Additionally, TMAO treatment induced cardiac hypertrophy and cardiac fibrosis in SD rats. Reducing TMAO synthesis by antibiotics (Abs) attenuated TAC-induced cardiac hypertrophy and fibrosis. Furthermore, pharmacological inhibition of Smad3 by SIS3 significantly reduced the expression of ANP and β-MHC, and cardiomyocyte cell size in TMAO-treated group. These data for the first time demonstrate that gut microbe-derived metabolite TMAO induces cardiac hypertrophy and fibrosis involving Smad3 signaling, suggesting that inhibition of gut microbes or generation of TMAO may become a potential target for the prevention and treatment of cardiac hypertrophy.

Overlapping and differential functions of ATF6\u03b1 versus ATF6\u03b2 in the mouse heart

Hemodynamic stress on the mammalian heart results in compensatory hypertrophy and activation of the unfolded protein response through activating transcription factor 6α (ATF6α) in cardiac myocytes, but the roles of ATF6α or the related transcription factor ATF6β in regulating this hypertrophic response are not well-understood. Here we examined the effects of loss of ATF6α or ATF6β on the cardiac response to pressure overload. Mice gene-deleted for Atf6 or Atf6b were subjected to 2 weeks of transverse aortic constriction, and each showed a significant reduction in hypertrophy with reduced expression of endoplasmic reticulum (ER) stress-associated proteins compared with controls. However, with long-term pressure overload both Atf6 and Atf6b null mice showed enhanced decompensation typified by increased heart weight, pulmonary edema and reduced function compared to control mice. Our subsequent studies using cardiac-specific transgenic mice expressing the transcriptionally active N-terminus of ATF6α or ATF6β revealed that these factors control overlapping gene expression networks that include numerous ER protein chaperones and ER associated degradation components. This work reveals previously unappreciated roles for ATF6α and ATF6β in regulating the pressure overload induced cardiac hypertrophic response and in controlling the expression of genes that condition the ER during hemodynamic stress.

Abstract 434: A Diet Rich in Oleic Acid, as Primary Lipid Source, Attenuates the Progressive Functional Decline in Hypertrophied Hearts During Pathological Stress

Two major dietary long chain fatty acids (LCFA), oleate and palmitate, display differing capacities to induce transcriptional activation of metabolic genes, lipid trafficking, substrate metabolism, and contractile function in the failing heart. Perfusing isolated failing rat hearts with the unsaturated LCFA, oleate, improves contractile function and restores intracellular lipid dynamics vs. failing rat hearts perfused with the saturated LCFA, palmitate. We therefore investigated whether chronically feeding rats a diet of either high-oleate (as triolein) or high-palmitate (as tripalmitin) could alter the development of heart failure in response to chronic pressure overload induced by transverse aortic constriction (TAC). Following TAC or sham surgery rats were randomized to a diet rich in either triolein or tripalmitin as primary fat source (65% carbohydrate, 20% protein and 15% fat (210 Kcal/kg)). TAC induced similar increases in LV mass independent of diet, however rats fed tripalmitin showed a significant reduction in survival and a decline in ejection fraction and fractional shortening as well as LV dilation, evident at 6 and 10 weeks post TAC (Figure). In contrast, triolein diet attenuated declining systolic function and LV dilation (Figure) and eliminated the otherwise expected elevation of plasma BNP, as seen with tripalmitin diet (50%, P<0.05) after 14 weeks of TAC, indicating differential effects of these fats on both the heart and whole body metabolism and physiology during cardiac stress. The results suggest that a diet rich in oleic acid affords benefits in attenuating the development of cardiac decompensation independent of changes in hypertrophic growth.