Pressure Overload-induced Heart Failure Research Articles

Depletion of β3-adrenergic receptor relieves pressure overload-induced cardiac hypertrophy and heart failure via enhancing innate immune response

Cardiac pressure overload is a crucial risk factor for cardiac hypertrophy and heart failure. Our previous study showed that depletion of the β3-adrenergic receptor (ADRB3) induced left ventricular diastolic dysfunction via potential regulation of energy metabolism and cardiac contraction. However, the effects of ADRB3 on pressure overload-induced heart failure remain unclear. In the present study, systemic ADRB3-knockout mice suffering from transverse aortic constriction (TAC) surgery were used to identify the effects of ADRB3 on pressure overload-induced heart failure. Compared to wild-type mice, ADRB3 depletion significantly improved the left ventricular ejection fraction, reduced left ventricular posterior wall thickness and interventricular septum thickness, and decreased the area of cardiomyocytes after TAC. RNA sequencing and bioinformatics analysis showed that ADRB3 depletion up-regulated 275 mRNAs and down-regulated 105 mRNAs in mice suffering TAC surgery. GO analysis, GO-tree analysis, and GSEA showed that ADRB3 depletion mainly enhanced the innate immune response of hearts in cardiac pressure overload mice. In addition, pathway analysis and Pathway-Act analysis presented that innate immune response-related pathways, including RIG-I-like receptor signaling pathway, antigen processing and presentation, Toll-like receptor signaling pathway, and cell adhesion molecules, were significantly enriched in ADRB3-KO-TAC mice. Ten hub genes were identified using protein-protein interaction network, MCODE, and cytoHubba analysis. Furthermore, the depletion and activation of ADRB3 validated the effects of ADRB3 on the innate immune response of hearts after TAC. In conclusion, ADRB3 depletion relieves pressure overload-induced cardiac hypertrophy and heart failure, and these effects could be explained by the enhancement of innate immune response.

Enhanced oxidative stress mediates pathological autophagy and necroptosis in cardiac myocytes in pressure overload induced heart failure in rats.

In cardiac myocytes in vitro, hydrogen peroxide induces autophagic cell death and necroptosis. Oxidative stress, myocyte autophagy and necroptosis coexist in heart failure (HF). In this study, we tested the hypothesis that excessive oxidative stress mediates pathological autophagy and necroptosis in myocytes in pressure overload-induced HF. HF was produced by chronic pressure overload induced by abdominal aortic constriction (AAC) in rats. Rats with AAC or sham operation were randomised to orally receive an antioxidant N-acetylcysteine (NAC) or placebo for 4weeks. Echocardiography was performed for the assessments of left ventricular (LV) structure and function. AAC rats exhibited decreased LV fractional shortening (FS) at 4weeks after surgery. NAC treatment attenuated decreased LV FS in AAC rats. In AAC rats, myocardial level of 8-hydroxydeoxyguanosine assessed by immunohistochemical staining, indicative of oxidative stress, was increased, LC3 II protein, a marker of autophagy, Beclin1 protein and Atg4b, Atg5, Atg7 and Atg12mRNA expression were markedly increased, RIP1, RIP3 and MLKL expression, indicative of necroptosis, was increased, and all of the alterations in AAC rats were prevented by the NAC treatment. NAC treatment also attenuated myocyte cross-sectional area and myocardial fibrosis in AAC rats. In conclusion, NAC treatment prevented the increases in oxidative stress, myocyte autophagy and necroptosis and the decrease in LV systolic function in pressure overload-induced HF. These findings suggest that enhanced oxidative stress mediates pathological autophagy and necroptosis in myocytes, leading to LV systolic dysfunction, and antioxidants may be of value to prevent HF through the inhibition of excessive autophagy and necroptosis.

Trimetazidine Attenuates Heart Failure by Improving Myocardial Metabolism via AMPK.

Energic deficiency of cardiomyocytes is a dominant cause of heart failure. An antianginal agent, trimetazidine improves the myocardial energetic supply. We presumed that trimetazidine protects the cardiomyocytes from the pressure overload-induced heart failure through improving the myocardial metabolism. C57BL/6 mice were subjected to transverse aortic constriction (TAC). After 4 weeks of TAC, heart failure was observed in mice manifested by an increased left ventricular (LV) chamber dimension, an impaired LV ejection fraction evaluated by echocardiography analysis, which were significantly restrained by the treatment of trimetazidine. Trimetazidine restored the mitochondrial morphology and function tested by cardiac transmission electron microscope and mitochondrial dynamic proteins analysis. Positron emission tomography showed that trimetazidine significantly elevated the glucose uptake in TAC mouse heart. Trimetazidine restrained the impairments of the insulin signaling in TAC mice and promoted the translocation of glucose transporter type IV (GLUT4) from the storage vesicle to membrane. However, these cardioprotective effects of trimetazidine in TAC mice were notably abolished by compound C (C.C), a specific AMPK inhibitor. The enlargement of neonatal rat cardiomyocyte induced by mechanical stretch, together with the increased expression of hypertrophy-associated proteins, mitochondria deformation and dysfunction were significantly ameliorated by trimetazidine. Trimetazidine enhanced the isolated cardiomyocyte glucose uptake in vitro. These benefits brought by trimetazidine were also removed with the presence of C.C. In conclusion, trimetazidine attenuated pressure overload-induced heart failure through improving myocardial mitochondrial function and glucose uptake via AMPK.

Zerumbone prevents pressure overload-induced left ventricular systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis

BackgroundCardiac hypertrophy and fibrosis are hallmarks of cardiac remodeling and are involved functionally in the development of heart failure (HF). However, it is unknown whether Zerumbone (Zer) prevents left ventricular (LV) systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis. PurposeThis study investigated the effect of Zer on cardiac hypertrophy and fibrosis in vitro and in vivo. Study Design/methodsIn primary cultured cardiac cells from neonatal rats, the effect of Zer on phenylephrine (PE)-induced hypertrophic responses and transforming growth factor beta (TGF-β)-induced fibrotic responses was observed. To determine whether Zer prevents the development of pressure overload-induced HF in vivo, a transverse aortic constriction (TAC) mouse model was utilized. Cardiac function was evaluated by echocardiography. The changes of cardiomyocyte surface area were observed using immunofluorescence staining and histological analysis (HE and WGA staining). Collagen synthesis and fibrosis formation were measured by scintillation counter and picrosirius staining, respectively. The total mRNA levels of genes associated with hypertrophy (ANF and BNP) and fibrosis (Postn and α-SMA) were measured by qRT-PCR. The protein expressions (Akt and α-SMA) were assessed by western blotting. ResultsZer significantly suppressed PE-induced increase in cell size, mRNA levels of ANF and BNP, and Akt phosphorylation in cardiomyocytes. The TGF-β-induced increase in proline incorporation, mRNA levels of Postn and α-SMA, and protein expression of α-SMA were decreased by Zer in cultured cardiac fibroblasts. In the TAC male C57BL/6 mice, echocardiography results demonstrated that Zer improved cardiac function by increasing LV fractional shortening and reducing LV wall thickness compared with the vehicle group. ZER significantly reduced the level of phosphorylated Akt both in cultured cardiomyocytes treated with PE and in the hearts of TAC. Finally, Zer inhibited the pressure overload-induced cardiac hypertrophy and cardiac fibrosis. ConclusionZer ameliorates pressure overload-induced LV dysfunction, at least in part by suppressing both cardiac hypertrophy and fibrosis.

Abstract P467: Cardiac-specific Deletion Of Voltage Dependent Anion Channel 2 Leads To Dilated Cardiomyopathy By Altering Calcium Homeostasis

Voltage dependent anion channel 2 (VDAC2) is a mitochondrial outer membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in cellular calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. Previous literature suggests that improving mitochondrial calcium uptake via VDAC2 rescues arrhythmia phenotypes in genetic models of impaired cellular calcium signaling. However, the direct role of VDAC2 in intracellular calcium signaling and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac-specific deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium during development causes severe impairment in excitation-contraction coupling by reducing mitochondrial calcium uptake (n=3, p<0.05) and thereby impairing intracellular calcium signaling. VDAC2 knock-out mice showed a significant reduction in RYR-mediated calcium release (F/F 0 ) and rate of calcium uptake by SERCA2a [tau(msec)] compared to control mice (N=3, WT=54, KO=38, p<0.0001 (F/F 0 ) and p<0.05 (tau)). We also observed adverse cardiac remodeling which progressed to severe dilated cardiomyopathy and death (N=6, p<0.0001). Reintroducing VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype evident from improvement in ejection fraction and fractional shortening (n=3, p<0.05). Improving mitochondrial calcium uptake via VDAC2 using a VDAC2 agonist efsevin, increased cardiac contractile force in a mouse model of pressure-overload induced heart failure (N=8, n=22, p<0.05). In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing mitochondrial and cellular calcium signaling. Through this role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.

Abstract P106: Development Of An Oral Drug Delivery System For The Cardio-protective Neuropeptide Alpha-calcitonin Gene Related Peptide

Alpha-calcitonin gene related peptide (α-CGRP) is a promising neuropeptide for the treatment of cardiovascular disease. We have developed an alginate biomaterial-based delivery system which increases the peptide’s bioavailability and have shown that subcutaneous delivery of alginate-CGRP microcapsules significantly improved cardiac function in pressure overload-induced heart failure in mice. The goal of this study is to develop efficient alginate microcapsule formulations for oral delivery of α-CGRP. An electrospray method was used to prepare four different formulations of alginate-CGRP microcapsules all of 200 μm diameter- i)- alginate-CGRP microcapsules, ii)- alginate-CGRP microcapsules with UV-light exposure, iii)- poly-L-ornithine (PLO) coated alginate-CGRP microcapsules, and iv)- PLO alginate-CGRP microcapsules with UV-light exposure. The stability of the microcapsules in the digestive tract was evaluated in deionized water, simulated gastric fluid (SGF; pH 1.2), and simulated intestinal fluid (SIF; pH 6.8). Over time, the size of all four microcapsule formulations remained almost unchanged in SGF, however all four of the microcapsule formulations swelled in presence of SIF. Compared to deionized water, the size of alginate-CGRP microcapsules after 6 h incubation in SIF increased by 1.7 fold. Since all four formulations yielded similar results, we chose to further study the alginate-CGRP microcapsules in vivo . To determine the bioactive nature of the released peptide, alginate microcapsules containing α-CGRP in doses of 1, 3, and 10 (mg/kg b.wt) were fed to male C57BL/6 mice via oral gavage. Systolic blood pressure (SBP) was subsequently measured by a tail-cuff method. α-CGRP microcapsules reduced SBP in a time-dependent manner. Alginate-CGRP at 1 and 3 mg/kg lowered the SBP by 25 mmHg for up to 4 h and 48 h, respectively. However, 10 mg/kg alginate-CGRP initially reduced SBP to undetectable levels which ultimately returned to baseline level by day 7. These studies indicate that alginate microcapsules can withstand the low pH of the stomach and the release of the peptide is bioactive in vivo . Thus, alginate microcapsules may provide an ideal formulation to deliver α-CGRP orally for the long-term treatment of cardiac diseases.

Interleukin-1 Receptor Antagonism Abrogates Acute Pressure Overload-Induced Murine Heart Failure

BackgroundRecent clinical trials have suggested that blockade of interleukin-1 (IL-1) can have a favorable impact on patients with myocardial infarction and heart failure. However, the mechanism of antagonism of this specific cytokine in mediating cardiac disease remains unclear. Hence, we sought to determine the influence of IL-1 blockade on acute hypertensive remodeling. MethodsTransverse aortic constriction was performed in C57BL mice with or without intraperitoneal administration of interleukin 1 receptor antagonism (IL-1Ra). Function, structure, and molecular diagnostics were subsequently performed and analyzed. ResultsSix weeks after transverse aortic constriction, a progressive decline of ejection fraction and increases in left ventricle mass and dimensions were effectively mitigated with IL-1Ra. Transverse aortic constriction resulted in an expected profile of hypertrophic markers including myosin heavy chain, atrial natriuretic peptide, and skeletal muscle actin, which were all significantly lower in IL-1Ra treated mice. Although trichrome staining 2 weeks after transverse aortic constriction demonstrated similar levels of fibrosis, IL-1ra–reduced expression of collagen-1, tissue inhibitor of metallopeptidase 1, and periostin. Investigating the angiogenic response to pressure overload, similar levels of vascular endothelial growth factor were observed, but IL-1Ra was associated with more stromal cell-derived factor-1. Immune cell infiltration (macrophages and lymphocytes) was also decreased in IL-1Ra treated mice. Similarly, cytokine concentrations of IL-1, IL-18, and IL-6 were all reduced in IL-1Ra–treated animals. ConclusionsInterleukin-1Ra prevents the progression toward heart failure associated with acute pressure overload. This functional response was associated with reductions in mediators of fibrosis, cellular infiltration, and cytokine production. These results provide mechanistic insight into recent clinical trials and could springboard future investigations in patients with pressure-overload–based cardiomyopathies.

Myeloid-Derived Growth Factor Protects Against Pressure Overload–Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca 2+ -ATPase Expression in Cardiomyocytes

Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation’s potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient ( Mydgf −/− ), and Mydgf bone marrow–chimeric or bone marrow–conditional transgenic mice with pressure overload–induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography–mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca 2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf −/− mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow–derived inflammatory cells attenuated pressure overload–induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein–coupled receptor agonist–induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca 2+ -ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf −/− mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca 2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf −/− mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload–induced hypertrophy and dysfunction. Pressure-overloaded Mydgf −/− mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload–induced heart failure.

Resistin deletion protects against heart failure injury by targeting DNA damage response.

Increased resistin (Retn) levels are associated with development of cardiovascular diseases. However, the role of Retn in heart failure (HF) is still unclear. Here we probed the functional and molecular mechanism underlying the beneficial effect of Retn deletion in HF. Wild-type (WT) and adipose tissue-specific Retn-knockout (RKO) mice were subjected to transverse aortic constriction (TAC)-induced HF. Cardiac function and haemodynamic changes were measured by echocardiography and left ventricular catheterization. Adipose tissue Retn deletion attenuated while Retn cardiac-selective overexpression, via a recombinant adeno-associated virus-9 vector, exacerbated TAC-induced hypertrophy, cardiac dysfunction, and myocardial fibrosis in WT and RKO mice. Mechanistically, we showed that Gadd45α was significantly increased in RKO HF mice while cardiac overexpression of Retn led to its downregulation. miR148b-3p directly targets Gadd45α and inhibits its expression. Retn overexpression upregulated miR148b-3p expression and triggered DNA damage response (DDR) in RKO-HF mice. Inhibition of miR148b-3p in vivo normalized Gadd45α expression, decreased DDR, and reversed cardiac dysfunction and fibrosis. In vitro Retn overexpression in adult mouse cardiomyocytes activated miR148b-3p and reduced Gadd45α expression. Gadd45α overexpression in H9C2-cardiomyoblasts protected against hydrogen peroxide- and Retn-induced DDR. These findings reveal that diminution in circulating Retn reduced myocardial fibrosis and apoptosis, and improved heart function in a mouse model of HF, at least in part, through attenuation of miR148b-3p and DDR. The results of this study indicate that controlling Retn levels may provide a potential therapeutic approach for treating pressure overload-induced HF.

Cardiac-specific deletion of voltage dependent anion channel 2 leads to dilated cardiomyopathy by altering calcium homeostasis

Voltage dependent anion channel 2 (VDAC2) is an outer mitochondrial membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. However, the specific role of VDAC2 in intracellular calcium dynamics and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac ventricular myocyte-specific developmental deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium causes severe impairment in excitation-contraction coupling by altering both intracellular and mitochondrial calcium signaling. We also observed adverse cardiac remodeling which progressed to severe cardiomyopathy and death. Reintroduction of VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype. Activation of VDAC2 by efsevin increased cardiac contractile force in a mouse model of pressure-overload induced heart failure. In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing cellular calcium signaling. Through this unique role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.

Left Ventricular SGLT1 Protein Expression Correlates with the Extent of Myocardial Nitro-Oxidative Stress in Rats with Pressure and Volume Overload-Induced Heart Failure.

Myocardial sodium-glucose cotransporter 1 (SGLT1) has been shown to be upregulated in humans with heart failure (HF) with or without diabetes. In vitro studies have linked SGLT1 to increased nitro-oxidative stress in cardiomyocytes. We aimed to assess the relation between left ventricular (LV) SGLT1 expression and the extent of nitro-oxidative stress in two non-diabetic rat models of chronic heart failure (HF) evoked by either pressure (TAC, n = 12) or volume overload (ACF, n = 12). Sham-operated animals (Sham-T and Sham-A, both n = 12) served as controls. Both TAC and ACF induced characteristic LV structural and functional remodeling. Western blotting revealed that LV SGLT1 protein expression was significantly upregulated in both HF models (both p < 0.01), whereas the phosphorylation of ERK1/2 was decreased only in ACF; AMPKα activity was significantly reduced in both models. The protein expression of the Nox4 NADPH oxidase isoform was increased in both TAC and ACF compared with respective controls (both p < 0.01), showing a strong positive correlation with SGLT1 expression (r = 0.855, p < 0.001; and r = 0.798, p = 0.001, respectively). Furthermore, SGLT1 protein expression positively correlated with the extent of myocardial nitro-oxidative stress in failing hearts assessed by 3-nitrotyrosin (r = 0.818, p = 0.006) and 4-hydroxy-2-nonenal (r = 0.733, p = 0.020) immunostaining. Therefore, LV SGLT1 protein expression was upregulated irrespective of the nature of chronic hemodynamic overload, and correlated significantly with the expression of Nox4 and with the level of myocardial nitro-oxidative stress, suggesting a pathophysiological role of SGLT1 in HF.

Drug repurposing to prevent pressure overload-induced cardiac hypertrophy and heart failure.

Drug repurposing to prevent pressure overload-induced cardiac hypertrophy and heart failure.

Multidrug resistance proteins preferentially regulate natriuretic peptide-driven cGMP signalling in the heart and vasculature.

cGMP underpins the bioactivity of NO and natriuretic peptides and is key to cardiovascular homeostasis. cGMP-driven responses are terminated primarily by PDEs, but cellular efflux via multidrug resistance proteins (MRPs) might contribute. Herein, the effect of pharmacological blockade of MRPs on cGMP signalling in the heart and vasculature was investigated in vitro and in vivo. Proliferation of human coronary artery smooth muscle cells (hCASMCs), vasorelaxation of murine aorta and reductions in mean arterial BP (MABP) in response to NO donors or natriuretic peptides were determined in the absence and presence of the MRP inhibitor MK571. The ability of MRP inhibition to reverse morphological and contractile deficits in a murine model of pressure overload-induced heart failure was also explored. MK571 attenuated hCASMC growth and enhanced the anti-proliferative effects of NO and atrial natriuretic peptide (ANP). MRP blockade caused concentration-dependent relaxations of murine aorta and augmented responses to ANP (and to a lesser extent NO). MK571 did not decrease MABP per se but enhanced the hypotensive actions of ANP and improved structural and functional indices of disease severity in experimental heart failure. These beneficial actions of MRP inhibition were associated with a greater intracellular:extracellular cGMP ratio in vitro and in vivo. MRP blockade promotes the cardiovascular functions of natriuretic peptides in vitro and in vivo, with more modest effects on NO. MRP inhibition may have therapeutic utility in cardiovascular diseases triggered by dysfunctional cGMP signalling, particularly those associated with altered natriuretic peptide bioactivity. 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.

Whole-Mount Kidney Clearing and Visualization Reveal Renal Sympathetic Hyperinnervation in Heart Failure Mice.

Developing a three-dimensional (3D) visualization of the kidney at the whole-mount scale is challenging. In the present study, we optimized mouse whole-mount kidney clearing, which improved the transparency ratio to over 90% based on organ-specific perfusion (OSP)-clear, unobstructed brain imaging cocktails and computational analysis (CUBIC). The optimized OSP-CUBIC-compatible 3D immunostaining and imaging simultaneously visualized the high-resolution 3D structure of the whole-mount renal microvascular, glomerulus, and accompanying wrapped traveling sympathetic nerves in mice. A mouse model of pressure overload-induced heart failure (HF) was then established by minimally invasive transverse aortic constriction (MTAC). Further 3D quantification revealed renal sympathetic hyperinnervation (6.80 ± 1.04% vs. 3.73 ± 0.60%, P < 0.05) in mice with HF. In conclusion, this newly developed whole-organ tissue clearing and imaging system provides comprehensive information at the whole-mount scale and has great potential for kidney research. Our data suggest that renal sympathetic hyperinnervation is involved in HF associated with renal dysfunction.

AntimiR-132 Attenuates Myocardial Hypertrophy in an Animal Model of Percutaneous Aortic Constriction

BackgroundPathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure. ObjectivesThis study aimed to establish a novel porcine model of pressure-overload–induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model. MethodsThis study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28. ResultsAt d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 μm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132–treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2). ConclusionsThe inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin.

The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice.

BackgroundMyocardial macrophages have key roles in cardiac remodeling and dysfunction. The gamma-aminobutyric acid subtype A (GABAA) receptor was recently found to be distributed in macrophages, allowing regulation of inflammatory responses to various diseases. This study aimed to clarify the role of GABAA receptor-mediated macrophage responses in pressure overload-induced heart failure.Methods and ResultsC57BL/6J mice underwent transverse aortic constriction for pressure-overload hypertrophy (POH) and were intraperitoneally treated with a specific GABAA receptor agonist (topiramate) or antagonist (bicuculline). Echocardiography, histology, and flow cytometry were performed to evaluate the causes and effects of myocardial hypertrophy and fibrosis. Activation of the GABAA receptor by topiramate reduced ejection fraction and fractional shortening, enlarged the end-diastolic and end-systolic left ventricular internal diameter, aggravated myocardial hypertrophy and fibrosis, and accelerated heart failure in response to pressure overload. Mechanistically, topiramate increased the number of Ly6Clow macrophages in the heart during POH and circulating Ly6Chigh classic monocyte infiltration in late-phase POH. Further, topiramate drove Ly6Clow macrophages toward MHCIIhigh macrophage polarization. As a result, Ly6Clow macrophages activated the amphiregulin-induced AKT/mTOR signaling pathway, and Ly6ClowMHCIIhigh macrophage polarization increased expression levels of osteopontin and TGF-β, which led to myocardial hypertrophy and fibrosis. Conversely, GABAA receptor blockage with bicuculline reversed these effects.ConclusionsControl of the GABAA receptor activity in monocytes/macrophages plays an important role in myocardial hypertrophy and fibrosis after POH. Blockade of the GABAA receptor has the potential to improve pressure overload-induced heart failure.

Artificial Neural Networks Indicate Cardiac TIMP1, JNK, and Collagen I &amp; III Predict Sex Hormone Status in Female Aortic‐Banded Yucatan Mini‐Swine in a Chamber‐Specific Manner

The prevalence of heart failure (HF) with preserved ejection fraction (HFpEF) is increased in older, postmenopausal women and often associated with an increase in cardiac fibrosis. Therefore, the goal of this study was to assess the role of female sex hormones on chamber-dependent differences i.e., left ventricle (LV) vs. right ventricle (RV), in extracellular matrix (ECM) remodeling and regulation in a mini-swine model of pressure overload-induced heart failure. We hypothesized molecular markers involved in the bioregulation of the cardiac ECM can predict experimental intervention combinations in a chamber-specific manner. An ovariectomy (OVX) model of surgical menopause was used in aortic-banded (AB) female Yucatan mini-swine (sex hormones X pressure-overload) divided into 4 groups: 1) Control, intact (CON-INT; n=6); 2) CON-OVX (n=5); 3) AB-INT (n=7;) and 4) AB-OVX (n=6). Seventy-seven input variables from both the LV and RV included: 1) mRNA levels of estrogen (isoforms 1, 2) and progesterone receptors; ERK/JNK signaling and regulation including MAPK isoforms 1, 3, 8, and 9, MAPKK isoforms 1, 2, 4, and 7, and dual specificity phosphatases (DUSP) isoforms 1, 4, 6, 9, and 10; matrix metalloproteinase (MMP) isoforms 1, 2, 3, 9, 13, and 14; tissue inhibitors of MMP (TIMP) isoforms 1, 2, and 4; the ECM components collagen (isoforms 1 and 3) and fibronectin; and 2) protein levels of ERK/JNK (total and phosphorylated), MMP14, TIMP2, and fibronectin. Missing data were mean imputed and the min-max normalization method was used for all measures. One-way ANOVA models were used to identify mRNA or protein targets associated with group status. Resulting molecular predictors were then used in an artificial neural network (ANN) model, with logistic activation function, composed of 1 hidden layer and 5 nodes. 5-fold cross-validation conditioned on group i.e., at least one observation from each of the four groups, and confusion matrices were used to test the developed ANN model. One observation from each group (n=4 total) was retained for later model testing with the remaining observations used for ANN development i.e., 84% training and 16% testing. One-way ANOVA models indicated TIMP1 mRNA and total JNK protein levels in the LV, and Collagen I and III mRNA levels in the RV, were associated with group status (p<0.05). These 4 molecular markers were then used to develop the ANN model (Figure 1). Cross-validation and confusion matrices indicate all 4 targets formed a linear relationship predictive of group with an accuracy of 70.8%. In conclusion, molecular mechanisms involved in the bioregulation of the ECM have analytical power to extrapolate sex hormone and aortic-banding status in a pre-clinical model of pressure overload-induced HF. Ongoing work will compare multiple activation functions to improve prediction accuracy and delineate non-linear relationships amongst these molecular ECM targets.

The effect of Guanxin Shutong capsule on alleviating the myocardial fibrosis in heart failure rats

Ethnopharmacological relevanceGuanxin Shutong (GXST) capsule is a renowned traditional Chinese medicine widely used for the treatment of cardiovascular diseases in the clinic. However, no pharmacological experimental studies of GXST has been reported on the treatment of pressure overload-induced heart failure. This study aimed to investigate the effects of GXST capsule on ameliorating myocardial fibrosis conditions in pressure overload-induced heart failure rats. Material and methodsRats were randomly divided into 6 groups: Normal group, Model group, GXST-treated group at a dose of 0.5 g/kg, 1 g/kg, 2 g/kg, respectively, and digoxin positive control group at a dose of 1 mg/kg. After 4 weeks of administration, cardiac function was evaluated by echocardiography. Cardiac injury and fibrotic conditions were evaluated by H&E staining, Masson staining, and Sirius Red staining. Myocardial fibrosis was evaluated by immunohistochemistry staining and Western blot. ResultsGXST significantly inhibited cardiac fibrosis, reduced the excessive deposition of collagen, and finally improved cardiac function. GXST reversed ventricular remodeling might be through the TGF-β/Smad3 pathway. ConclusionGXST capsule demonstrated a strong anti-fibrosis effect in heart failure rats by inhibiting the TGF-β/Smad3 signaling pathway.

Qindan Capsule Attenuates Myocardial Hypertrophy and Fibrosis in Pressure Overload-Induced Mice Involving mTOR and TGF-β1/Smad Signaling Pathway Inhibition.

Qindan capsule (QC), a traditional Chinese medicine compound, has been used to treat hypertension in the clinic for over 30 years. It is still not known about the effects of QC on pressure overload-induced cardiac remodeling. Hence, this study aims to investigate the effects of QC on pressure overload-induced cardiac hypertrophy, fibrosis, and heart failure in mice and to determine the possible mechanisms. Transverse aortic constriction (TAC) surgery was used to induce cardiac hypertrophy and heart failure in C57BL/6 mice. Mice were treated with QC or losartan for 8 weeks after TAC surgery. Cardiac function indexes were evaluated with transthoracic echocardiography. Cardiac pathology was detected using HE and Masson's trichrome staining. Cardiomyocyte ultrastructure was detected using transmission electron microscopy. Hypertrophy-related fetal gene expression was investigated using real-time RT-PCR. The expression of 8-OHdG and the concentration of MDA and Ang-II were assessed by immunohistochemistry stain and ELISA assay, respectively. The total and phosphorylated protein levels of mTOR, p70S6K, 4EBP1, Smad2, and Smad3 and the expression of TGF-β1 and collagen I were measured using western blot. The results showed that low- and high-dose QC improved pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction. QC inhibited ANP, BNP, and β-MHC mRNA expression in failing hearts. QC improved myocardial ultrastructure after TAC surgery. Furthermore, QC downregulated the expression of 8-OHdG and the concentration of MDA, 15-F2t-IsoP, and Ang-II in heart tissues after TAC surgery. We also found that QC inhibited the phosphorylation of mTOR, p70S6K, and 4EBP1 and the expression of TGF-β1, p-Smad2, p-Smad3, and collagen I in pressure overload-induced failing hearts. These data indicate that QC has direct benefic effects on pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction. The protective effects of QC involve prevention of increased oxidative stress injury and Ang-II levels and inhibition of mTOR and TGF-β1/Smad pathways in failing hearts.

CircSnx12 Is Involved in Ferroptosis During Heart Failure by Targeting miR-224-5p.

Circular RNA (circRNA) is a subclass of non-coding RNAs that enables the circular transcripts resistant to the exonuclease digestion. Iron homeostasis is essential for the body to maintain normal physiological functions. At present, the relationship among circRNA, iron metabolism and heart failure remains largely unknown. This study aimed to explore the regulatory mechanism of circRNA and iron metabolism in heart failure. We obtained circRNA, miRNA and mRNA data from public databases and built a ceRNA network. The prediction results were verified in the myocardial tissues of pressure overload-induced heart failure mice through the use of histopathological staining methods, iron and malondialdehyde (MDA) measurement tests, quantitative real-time PCR (qRT-PCR), Western blot analysis and luciferase reporter assay. A total of 4 genes related to iron metabolism and oxidative stress were identified, and a ceRNA network involving 7 circRNAs, 7 miRNAs, and 4 mRNAs was constructed using bioinformatics tools. The results of qRT-PCR and Western blot analyses indicated that the expression level of FTH1 was similar with that predicted by bioinformatics analysis. Echocardiographic measurement showed that heart failure mice have lower fractional shortening and ejection fraction. Moreover, the myocardium of heart failure mice displayed obvious fibrosis as well as increased levels of iron and MDA compared to control mice. Besides, circSnx12 could act as an endogenous sponge to bind with miR-224-5p, and the 3'UTR region of FTH1 also had miRNA binding sites. A circRNA-miRNA-mRNA regulatory network was successfully constructed by identifying differentially expressed genes related to iron metabolism. This new approach reveals potential circRNA targets for the treatment of heart failure.