Pressure-overloaded Rat Research Articles

Impact of Right Ventricular Pressure Overload on Myocardial Stiffness Assessed by Natural Wave Imaging.

Right ventricular (RV) hemodynamic performance determines the prognosis of patients with RV pressure overload. Using ultrafast ultrasound, natural wave velocity (NWV) induced by cardiac valve closure was proposed as a new surrogate to quantify myocardial stiffness. This study aimed to assess RV NWV in rodent models and children with RV pressure overload vs control subjects and to correlate NWV with RV hemodynamic parameters. Six-week-old rats were randomized to pulmonary artery banding (n=6), Sugen hypoxia-induced pulmonary arterial hypertension (n=7), or sham (n=6) groups. They underwent natural wave imaging, echocardiography, and hemodynamic assessment at baseline and 6weeks postoperatively. The authors analyzed NWV after tricuspid and after pulmonary valve closure (TVC and PVC, respectively). Conductance catheters were used to generate pressure-volume loops. In parallel, the authors prospectively recruited 14 children (7 RV pressure overload; 7 age-matched control subjects) and compared RV NWV with echocardiographic and invasive hemodynamic parameters. NWV significantly increased in RV pressure overload rat models (4.99 ± 0.27m/s after TVC and 5.03 ± 0.32m/s after PVC in pulmonary artery banding at 6weeks; 4.89 ± 0.26m/s after TVC and 4.84 ± 0.30m/s after PVC in Sugen hypoxia at 6weeks) compared with control subjects (2.83 ± 0.15m/s after TVC and 2.72 ± 0.34m/s after PVC). NWV after TVC correlated with both systolic and diastolic parameters including RV dP/dtmax (r=0.75; P< 0.005) and RV Ees (r=0.81; P< 0.005). NWV after PVC correlated with both diastolic and systolic parameters and notably with RV end-diastolic pressure (r=0.65; P< 0.01). In children, NWV after both right valves closure in RV pressure overload were higher than in healthy volunteers (P< 0.01). NWV after PVC correlated with RV E/E' (r=0.81; P=0.008) and with RV chamber stiffness (r=0.97; P=0.03). Both RV early-systolic and early-diastolic myocardial stiffness show significant increase in response to pressure overload. Based on physiology and our observations, early-systolic myocardial stiffness may reflect contractility, whereas early-diastolic myocardial stiffness might be indicative of diastolic function.

Curcumin alone not combined with piperine exerts cardioprotective effects in pressure-overload rats by increasing glucagon-like peptide-1 receptor signaling and additional properties

Background and aimCurcumin has shown significant cardiovascular protective effects. However, the low bioavailability limits its practical application. As a natural bioenhancer, piperine is expected to increase the bioavailability of curcumin, and then, enhance its beneficial effects. Meanwhile, glucagon-like peptide-1 receptor (GLP-1R) signaling has been confirmed to be involved in the regulation of cardiovascular events. Herein, this study aimed to investigate whether piperine could enhance the beneficial effects of curcumin in pressure-overload rats and whether GLP-1/GLP-1R signaling is involved in their action mechanism. Methods and resultsMale Sprague-Dawley (SD) adult rats were subjected to abdominal aortic constriction (AAC) surgery to 16 weeks, which elevated blood pressure and induced cardiac hypertrophy. Curcumin (100 mg/kg/day) or/and piperine (20 mg/kg/day) were orally given for 4 weeks from the 17th week after AAC surgery. Curcumin alone treatment significantly lowered blood pressure, ameliorated cardiovascular remodeling, improved left ventricular performance and endothelium-dependent vascular relaxation. Accompanied by these results, GLP-1R expression in the myocardium was up-regulated. Piperine alone also exhibited a certain positive inotropic cardiac activity, but the effect was less significant. The combination of curcumin and piperine didn't show enhanced cardiovascular protective effects relative to curcumin, along with decreased fasting serum GLP-1 level and GLP-1R expression in the myocardium. ConclusionCurcumin exerts cardiovascular protective effects in pressure-overload rats by upregulating GLP-1R, and inhibiting of GLP-1/GLP-1R signaling pathway may be related to the limited beneficial effects of combination of curcumin and piperine. This study provides a new thinking that oral administration of some spices may affect cardiovascular function by modulating the GLP-1/GLP-1R system in a mode of gut-heart axis regulation.

Cardiac sympathetic overdrive, M2 macrophage activation and fibroblast heterogeneity are associated with cardiac remodeling in a chronic pressure overload rat model of HFpEF.

Heart failure with preserved ejection fraction (HFpEF) is a multifaceted pathogenesis disease and the exact mechanisms driving HFpEF have not been completely elucidated. Pressure overload hypertrophy (POH) related fibroblasts and M2 macrophages in HFpEF myocardium have been recently identified and are now of great interest. Sympathetic overdrive has also been implicated in HFpEF. This study is designed to dynamically observe the potential roles of aforementioned mechanisms in pathological remodeling and cardiac dysfunction in chronic PO rats. Surgical constriction of the abdominal aorta was used for induction of HFpEF. Echocardiography, electrocardiogram, hemodynamic measurement, hematoxylin and eosin staining, Masson staining, immunohistochemistry and immunofluorescence were performed to assess the changes in heart dysfunction, cardiac remodeling and driving mechanisms at different time points (2, 18, 24weeks). The PO induced HFpEF model was well established, which was confirmed by the persistent increase in carotid artery systolic and diastolic blood pressure, and left ventricle hypertrophy at the corresponding postoperative stage. Meanwhile, PO hypertrophy gradually developed into HFpEF, associated with QT and QTc intervals prolongation, normal systolic (EF was maintained at >50%) but impaired diastolic function (increasing LVEDP and LV -dP/dtmin, abnormal E/A ratio), increased myocytes size, and observed relatively slight inflammatory infiltration but robust reactive fibrosis. IHC staining further confirmed that macrophages (CD68) but not neutrophils (MPO) or T cells (CD3) accounted for a predominant proportion of infiltrating cells. Mechanistically, we found that the infiltrating macrophages in the heart expressed high levels of CD206 which was simultaneously adjacent to POH fibroblasts appeared to overexpression of α-SMA in PO rats at late stages. Interestingly, we distinguished two different POHF sub-populations during PO induced HFpEF development, according to non overlapping signals of α-SMA and PDGFRα/β proteins. Additionally, PO led to a pronounced exaggeration in sympathetic fibers at all time points. These findings suggest that the establishing model here begins with cardiac sympathetic overdrive, subsequently along with immune cells especially M2 macrophage accumulation and fibroblast heterogeneity at later stages is associated with the development of cardiac maladaptive remodeling and diastolic dysfunction thus further progression to HFpEF.

Shen'ge Formula Protects Cardiac Function in Rats with Pressure Overload-Induced Heart Failure.

In China, Shen'ge formula (SGF), a Traditional Chinese Medicine blend crafted from ginseng and gecko, holds a revered place in the treatment of cardiovascular diseases. However, despite its prevalent use, the precise cardioprotective mechanisms of SGF remain largely uncharted. This study aims to fill this gap by delving deeper into SGF's therapeutic potential and underlying action mechanism, thus giving its traditional use a solid scientific grounding. In this study, rats were subjected to abdominal aortic constriction (AAC) to generate pressure overload. Following AAC, we administered SGF and bisoprolol intragastrically at specified doses for two distinct durations: 8 and 24 weeks. The cardiac function post-treatment was thoroughly analyzed using echocardiography and histological examinations, offering insights into SGF's influence on vital cardiovascular metrics, and signaling pathways central to cardiac health. SGF exhibited promising results, significantly enhanced cardiac functions over both 8 and 24-week periods, evidenced by improved ejection fraction and fractional shortening while moderating left ventricular parameters. Noteworthy was SGF's role in the significant mitigation of myocardial hypertrophy and in fostering the expression of vital proteins essential for heart health by the 24-week mark. This intervention markedly altered the dynamics of the Akt/HIF-1α/p53 pathway, inhibiting detrimental processes while promoting protective mechanisms. Our research casts SGF in a promising light as a cardioprotective agent in heart failure conditions induced by pressure overload in rats. Central to this protective shield is the modulation of the Akt/HIF-1α/p53 pathway, pointing to a therapeutic trajectory that leverages HIF-1α promotion and p53 nuclear transport inhibition.

Vagus nerve stimulation alleviates cardiac dysfunction and inflammatory markers during heart failure in rats

Vagus nerve stimulation (VNS) is under clinical investigation as a therapy for heart failure with reduced ejection fraction (HFrEF). This study aimed to investigate its therapeutic effects on three main components of heart failure: cardiac function, cardiac remodeling and central neuroinflammation using a pressure overload (PO) rat model. Male Sprague-Dawley rats were divided into four groups: PO, PO + VNS, PO + VNS sham, and controls. All rats, except controls, underwent a PO surgery to constrict the thoracic aorta (~50 %) to induce HFrEF. Open loop VNS therapy was continuously administered to PO + VNS rats at 20 Hz, 1.0 mA for 60 days. Evaluation of cardiac function and structure via echocardiograms showed decreases in stroke volume and relative ejection fraction and increases in the internal diameter of the left ventricle during systole and diastole in PO rats (p < 0.05). However, these PO-induced adverse changes were alleviated with VNS therapy. Additionally, PO rats exhibited significant increases in myocyte cross sectional areas indicating hypertrophy, along with significant increases in myocardial fibrosis and apoptosis, all of which were reversed by VNS therapy (p < 0.05). Furthermore, VNS mitigated microglial activation in two central autonomic nuclei: the paraventricular nucleus of the hypothalamus and locus coeruleus. These findings demonstrate that when VNS therapy is initiated at an early stage of HFrEF progression (<10 % reduction in relative ejection fraction), the supplementation of vagal activity is effective in restoring multi organ homeostasis in a PO model.

Activation of SIRT1 by Resveratrol Alleviates Pressure Overload-Induced Cardiac Hypertrophy via Suppression of TGF-β1 Signaling

Introduction: Silent information regulator 1 (SIRT1) has been extensively investigated in the cardiovascular system and has been shown to play a pivotal role in mediating cell death/survival, energy production, and oxidative stress. However, the functional role of SIRT1 in pressure overload-induced cardiac hypertrophy and dysfunction remains unclear. Resveratrol (Rsv), a widely used activator of SIRT1, has been reported to protect against cardiovascular disease. We here examine whether activation of SIRT1 by Rsv attenuate pressure overload-induced cardiac hypertrophy and to identify the underlying molecular mechanisms. Methods: In vivo, rat model of pressure overload-induced myocardial hypertrophy was established by abdominal aorta constriction (AAC) procedure. In vitro, Angiotensin II (Ang II) was applied to induce hypertrophy in cultured neonatal rat cardiomyocytes (NCMs). Hemodynamics and histological analyses of the heart were evaluated. The expression of SIRT1, transforming growth factor-β1 (TGF-β1)/phosphorylated (p)-small mother against decapentaplegic (Smad)3 and hypertrophic markers were determined by immunofluorescence, real-time PCR, and Western blotting techniques. Results: In the current study, Rsv treatment improved left ventricular function and reduced left ventricular hypertrophy and cardiac fibrosis significantly in the pressure overload rats. The expression of SIRT1 was significantly reduced, while the expression of TGF-β1/p-Smad3 was significantly enhanced in AAC afflicted rat heart. Strikingly, treatment with Rsv restored the expressions of SIRT1 and TGF-β1/p-Smad3 under AAC influence. However, SIRT1 inhibitor Sirtinol (Snl) markedly prevented the effects of Rsv, which suggest that SIRT1 signaling pathway was involved in the cardiac protective effect of Rsv. In vitro studies performed in Ang II-induced hypertrophy in NCMs confirmed the cardiac protective effect of Rsv. Furthermore, the study presented that SIRT1 negatively correlated with the cardiac hypertrophy, cardiac fibrosis, and the TGF-β1/p-Smad3 expression. Conclusions: Taken together, these results indicated that activation of SIRT1 by Rsv attenuates cardiac hypertrophy, cardiac fibrosis, and improves cardiac function possibly via regulation of the TGF-β1/p-Smad3 signaling pathway. Our study may provide a potential therapeutic strategy for cardiac hypertrophy.

Cardioprotective effects of early intervention with sacubitril/valsartan on pressure overloaded rat hearts

Left ventricular remodeling due to pressure overload is associated with poor prognosis. Sacubitril/valsartan is the first-in-class Angiotensin Receptor Neprilysin Inhibitor and has been demonstrated to have superior beneficial effects in the settings of heart failure. The aim of this study was to determine whether sacubitril/valsartan has cardioprotective effect in the early intervention of pressure overloaded hearts and whether it is superior to valsartan alone. We induced persistent left ventricular pressure overload in rats by ascending aortic constriction surgery and orally administrated sacubitril/valsartan, valsartan, or vehicle one week post operation for 10 weeks. We also determined the effects of sacubitril/valsartan over valsartan on adult ventricular myocytes and fibroblasts that were isolated from healthy rats and treated in culture. We found that early intervention with sacubitril/valsartan is superior to valsartan in reducing pressure overload-induced ventricular fibrosis and in reducing angiotensin II-induced adult ventricular fibroblast activation. While neither sacubitril/valsartan nor valsartan changes cardiac hypertrophy development, early intervention with sacubitril/valsartan protects ventricular myocytes from mitochondrial dysfunction and is superior to valsartan in reducing mitochondrial oxidative stress in response to persistent left ventricular pressure overload. In conclusion, our findings demonstrate that sacubitril/valsartan has a superior cardioprotective effect over valsartan in the early intervention of pressure overloaded hearts, which is independent of the reduction of left ventricular afterload. Our study provides evidence in support of potential benefits of the use of sacubitril/valsartan in patients with resistant hypertension or in patients with severe aortic stenosis.

Angiotensin II-Induced Vasoconstriction via Rho Kinase Activation in Pressure-Overloaded Rat Thoracic Aortas.

Angiotensin II (Ang II) induces vasoconstriction through myosin light chain (MLC) kinase activation and MLC phosphatase inactivation via phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) by Rho kinase. However, the detailed mechanism underlying Rho kinase activation by Ang II is still unknown. We investigated the mechanism of Ang II-induced vasoconstriction mediated by Rho kinase in pressure-overloaded rat thoracic aortas. Pressure-overloaded rats were produced by coarctation of the suprarenal abdominal aorta in four-week-old male Wistar rats. The contractile response to Ang II was significantly enhanced in the pressure-overloaded rats. Ang II-induced vasoconstriction was attenuated by inhibitors of Rho kinase, extracellular signal-regulated kinase 1 and 2 (Erk1/2), and epidermal growth factor receptor (EGFR) in both the sham-operated and pressure-overloaded rats. The Ang II-induced vasoconstriction was attenuated by a Janus kinase 2 (JAK2) inhibitor in only the pressure-overloaded rats. The protein levels of MYPT1 and JAK2 increased only in the pressure-overloaded rat thoracic aortas. These results suggested that Ang II-induced contraction is mediated by Rho kinase activation via EGFR, Erk1/2, and JAK2 in pressure-overloaded rat thoracic aortas. Moreover, Ang II-induced contraction was enhanced in pressure-overloaded rats probably because the protein levels of MYPT1 and JAK2 increased in the thoracic aortas.

Alterations in pore-forming subunits of adenosine triphosphate-sensitive potassium channels in pressure overload rat cardiomyocytes.

Adenosine triphosphate (ATP)-sensitive potassium channels (KATP) link cellular metabolic state and electrical activity of cardiomyocytes. Pharmacological studies indicated the involvement of KATP in pressure overload-induced left ventricular hypertrophy, but the alteration of pore-forming subunits, Kir6.1 and Kir6.2, at membranes and subcellular fractions is unclear. The objective of this study was to investigate the changes in the distribution and levels of Kir6.1 and Kir6.2 in rat cardiomyocytes responding to chronic pressure overload. Male Wistar rats were separated into a sham-operated group (sham) and a pressure overload or transverse aortic constriction (TAC) group. Echocardiography was performed to assess cardiac structure and functions at the 4th month after operation. Ventricular cardiomyocytes in both groups were isolated and then subjected to extract protein into cytoplasm, organelle membrane, and plasma membrane fractions. Immunoblotting and immunohistochemistry techniques were performed to detect and measure Kir6.1 and Kir6.2 protein levels. Echocardiographic parameters showed left ventricular hypertrophy with hypercontractility in TAC rats. The immunoblotting showed the presence of Kir6.1 and Kir6.2 at plasma membranes and only Kir6.1 at organelle membranes. Significantly, Kir6.1 and Kir6.2 levels were decreased in the plasma membrane fraction of the TAC group (n = 8 and 6 for Kir6.1 and Kir6.2, respectively), whereas Kir6.1 in the organelle membrane fraction tended to be higher in TAC but did not reach statistical significance (n = 7). These results seemed to relate to a left ventricular immunohistochemistry study, which showed the trend of decreased staining of Kir6.1 and Kir6.2 in pressure overload left ventricular tissue. In conclusion, both Kir6.1 and Kir6.2 plasma membrane subunits were decreased significantly in pressure overload-induced left ventricular hypertrophy.

Rutaecarpine Ameliorates Pressure Overload Cardiac Hypertrophy by Suppression of Calcineurin and Angiotensin II

Cardiac hypertrophy is a major pathological process to result in heart failure and sudden death. Rutaecarpine, a pentacyclic indolopyridoquinazolinone alkaloid extracted from Evodia rutaecarpa with multiple pharmacological activities, yet the underlying protective effects and the mechanisms on cardiac hypertrophy remain unclear. This study aimed to evaluate the potential effects of rutaecarpine on pressure overload cardiac hypertrophy. Cardiac hypertrophy in rat was developed by abdominal aortic constriction (AAC) for 4 weeks, which was improved by rutaecarpine supplementation (20 or 40 mg/kg/day, i.g.) for another 4 weeks. The level of angiotensin II was increased; the mRNA expression and the activity of calcineurin in the left ventricular tissue were augmented following cardiac hypertrophy. Rutaecarpine administration decreased angiotensin II content and reduced calcineurin expression and activity. Noteworthily, in angiotensin II-induced cardiomyocytes, rutaecarpine ameliorated the hypertrophic effects in a dose-dependent manner and downregulated the increased mRNA expression and activity of calcineurin. In conclusion, rutaecarpine can improve cardiac hypertrophy in pressure overload rats, which may be related to the inhibition of angiotensin II-calcineurin signal pathway.

Hydrogen-rich saline mitigates pressure overload-induced cardiac hypertrophy and atrial fibrillation in rats via the JAK-STAT signalling pathway.

ObjectiveTo investigate if hydrogen-rich saline (HRS), which has been shown to have antioxidant and anti-inflammatory properties, could mitigate cardiac remodelling and reduce the incidence of atrial fibrillation (AF) in the rat model of cardiac hypertrophy.MethodsPressure overload was induced in rats by abdominal aortic constriction (AAC). The animals were separated into four groups: sham; AAC group; AAC plus low dose HRS (LHRS); AAC plus high dose HRS (HHRS). The sham and AAC groups received normal saline intraperitoneally and the LHRS and HHRS groups received 3 or 6 ml/kg HRS daily for six weeks, respectively. In vitro research was also performed using cardiotrophin-1 (CT-1)-induced hypertrophy of cultured neonatal rat cardiomyocytes.ResultsCardiac hypertrophy was successfully induced by AAC and low and high dose HRS mitigated the pressure overload as shown by lower heart and atrial weights in these treatment groups. AF incidence and duration of the HRS groups were also significantly lower in the HRS groups compared with the AAC group. Atrial fibrosis was also reduced in the HRS groups and the JAK-STAT signalling pathway was down-regulated. In vitro experiments showed that hydrogen-rich medium mitigated the CT-1-induced cardiomyocyte hypertrophy with a similar effect as the JAK specific antagonists AG490.ConclusionsHRS was found to mitigate cardiac hypertrophy induced by pressure overload in rats and reduce atrial fibrosis and AF which was possibly achieved via inhibition of the JAK-STAT signalling pathway.

Histone Deacetylase Inhibition Attenuates Aortic Remodeling in Rats under Pressure Overload.

The use of histone deacetylase (HDAC) inhibitor is a novel therapeutic strategy for cardiovascular disease. Studies have shown that many HDAC inhibitors have the ability to reduce the aortic remodeling in various animal models. We hypothesized that the HDAC inhibitor, MGCD0103 (MGCD), attenuates aortic remodeling in rats under pressure overload-induced by transverse aortic constriction (TAC). The aortic ring tension analysis was conducted using the thoracic aorta. Sections of the aorta were visualized after hematoxylin and eosin, trichrome, and Verhoeff-van Gieson staining, and immunohistochemistry. The expression of genes related to aortic remodeling (αSMA, Mmp2, and Mmp9) and angiotensin receptors (Agtr1 and Agtr2) was determined by quantitative real-time polymerase chain reaction. There was a significant decrease in relaxation of the aorta when treated with MGCD. Fibrosis of the aortic wall and expression of angiotensin receptors increased in TAC rats, which was attenuated by MGCD. These results indicate that MGCD, an HDAC inhibitor, attenuates aortic remodeling in rats with TAC-induced pressure overload rats and may serve as a potential therapeutic target of antiaortic remodeling in pressure overload-induced hypertension-related diseases.

Soluble St2 Induces Cardiac Fibroblast Activation and Collagen Synthesis via Neuropilin-1.

Circulating levels of soluble interleukin 1 receptor-like 1 (sST2) are increased in heart failure and associated with poor outcome, likely because of the activation of inflammation and fibrosis. We investigated the pathogenic role of sST2 as an inductor of cardiac fibroblasts activation and collagen synthesis. The effects of sST2 on human cardiac fibroblasts was assessed using proteomics and immunodetection approaches to evidence the upregulation of neuropilin-1 (NRP-1), a regulator of the profibrotic transforming growth factor (TGF)-β1. In parallel, sST2 increased fibroblast activation, collagen and fibrosis mediators. Pharmacological inhibition of nuclear factor-kappa B (NF-κB) restored NRP-1 levels and blocked profibrotic effects induced by sST2. In NRP-1 knockdown cells, sST2 failed to induce fibroblast activation and collagen synthesis. Exogenous NRP-1 enhanced cardiac fibroblast activation and collagen synthesis via NF-κB. In a pressure overload rat model, sST2 was elevated in association with cardiac fibrosis and was positively correlated with NRP-1 expression. Our study shows that sST2 induces human cardiac fibroblasts activation, as well as the synthesis of collagen and profibrotic molecules. These effects are mediated by NRP-1. The blockade of NF-κB restored NRP-1 expression, improving the profibrotic status induced by sST2. These results show a new pathogenic role for sST2 and its mediator, NRP-1, as cardiac fibroblast activators contributing to cardiac fibrosis.

Toxic effects of combination of Aconiti Lateralis Radix Praeparata with Trichosanthis Fructus on inflammatory response and myocardial fibrosis in pressure overload rats based on β2-AR/PKA signal

To study the effects of combination of Aconiti Lateralis Radix Praeparata( Fuzi) with Trichosanthis Fructus( Gualou) on cardiac function,electrocardiogram,inflammatory response and myocardial fibrosis in pressure overload( PO) rats,and further explore the mechanism based on β2-AR/PKA signaling. PO rat model was established by constricting the abdominal aorta. Twelve weeks after the operation,these rats were randomly divided into model goup( PO),low dose Fuzi group( FL,5. 4 g·kg-1·d-1),Gualou group( GL,5. 4 g·kg-1·d-1),Fuzi and Gualou combination group( FG,5. 4 g·kg-1·d-1+5. 4 g·kg-1·d-1) and high dose Fuzi group( FH,10. 8 g·kg-1·d-1). At the same time,sham operation group was set. After intervention for 6 weeks,carotid blood pressure,cardiac function,electrocardiogram and heart mass index were measured. HE staining was used to observe the inflammatory response in the rat heart and kidney. Masson staining was used to determine the myocardial fibrosis. Western blot was used to detect the protein expression of β2-AR and PKA. As compared with sham operation group,the blood pressure and heart mass index were obviously increased in PO model group,but there was no significant difference in various treatment groups in the above indexes. As compared with PO model group,FH treatment significantly increased the ejection fraction( EF) and GL treatment effectively enhanced the cardiac output( CO),but other treatment groups had no significant effect on these parameters. Moreover,FG treatment can synergistically attenuate QT and QTc internal prolongation,but it also aggravated inflammatory response in the heart and kidney tissues and promoted myocardial fibrosis as compared to FZ or GL alone treatment,with toxic effects equivalent to FH treatment group. Following FG and FH treatment,simultaneously,β2-AR and PKA protein levels were significantly elevated,indicating that the increasing toxicity of FG could be associated with activation of β2-AR/PKA signaling. These results suggested that combination of FZ and GL could synergistically enhance toxicity of FZ in special pathological states such as pressure overload,and caution should be taken in clinical application.

Abstract P2017: Angiotensin-Induced Constriction via Rho Kinase Activation in Pressure-Overloaded Rat Thoracic Aortas

Activation of the angiotensin II (Ang II) receptor increases myosin light chain (MLC) kinase activity and causes inactivation of MLC phosphatase thorough myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation by Rho kinase. This results in MLC phosphorylation and subsequent smooth muscle constriction. However, the detailed mechanism of Rho kinase activation by Ang II is still unknown. The aim of this study was to clarify whether Ang II-induced constriction in pressure-overloaded rat thoracic aortas is mediated by Rho kinase activation through epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase 1 and 2 (Erk1/2), and janus kinase (JAK). Pressure-overload in the rat thoracic aortas was produced by suprarenal abdominal aortic coarctation. After 4 weeks, the thoracic aortas were excised for performing organ chamber experiments. Ang II was cumulatively added to reach a final concentration of a 0.1 – 1 μM. The inhibitors were added to 15 min prior to Ang II, and protein levels were measured by Western blotting. The contractile response to Ang II was significantly enhanced in the pressure-overloaded rats as compared to sham-operated ones [E max (% 40 mM KCl-induced constriction): sham; 44.6 ± 2.4, pressure-overload; 61.6 ± 2.2]. Ang II-induced constriction was attenuated by inhibitors of Rho kinase (E max : sham; 14.2 ± 5.3, pressure-overload; 6.8 ± 0.6), Erk1/2 (E max : sham; 21.2 ± 3.5, pressure-overload; 33.7 ± 3.5), and EGFR (E max : sham; 7.8 ± 2.0, pressure-overload; 26.6 ± 3.0) in both the sham-operated and pressure-overloaded rats. The Ang II-induced constriction was attenuated by JAK inhibitor (E max : sham; 41.9 ± 4.8, pressure-overload; 45.1 ± 4.2) in only pressure-overloaded rats. The protein levels of MYPT1 and JAK exhibited to increase in the pressure-overloaded rat thoracic aortas (MYPT1: sham; 1.0 ± 0.1, pressure-overload; 1.3 ± 0.1, JAK: sham; 1.0 ± 0.1, pressure-overload; 1.2 ± 0.1). These data suggest that Ang II-induced constriction is mediated by Rho kinase activation via EGFR, Erk1/2, and JAK in pressure-overloaded rat thoracic aortas. Moreover, Ang II-induced constriction was enhanced in pressure-overloaded rats probably because the protein levels of MYPT1 and JAK tended to increase in the thoracic aortas.

Abstract 753: Exercise Capacity, Cardiac fibrosis and Left Ventricular Remodeling in a Rat Model of Chronic Pressure Overload: Serial Echocardiography, Pressure-Volume Analysis and Gene Expression Profiling

Background: Pressure overload (PO) left ventricular (LV) hypertrophy is a known precursor of heart failure with preserved ejection fraction (EF). The aims of this study were to establish a reliable model of chronic PO in rats and verify the pathophysiological features of this model by evaluating cardiac function using serial echocardiography and a pressure-volume analysis. Methods: Constriction of the abdominal aorta was used to induce PO LV hypertrophy (n = 40) in rats. Twenty sham rats were compared with PO rats. Serial echo studies and exercise were performed at 2-week intervals, and invasive hemodynamic examination by a pressure-volume catheter system was performed 12 weeks after the procedure. The gene expression profiles of the left ventricle (LV) 12 weeks after the procedure were analyzed by DNA chip technology. Results: Serial echo revealed that the LV wall thickness began to increase after the PO and showed progressive increasing until the 8th week (LV septal wall thickness at 8 weeks 1.4mm±0.1mm for PO vs 0.6mm ±0.05mm for Sham, p &lt;0.01). LV dimension was comparable increased until 14 th week (LV end-systolic dimension at 14 weeks, 4.91±0.50 mm 4.71±0.25 mm for PO vs Sham; LV end-diastolic dimension, 9.02±0.42 mm vs. 8.71±0.47 mm for PO vs Sham, p&gt;0.05). The LV ejection fraction showed similar between groups until the 14th week (70.0±2.2% vs. 69.1±3.1% for PO vs. Sham). In hemodynamic analyses, the LV end-diastolic pressure and the end-diastolic pressure-volume relationship slope were greater in the PO group than sham group. When we compared LV remodeling and exercise capacity, cardiac fibrosis and exercise intolerance developed in the PO group but not in the sham group (exercise duration, 234.0 ± 80.3 vs. 597.8 ± 49.0 seconds, p &lt; 0.05, respectively). Transcriptional profiling of cardiac apical tissues revealed that gene expression related to the cardiac fibrosis, cytoskeletal pathway and G-protein signaling genes were enriched in the PO group. Conclusions: We established a small animal model of chronic PO and verified its pathophysiological features. Cardiac fibrosis and cytoskeletal pathway were important pathways in the PO group and influenced exercise capacity. This model may provide a useful tool for future research on PO heart failure.

Abstract 186: Superiority of Sacubitril/Valsartan Over Valsartan Alone in Attenuating Pressure Overload-induced Cardiac Fibrosis and Oxidative Stress

Background: Sacubitril/valsartan, a first-in-class Angiotensin Receptor-Neprilysin Inhibitor (ARNi), is superior to valsartan in reducing blood pressure in hypertensive patients. However, cardiac effects of sacubitril/valsartan in hypertension treatment and its potential superiority over valsartan are unclear. This study was designed to investigate effects of sacubitril/valsartan on cardiac structural remodeling and oxidative stress using a pressure overload rat model induced by ascending aortic constriction (AAC) and to address whether direct regulation of cardiac myocytes and/or fibroblasts is involved in the cardiac effects independent of afterload changes. Methods and Results: 1 week after AAC surgery, adult male Sprague-Dawley rats were randomized for 10 weeks treatment with vehicle, valsartan, or sacubitril/valsartan. Sacubitril/valsartan was superior to valsartan in inhibiting ventricular fibrosis, while both equally inhibited cardiomyocyte hypertrophy at the cellular level. These findings were supported by in vitro experiments showing that combined LBQ657 (sacubitrilat, active form of sacubitril) and valsartan was superior to valsartan alone in inhibiting angiotensin II (Ang II)-induced adult ventricular fibroblast activation, but not adult cardiomyocyte hypertrophy. In adult cardiomyocytes LBQ657 effectively inhibited Ang II-induced mitochondrial superoxide, and combined LBQ657 and valsartan was superior to valsartan or LBQ657 alone. Importantly, sacubitril/valsartan was also superior to valsartan in reducing mitochondrial superoxide in cardiomyocytes from pressure-overloaded hearts and improved cardiomyocyte mitochondrial maximal and spare respiration capacity. Conclusions: These findings provide direct evidence and novel mechanistic insights on the superior cardioprotective effects of sacubitril/valsartan in the setting of pressure overload: independent of afterload reduction, sacubitril/valsartan is superior to valsartan in attenuating cardiac fibrosis and myocyte oxidative stress and improves myocyte mitochondrial function in rats. Our study thus suggests a greater therapeutic effect of sacubitril/valsartan in the early stage of hypertensive heart disease before heart failure.

PKA/β2-AR-Gs/Gi signaling pathway is associated with anti-inflammatory and pro-apoptotic effects of Fuzi and Banxia combination on rats subjected to pressure overload

Ethnopharmacological relevanceEither Aconite Lateralis Radix Praeparata (Fuzi) or Pinelliae Rhizoma (Banxia) exerts anti-inflammatory activity and their combination has long been used in China for treating cardiovascular diseases. However, combination of two drugs is controversially prohibited in clinical prescriptions because it serves a representative incompatible pairs in “eighteen antagonisms”. Up to date, whether the combination of Fuzi and Banxia could be used for treating heart failure with preserved ejection fraction (HFpEF) especially charactered by systemic inflammation and the potential mechanisms have not been elucidated. Aim of the studyThe pros and cons of Fuzi in combination with Banxia were evaluated in pressure overload (PO) rat models of HF in vivo. Materials and methodsMale Sprague Dawley rats were subjected to abdominal aorta constriction or sham-operated procedure. From week 12, rats were administered with low dose Fuzi (5.4 g kg−1 d−1), Banxia (5.4 g kg−1 d−1), combination (5.4 g kg−1 d−1 + 5.4 g kg−1 d−1), high dose Fuzi (10.8 g kg−1 d−1) or with vehicle (n = 15 per group) orally for additional 6 weeks. ResultsFuzi alone treatment led to exaggerated cardiac-renal response to PO, and occurred dramatically at high dose as manifested by markedly exacerbated cardiac-renal inflammation and myocardial fibrosis. Further studies revealed that cardiotoxicity of Fuzi may be associated with highly expression levels of β2-AR and PKA. In contrast, coadministration of Fuzi and Banxia restored cardiac function, as indicated by relieving inflammation and fibrosis as well as normalizing electrocardiogram parameters, which were accompanied by PKA down-regulation. More importantly, both high dose Fuzi and combination treatment enhanced induction of apoptosis, which could be partially associated with inhibition of β2-AR-Gi signaling. ConclusionThus, combination of Fuzi and Banxia elicited concurrent protective and toxic effects in PO induced HF. The protective effect appeared to predominate and was associated with suppression of PKA/β2-AR-Gs signaling pathway. Unlike the eighteen antagonisms theory where Fuzi and Banxia combination was considered incompatible, in the present study, this herb pairs appeared to be benefit, and probably had potential therapeutic prospect in treating HFpEF and diseases associated with inflammation.

Angiotensin II-induced constriction via Rho kinase activation in pressure-overloaded rat thoracic aortas

Angiotensin II-induced constriction via Rho kinase activation in pressure-overloaded rat thoracic aortas

Chronic Ethanol Administration Prevents Compensatory Cardiac Hypertrophy in Pressure Overload.

Alcohol is among the most commonly abused drugs worldwide and affects many organ systems, including the heart. Alcoholic cardiomyopathy is characterized by a dilated cardiac phenotype with extensive hypertrophy and extracellular matrix (ECM) remodeling. We have previously shown that chronic ethanol (EtOH) administration accelerates the progression to heart failure in a rat model of volume overload. However, the mechanism by which this decompensation occurs is unknown. For this study, we hypothesized that chronic EtOH administration would prevent compensatory hypertrophy and cardiac remodeling in a rodent model of pressure overload (PO). Abdominal aortic constriction was used to create PO in 8-week-old male Wistar rats. Alcohol administration was performed via chronic intermittent EtOH vapor inhalation for 2weeks prior to surgery and for the duration of the 8-week study. Echocardiography measurements were taken to assess ventricular functional and structural changes. PO increased posterior wall thickness and the hypertrophic markers, atrial and B-type natriuretic peptides (ANP and BNP). With the added stressor of EtOH, wall thickness, ANP, and BNP decreased in PO animals. The combination of PO and EtOH resulted in increased wall stress compared to PO alone. PO also caused increased expression of collagen I and III, whereas EtOH alone only increased collagen III. The combined stresses of PO and EtOH led to an increase in collagen I expression, but collagen III did not change, resulting in an increased collagen I/III ratio in the PO rats treated with EtOH. Lastly, Notch1 expression was significantly increased only in the PO rats treated with EtOH. Our data indicate that chronic EtOH may limit the cardiac hypertrophy induced by PO which may be associated with a Notch1 mechanism, resulting in increased wall stress and altered ECM profile.