Cardiomyocyte Cross-sectional Area Research Articles

Sexual dimorphism in right ventricular adaptation to pressure overload involves differential angiogenic response.

This study investigated the sexual dimorphism in right ventricle (RV) remodeling in right heart failure susceptible Fischer CDF rats using the pulmonary artery banding (PAB) model. Echocardiography and hemodynamic measurements were performed in adult male and female Fischer CDF rats at 1- or 2-weeks post-PAB. RV systolic pressure and RV hypertrophy were significantly elevated in PAB rats compared to sham control at 1- and 2-weeks post-PAB; however, no differences were observed between male and female rats. Increase in cardiomyocyte cross-sectional area and RV end-diastolic diameter was observed in male rats compared to female rats at 2-weeks post-PAB. Conversely, higher fractional area change and cardiac index were observed in female rats compared to male rats at 2-weeks post-PAB. To explore the mechanisms, a focused PCR array was performed and higher expression of angiogenic genes, including sphingosine kinase-1 (Sphk1), was observed in the RV of female rats compared to male rats. Consistent with the higher angiogenic gene expression, female rats had a higher RV vascular density at 2-weeks post-PAB compared to male rats. Female RV endothelial cells (RVEC) had better angiogenic ability compared to male cells that was potentiated by estradiol. Furthermore, effect of estradiol on RVEC was inhibited by Sphk1 inhibitor (PF-543). Together, female Fischer CDF rats develop adaptive RV remodeling post-PAB compared to mal-adaptive remodeling in male rats. Moreover, the adaptive remodeling in female rats is associated with better RV angiogenic response that may result from better angiogenic ability of female RVEC and proangiogenic effects of estradiol through Sphk1.

Baicalin Mitigates Cardiac Hypertrophy and Fibrosis by Inhibiting the p85a Subunit of PI3K.

Background: Heart failure (HF) is a serious public health concern. Baicalin is one of the major active ingredients of a traditional Chinese herbal medicine, Huang Qin, which is used to treat patients with chest pain or cardiac discomfort. However, the underlying mechanism(s) of the cardioprotective effect of baicalin are still not fully understood. Methods: Isoprenaline injection or transverse aortic constriction-induced animal models and isoprenaline or angiotensin 2 administration-induced cell models of heart failure were established. Baicalin (15 mg/kg/day or 25 mg/kg/day) was administered in vivo, and 10 μM baicalin was administered in vitro. Potential pharmacological targets of baicalin and genes related to heart failure were identified via different databases, which suggested that PI3K-Akt may be involved in the effects of baicalin. Molecular docking was carried out to reveal the effect of baicalin on p85a. Results: We observed significant antihypertrophic and antifibrotic effects of baicalin both in vivo and in vitro. The mean cross-sectional area of cardiomyocytes recovered from 390 μm2 in the HF group to 195 μm2 in the baicalin-treated group. The area of fibrosis was reduced from 2.8-fold in the HF group to 1.62-fold in the baicalin-treated group. Baicalin displayed a significant cardioprotective effect via the inhibition of the PI3K signaling pathway by binding with five amino acid residues of the p85a regulatory subunit of PI3K. The combination treatment of baicalin and an inhibitor of PI3K p110 demonstrated a stronger cardioprotective effect. The mean ejection fraction increased from 54% in the baicalin-treated group to 67% in the combination treatment group. Conclusions: Our work identified baicalin as a new active herbal ingredient that is able to treat isoprenaline-induced heart dysfunction and suggests that p85a is a pharmacological target. These findings reveal the significant potential of baicalin combined with an inhibitor of PI3K p110 for the treatment of heart failure and support more clinical trials in the future.

Astragali Radix-Notoginseng Radix et Rhizoma medicine pair prevents cardiac remodeling by improving mitochondrial dynamic balance.

Astragali Radix (AR) and Notoginseng Radix et Rhizoma (NR) are frequently employed in cardiovascular disease treatment. However, the efficacy of the AR-NR medicine pair (AN) in improving cardiac remodeling and its underlying mechanism remains unclear. This study aimed to evaluate AN's cardioprotective effect and potential mechanism on cardiac remodeling using transverse aortic constriction (TAC) in mice and angiotensin II (Ang II)-induced neonatal rat cardiomyocytes (NRCMs) and fibroblasts in vitro. High-performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS) characterized 23 main components of AN. AN significantly improved cardiac function in the TAC-induced mice. Furthermore, AN considerably reduced the serum levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), cardiac troponin T (CTn-T), and interleukin-6 (IL-6) and mitigated inflammatory cell infiltration. Post-AN treatment, TAC-induced heart size approached normal. AN decreased cardiomyocyte cross-sectional area and attenuated the upregulation of cardiac hypertrophy marker genes (ANP, BNP, and MYH7) in vivo and in vitro. Concurrently, AN alleviated collagen deposition in TAC-induced mice. AN also reduced the expression of fibrosis-related indicators (COL1A1 and COL3A1) and inhibited the activation of the transforming growth factor-β1 (TGF-β1)/mothers against decapentaplegic homolog 3 (Smad3) pathway. Thus, AN improved TAC-induced cardiac remodeling. Moreover, AN downregulated p-dynamin-related protein (Drp1) (Ser616) expression and upregulated mitogen 2 (MFN-2) and optic atrophy 1 (OPA1) expression in vivo and in vitro, thereby restoring mitochondrial fusion and fission balance. In conclusion, AN improves cardiac remodeling by regulating mitochondrial dynamic balance, providing experimental data for the rational application of Chinese medicine prescriptions with AN as the main component in clinical practice.

CardiLect: A combined cross-species lectin histochemistry protocol for the automated analysis of cardiac remodelling.

Cardiac remodelling, a crucial aspect of heart failure, is commonly investigated in preclinical models by quantifying cardiomyocyte cross-sectional area (CSA) and microvascular density (MVD) via histological methods, such as immunohistochemistry. To achieve this, optimized protocols are needed, and the species specificity is dependent on the antibody used. Lectin histochemistry offers several advantages compared to antibody-based immunohistochemistry, including as cost-effectiveness and cross-species applicability. Direct comparisons between the two methods are lacking from the literature. In this study, we compared antibody- and lectin-based methods for the histological assessment of cardiomyocyte CSA (with the use of anti-laminin and wheat germ agglutinin [WGA]) and microvascular density (utilizing anti-CD31 and isolectin B4 [ILB4]) using different embedding and antigen/carbohydrate retrieval techniques. Here, we describe a detailed, easy-to-use combined lectin histochemistry protocol (WGA and ILB4, 'CardiLect' protocol) for the histological assessment of cardiac remodelling. The lectin-based approach has been evaluated on a cross-species basis, and its efficacy has been demonstrated in zebrafish, rodents, large animals and human samples. We provide an ImageJ script ('CardiLect Analyser') for automated image analysis, validated in a preclinical heart failure model by correlating histological parameters with echocardiographic findings. CSA showed a significant positive correlation with left ventricular (LV) mass (P=0.0098, rS=0.7545) and significant negative correlation with markers of systolic function, such as ejection fraction (EF) (P=0.0402, rS=-0.6364). Microvascular density showed significant negative correlation with LV mass (P=0.0055, rS=-0.7622) and significant positive correlation with EF (P=0.0106, rS=0.7203). The described combined lectin histochemistry protocol with the provided ImageJ script is an easy-to-use, cost-effective, cross-species approach for the histological assessment of cardiac remodelling.

The impact of X-rays on cardiac hydrometabolism and the regulatory role of AS-IV

BackgroundRadiation-induced cardiac injury has emerged as a significant pathological entity, with many studies focusing on the fibrotic changes in myocardial tissue. However, these do not offer solutions for the clinical prevention and treatment of radiation-induced heart disease. Regulating hydrometabolism presents a potential therapeutic target for the management of cardiovascular diseases. This research seeks to explore the impacts of irradiation on cardiac hydrometabolism and its regulatory mechanisms. MethodsThe impact of X-ray radiation on cardiac and cardiomyocyte hydrometabolism was studied through in vivo and in vitro experiments, examining the pharmacological effects and mechanisms of PX-478 and AS-IV interventions in cardiomyocytes. Results28 days after direct chest irradiation with 20 Gy X-rays, C57BL/6 mice exhibited an increased heart wet-to-dry weight ratio, significant enlargement of cardiomyocyte cross-sectional area, and elevated protein expression of HIF-1α, AQP1, AQP4, Cx43, Caspase3, and Bax, with decreased expression of Bcl-2. Irradiation with 6 Gy X-rays induced edema and damage in AC16 and HL-1 cardiomyocytes at 24, 48, and 72 h, with increased expression of HIF-1α, AQP1, AQP4, and Cx43 proteins post-radiation. Inhibition of HIF-1α ameliorated edema and apoptosis in AC16 and HL-1 cardiomyocytes, reducing the expression of HIF-1α, AQP1, AQP4, and Cx43 proteins. AS-IV demonstrated strong binding affinity with HIF-1α, and successfully attenuated the expression levels of HIF-1α, AQP1, AQP4, and Cx43 proteins, alleviating edema, mitochondrial swelling, and apoptosis in AC16 and HL-1 cardiomyocytes. Furthermore, AS-IV improved cardiomyocyte edema by restoring the activity of Na/K-ATPase. ConclusionAberrant activation of the HIF-1α/AQPs/Cx43 axis is a key mechanism in X-ray-induced cardiomyocyte edema and damage. AS-IV can ameliorate X-ray induced cardiac damage by regulating hydrometabolism.

A newly developed small-molecule ErbB4 agonist reduces reactive cardiac fibrosis and adverse ventricular remodelling after myocardial infarction in a sex-specific manner

Abstract Background The beneficial effects of the neuregulin/ErbB pathway on the diseased heart have been well-established. The anti-fibrotic and cardioprotective aspects of neuregulin are largely attributable to the activation of the ErbB4 receptor. We recently developed a small-molecule selective ErbB4 agonist, which reduced collagen production in cultured fibroblasts and prevented oxidative-stress induced cardiomyocyte death in vitro. Purpose In order to evaluate the in vivo effects of this small-molecule ErbB4 agonist (referred to as compound, Cpd), we assessed its effect on left ventricular remodelling, cardiac fibrosis and cardiac function in a murine model of myocardial infarction (MI). Since there is a potential interaction between ErbB4 and the oestrogen receptor, we also aimed to evaluate whether these effects were sex-dependent. We hypothesised that Cpd would attenuate left ventricular remodelling, reduce cardiac fibrosis and help retain cardiac function in a sex-specific manner. Methods Mice were divided by sex and then further divided into three experimental groups: a sham group, an MI group receiving Cpd (MI/Cpd), and a control MI group receiving vehicle only (MI/Veh). MI was induced by ligating the left descending coronary artery. Seven days after MI, mice started treatment with either Cpd (2mg/kg/d) or its vehicle through subcutaneous osmotic minipumps. Cardiac ultrasound was performed weekly until mice were sacrificed after four weeks of treatment. Hearts were excised and stained with Masson’s trichrome to assess reactive cardiac fibrosis in the myocardium of the left ventricle, located remote from the infarction scar. A WGA-isolectinB4-DAPI immunofluorescent stain was performed to assess cardiomyocyte cross-sectional area in order to evaluate cardiomyocyte hypertrophy. Results In females (n=25), Cpd significantly decreased left ventricular end-diastolic volume compared to vehicle (LVEDV, Fig.1A, 80±23 µl vs.108±29 µl, p=0.045) and induced a slightly increased left ventricular ejection fraction (LVEF, Fig.1B, 32±12% vs. 22±9%, p=0.086). In addition, Cpd significantly reduced reactive interstitial fibrosis myocardium (Fig.1C, fibrotic area, 1.4±1.0% vs. vehicle: 3.8±3.1%, p=0.0363). In males (n=30), Cpd had no significant effects on either of the parameters mentioned above, (Fig.1E-G). There was no significant treatment effect in either group on cardiomyocyte cross sectional area (Fig.1D, Fig.1H). Conclusion Treatment with the novel small-molecule ErbB4 agonist attenuates left ventricular dilation and reactive interstitial fibrosis after MI in female mice, but not in males. These data support the premise that small molecule-induced ErbB4 activation is a potential new therapy for heart failure, especially in female patients. The mechanisms underlying the sex-dependent effects warrant further exploration.Figure 1

The role of IL15 in angiotensin II-mediated cardiac remodelling

Abstract Hypertension (HTN) mediated target organ damage, such as cardiac remodelling, is a major cause of death and disability. Cytokines are important regulators of cardiac function, inflammation, and remodelling in cardiovascular disease. Increased levels of interleukin 15 (IL15) have been noted in HTN, but its role in HTN heart disease remains unknown. This study investigates the role of the IL15 axis in the pathogenesis of cardiac remodelling in HTN. Male 12-week-old IL15 receptor α knock-out (IL15RAKO), IL15 knock-out (IL15KO) and matching wild type (WT) mice were administered vehicle or angiotensin II (AngII) (490 ng/min/kg) for 2 weeks by osmotic minipump. Blood pressure was measured by tail cuff (n=5-8). Cardiac function was analysed by echocardiography (n=3-4). Cardiac infiltrating immune cells were analysed by flow cytometry (n=2-3 for IL15RAKO, n=6-10 for IL15KO). Cardiac remodelling was assessed by picrosirius red, WGA staining (n=5-8) and expression of markers of remodelling using RT-qPCR and western blots (n=5-8). Data are presented as mean±SEM and analysed using two-way ANOVA. AngII infusion caused a significant increase in systolic blood pressure in WT and IL15KO animals. In contrast, in IL15RAKO, the response to AngII was blunted. This was in line with a decrease in ejection fraction, however only WT mice had an increase in global longitudinal strain (-20.3±2.0 vs -10.9±1.2, p=0.0039 in WT and 19.9±0.8 vs -16.0±2.1, p=0.1878 in IL15RAKO). HTN was associated with increased cardiac IL15 protein levels in mice. The heart-to-body ratio, along with cardiomyocyte cross-sectional, were significantly increased in WT, but not in IL15RAKO HTN mice. Furthermore, histological analysis showed significantly higher cardiac collagen accumulation in WT but not IL15RAKO upon AngII infusion (5.8±1.0% in WTvvs. 2.1±0.5% in IL15RAKO, p=0.0021). This was in line with a higher expression of col1a1 in WT but not IL15RAKO HTN hearts (20.0±6.0 vs 4.5±1.7, p=0.0173). Similarly, the FN1 protein level was significantly increased in WT but not IL15RAKO AngII-treated mice. These changes were associated with a higher number of Cd11b+ macrophages accumulated in WT hypertensive hearts only. In contrast to IL15RAKO, mice lacking IL15 showed similar increases in global longitudinal strain, heart hypertrophy, and cardiomyocyte cross-sectional area as WT mice upon AngII infusion. Similarly, IL15KO hypertensive hearts showed a significant increase of col1a1 and FN1 in comparison to vehicle-treated mice. A similar level of Cd11b+ macrophages was found in hypertensive IL15KO and WT mice's hearts. In conclusion, IL15RA, but not IL15, plays a critical role in cardiac remodelling mediated by AngII infusion. This suggests that the IL15-IL15RA axis may play an important yet complex role in hypertensive heart disease.

Matairesinol blunts adverse cardiac remodeling and heart failure induced by pressure overload by regulating Prdx1 and PI3K/AKT/FOXO1 signaling

BackgroundPathological cardiac remodeling is a critical process leading to heart failure, characterized primarily by inflammation and apoptosis. Matairesinol (Mat), a key chemical component of Podocarpus macrophyllus resin, exhibits a wide range of pharmacological activities, including anti-hydatid, antioxidant, antitumor, and anti-inflammatory effects. PurposeThis study aims to investigate whether Matairesinol alleviate cardiac hypertrophy and remodeling caused by pressure overload and to elucidate its mechanism of action. MethodsAn in vitro pressure loading model was established using neonatal rat cardiomyocytes treated with angiotensin Ⅱ, while an in vivo model was created using C57 mice subjected to transverse aortic constriction (TAC). To activate the PI3K/Akt/FoxO1 pathway, Ys-49 was employed. Moreover, small interfering RNA (siRNA) and short hairpin RNA (shRNA) were utilized to silence Prdx1 expression both in vitro and in vivo. Various techniques, including echocardiography, wheat germ agglutinin (WGA) staining, HE staining, PSR staining, and Masson trichrome staining, were used to assess cardiac function, cardiomyocyte cross-sectional area, and fibrosis levels in rats. Apoptosis in myocardial tissue and in vitro was detected by TUNEL assay, while reactive oxygen species (ROS) content in tissues and cells was measured using DHE staining. Furthermore, the affinity of Prdx1 with Mat and PI3K was analyzed using computer-simulated molecular docking. Western blotting and RT-PCR were utilized to evaluate Prdx1 levels and proteins related to apoptosis and oxidative stress, as well as the mRNA levels of cardiac hypertrophy and fibrosis-related indicators. ResultsMat significantly alleviated cardiac hypertrophy and fibrosis induced by TAC, preserved cardiac function, and markedly reduced cardiomyocyte apoptosis and oxidative damage. In vitro, mat attenuated ang Ⅱ - induced hypertrophy of nrvms and activation of neonatal rat fibroblasts. Notably, activation of the PI3K/Akt/FoxO1 pathway and downregulation of Prdx1 expression were observed in TAC mice; however, these effects were reversed by Mat treatment. Furthermore, Prdx1 knockdown activated the PI3K/Akt/FoxO1 pathway, leading to exacerbation of the disease. Molecular docking indicated that Molecular docking indicated that Mat upregulated Prdx1 expression by binding to it, thereby inhibiting the PI3K/Akt/FoxO1 pathway and protecting the heart by restoring Prdx1 expression levels. ConclusionMatairesinol alleviates pressure overload-induced cardiac remodeling both in vivo and in vitro by upregulating Prdx1 expression and inhibiting the PI3K/Akt/FoxO1 pathway. This study highlights the therapeutic potential of Matairesinol in the treatment of cardiac hypertrophy and remodeling, providing a promising avenue for future research and clinical application.

Left Ventricular Systolic Dysfunction in NBCe1-B/C-Knockout Mice.

Congenital proximal renal tubular acidosis (pRTA) is a rare systemic disease caused by mutations in the SLC4A4 gene that encodes the electrogenic sodium bicarbonate cotransporter, NBCe1. The major NBCe1 protein variants are designated NBCe1-A, NBCe1-B, and NBCe1-C. NBCe1-A expression is kidney-specific, NBCe1-B is broadly expressed and is the only NBCe1 variant expressed in the heart, and NBCe1-C is a splice variant of NBCe1-B that is expressed in the brain. No cardiac manifestations have been reported from patients with pRTA, but studies in adult rats with virally induced reduction in cardiac NBCe1-B expression indicate that NBCe1-B loss leads to cardiac hypertrophy and prolonged QT intervals in rodents. NBCe1-null mice die shortly after weaning, so the consequence of congenital, global NBCe1 loss on the heart is unknown. To circumvent this issue, we characterized the cardiac function of NBCe1-B/C-null (KOb/c) mice that survive up to 2 months of age and which, due to the uninterrupted expression of NBCe1-A, do not exhibit the confounding acidemia of the globally null mice. In contrast to the viral knockdown model, cardiac hypertrophy was not present in KOb/c mice as assessed by heart-weight-to-body-weight ratios and cardiomyocyte cross-sectional area. However, echocardiographic analysis revealed reduced left ventricular ejection fraction, and intraventricular pressure-volume measurements demonstrated reduced load-independent contractility. We also observed increased QT length variation in KOb/c mice. Finally, using the calcium indicator Fura-2 AM, we observed a significant reduction in the amplitude of Ca2+ transients in paced KOb/c cardiomyocytes. These data indicate that congenital, global absence of NBCe1-B/C leads to impaired cardiac contractility and increased QT length variation in juvenile mice. It remains to be determined whether the cardiac phenotype in KOb/c mice is influenced by the absence of NBCe1-B/C from neuronal and endocrine tissues.

Potential cardioprotective effect of Vitamin D and sodium-glucose transport protein 2 inhibitor in improving cardiac hypertrophy and fibrosis in Type 2 diabetic rats

Background: Diabetes mellitus (DM) is a major risk factor for cardiovascular diseases. The progression of myocardial abnormalities due to DM occurs slowly but is progressive and asymptomatic. Sodium-glucose transport protein 2 inhibitors (SGLT-2i) and Vitamin D have potential cardioprotective properties that inhibit cardiomyocyte fibrosis and hypertrophy, which are early structural changes that occur in the heart of DM patients. Aim: The study aimed to determine the potential protective effects of SGLT-2i and Vitamin D administration on cardiac hypertrophy and fibrosis in Type 2 diabetic rats.Methods: This is an experimental study with a post-test-only control group design. Thirty-two male Wistar rats were given a high-fat/high-glucose (HF/HG) diet. After 3 weeks, rats were given an injection of streptozotocin (STZ 35 mg/kg) to induce pancreatic damage. The diabetic rats were then divided into four groups (n = 8 per group): untreated diabetic group (HF/HG/STZ), the diabetic group treated with empagliflozin (EMPA) 10 mg/kg body weight (BW) (HF/HG/ STZ+EMPA), the diabetic group treated with Vitamin D 225 IU/day (HF/HG/STZ+VitD), and the diabetic group treated with a combination of EMPA 10 mg/kg BW and Vitamin D 225 IU/day (HF/ HG/STZ+EMPA+VitD). Treatments were given by oral gavage for 8 weeks. Left ventricular biopsy was performed at week 13 to examine collagen deposition, the cardiomyocyte cross-sectional area (CSA), and the mRNA expression of β-myosin heavy chain (β-MHC) and transforming growth factor-β (TGF-β). All the obtained data were analyzed statistically. Results: The administration of EMPA, Vitamin D, and combination therapy of EMPA and Vitamin D reduced the mRNA expression of β-MHC and TGF-β in diabetic rats compared to the untreated diabetic group. The administration of EMPA, Vitamin D, and combination therapy also resulted in a decrease in both the cardiomyocyte CSA and collagen deposition. Compared to monotherapy, combination therapy led to significantly better parameter reduction. Conclusion: Administration of EMPA, Vitamin D, and combination therapy improved cardiac hypertrophy and fibrosis in type 2 diabetic rats.Relevance for Patients: The combination of Vitamin D and SGLT-2i may be proposed as a cardioprotective strategy and preventive measure to reduce the incidence of cardiovascular disease in patients with Type 2 DM.

MiR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice

ObjectiveCardiac fibrosis is an important contributor to atrial fibrillation (AF). Our aim was to identify biomarkers for AF using bioinformatics methods and explore the regulatory mechanism of miR-450a-2-3p in cardiac fibrosis in mice.MethodsTwo datasets, GSE115574 and GSE79768, were obtained from the Gene Expression Omnibus (GEO) database and subsequently merged for further analysis. Differential gene expression analysis was performed to identify differentially expressed genes (DEGs) and miR-450a-2-3p-related differentially expressed genes (MRDEGs). To investigate the underlying mechanism of cardiac fibrosis, a mouse model was established by treating mice with isoproterenol (ISO) and the miR-450a-2-3p agomir.ResultsA total of 127 DEGs and 31 MRDEGs were identified and subjected to Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the functions and pathways involved in AF. In the animal model, histological analysis using HE and Masson staining, as well as quantification of the collagen volume fraction (CVF), was performed. The increased expression of α-smooth muscle actin (α-SMA), collagen type I (COL1), collagen type III (COL3), and extracellular signal-regulated kinase 1/2 (ERK(1/2)) at both the transcriptional and translational levels indicated the significant development of myocardial fibrosis in mice induced with isoproterenol (ISO). In addition, the cross-sectional area of cardiomyocytes and the expression of atrial natriuretic peptide (NPPA) and brain natriuretic peptide (NPPB) were increased in the ISO group compared with the control group. However, after overexpression of the miR-450a-2-3p agomir through caudal vein injection, there was a notable improvement in cardiac morphology in the treated group. The expression levels of α-SMA, COL1, COL3, ERK(1/2), NPPA, and NPPB were also significantly decreased.ConclusionOur study reveals the mechanistic connection between ISO-induced myocardial fibrosis and the miR-450a-2-3p/ERK(1/2) signaling pathway, highlighting its role in the development of cardiac fibrosis. Modulating miR-450a-2-3p expression and inhibiting ERK(1/2) activation are promising approaches for therapeutic intervention in patients with AF.

Abstract Mo014: Cardioactive molecules identified by functional in vivo screening prevent anthracycline cardiotoxicity

Introduction: Promotion of cardiomyocyte survival upon insult is mandatory to allow maintenance of ventricular function and dam development of severe heart dysfunction. Recently, through an in vivo functional screening of the mouse secretome we identified potent mediators of cardiomyocyte survival among 1200 soluble factors expressed by AAV vectors. The cardiac overexpression of the most effective cytokines (Chrdl1, Fam3c and Fam3b) maintained cardiac function after myocardial infarction by blunting oxidative damage, decreasing cell death, promoting autophagy, and inhibiting myofibroblast activation (Ruozi, Bortolotti et al. 2022. Sci Transl Med 14, eabo0699) Aim: We investigated the effect of cardioactive proteins in a mouse anthracycline model of cardiotoxicity. Material and Methods: Mimicking clinical chronic therapeutic regimens, doxorubicin (24 mg/kg) was administered to C57/BL6 female mice one week after AAV9-driven cardiac overexpression of Chrdl1, Fam3b or Fam3c. Echocardiography was performed after 6 and 8 weeks and was followed by histology and molecular analysis. Apoptosis, autophagy and mitochondrial function was analysed in primary cardiomyocytes along with pathway investigation. Results: AAV9-mediated overexpression of Chrdl1, Fam3b and Fam3c markedly improved animal survival. Echocardiographic assessment revealed significant reduction of left ventricular ejection fraction and marked heart dilatation in control animals, while all treated animals maintained functional and anatomical parameters. Treated mice showed normal cardiac mass and cardiomyocyte cross-sectional area, reduced interstitial collagen deposition and decreased expression of profibrotic genes. In both rodents and human cardiomyocytes, Chrdl1, Fam3b and Fam3c factors decreased doxorubicin induced apoptosis, induced protective macroautophagy and preserved mitochondrial morphology. Chrdl1 prevented doxorubicin-mediated BMP upregulation and autophagy engulfment through the inhibition of a detrimental molecular pathway downstream the BMP receptor. Conclusions: The development of biotherapeutics that act as pro-survival factors for cardiomyocytes offers significant translational perspectives in cardio-oncology.

Abstract Tu052: RPN6-Serine14 Phosphorylation by PKA Protects Against Systolic Overload-Induced Cardiac Remodeling and Heart Failure

The ubiquitin-proteasome system is the main proteolytic pathway for quality control (PQC) and regulatory degradation of proteins in living cells. Systolic overload, as seen in hypertension and aortic stenosis, is a leading cause of heart failure (HF). Proteasome functional insufficiency is implicated in HF caused by systolic overload. Previous studies have established that Ser14-RPN6 phosphorylation (pS14-RPN6) mediates the activation of 26S proteasomes by protein kinase A. However, the pathophysiological significance of pS14-RPN6 in systolic overload remains to be established. We hypothesize that pS14-RPN6 helps maintain proteostasis and protects the heart during systolic overload. We observed a significant increase in myocardial pS14-RPN6 and ubiquitin conjugates in human HF of ischemic or non-ischemic etiologies (p=0.020, p=0.034) and similarly in wild type (WT) mice after 2 weeks of transverse aortic constriction (TAC) (p<0.0001, p=0.001). To investigate the role of pS14-RPN6 in vivo, our lab created Rpn6 S14A (S14A) knock-in mice where Ser14-Rpn6 phosphorylation is blocked. Here we have compared the responses to TAC between WT and S14A mice. We found that TAC-induced increases in myocardial proteasome peptidase activities were significantly attenuated, and accumulation of ubiquitin conjugates exacerbated in S14A mice (p<0.0001; p<0.0005). Serial echocardiography revealed that S14A mice displayed significantly greater left ventricular (LV) end-diastolic posterior wall thickness and LV mass to body weight ratios at 1- and 12-week post-TAC (p=0.024, 0.0003; p=0.005, 0.01), and lower ejection fraction at 12-week post-TAC than WT mice (p=0.035). S14A mice also showed greater heart weight to tibial length (TL) and ventricular weight to TL ratios at 1 and 12-week post-TAC (1-week, p=0.043, 0.039; 12-week, p=0.007, 0.006). Wheat germ agglutinin and picrosirius red staining indicated a greater cardiomyocyte cross-sectional area and cardiac fibrosis in 12-week post-TAC S14A mice vs. WT mice (p<0.0001; p=0.005). These novel findings demonstrate compellingly that myocardial pS14-RPN6 helps maintain proteostasis, curtails cardiac hypertrophy and delays heart failure occurrence during systolic overload

Dietary Branched Chain Amino Acids Modify Post-Infarct Cardiac Remodeling and Function in the Murine Heart.

Branch-chain amino acids (BCAA) are markedly elevated in the heart following myocardial infarction (MI) in both humans and animal models. Nevertheless, it remains unclear whether dietary BCAA levels influence post-MI remodeling. We hypothesize that lowering dietary BCAA levels prevents adverse cardiac remodeling after MI. To assess whether altering dietary BCAA levels would impact circulating BCAA concentrations, mice were fed a low (1/3×), normal (1×), or high (2×) BCAA diet over a 7-day period. We found that mice fed the low BCAA diet had >2-fold lower circulating BCAA concentrations when compared with normal and high BCAA diet feeding strategies; notably, the high BCAA diet did not further increase BCAA levels over the normal chow diet. To investigate the impact of dietary BCAAs on cardiac remodeling and function after MI, male and female mice were fed either the low or high BCAA diet for 2 wk prior to MI and for 4 wk after MI. Although body weights or heart masses were not different in female mice fed the custom diets, male mice fed the high BCAA diet had significantly higher body and heart masses than those on the low BCAA diet. Echocardiographic assessments revealed that the low BCAA diet preserved stroke volume and cardiac output for the duration of the study, while the high BCAA diet led to progressive decreases in cardiac function. Although no discernible differences in cardiac fibrosis, scar collagen topography, or cardiomyocyte cross-sectional area were found between the dietary groups, male mice fed the high BCAA diet showed longer cardiomyocytes and higher capillary density compared with the low BCAA group. Provision of a diet low in BCAAs to mice mitigates eccentric cardiomyocyte remodeling and loss of cardiac function after MI, with dietary effects more prominent in males.

Fenofibrate reduces cardiac remodeling by mitochondrial dynamics preservation in a renovascular model of cardiac hypertrophy

Fenofibrate, a PPAR-α agonist clinically used to lower serum lipid levels, reduces cardiac remodeling and improves cardiac function. However, its mechanism of action is not completely elucidated. In this study we examined the effect of fenofibrate on mitochondria in a rat model of renovascular hypertension, focusing on mediators controlling mitochondrial dynamics and autophagy.Rats with two-kidney one-clip (2K1C) hypertension were treated with fenofibrate 150 mg/kg/day (2K1C-FFB) or vehicle (2K1C-VEH) for 8 weeks. Systolic blood pressure and cardiac functional were in-vivo assessed, while cardiomyocyte size and protein expression of mediators of cardiac hypertrophy and mitochondrial dynamics were ex-vivo examined by histological and Western blot analyses.Fenofibrate treatment counteracted the development of hypertension and the increase of left ventricular mass, relative wall thickness and cross-sectional area of cardiomyocytes. Furthermore, fenofibrate re-balanced the expression Mfn2, Drp1 and Parkin, regulators of fusion, fission, mitophagy respectively. Regarding autophagy, the LC3-II/LC3-I ratio was increased in 2K1C-VEH and 2K1C-FFB, whereas the autophagy was increased only in 2K1C-FFB.In cultured H9C2 cardiomyoblasts, fenofibrate reversed the Ang II-induced mRNA up-regulation of hypertrophy markers Nppa and Myh7, accumulation of reactive oxygen species and depolarization of the mitochondrial membrane exerting protection mediated by up-regulation of the Uncoupling protein 2.Our results indicate that fenofibrate acts directly on cardiomyocytes and counteracts the pressure overload-induced cardiac maladaptive remodeling. This study reveals a so far hidden mechanism involving mitochondrial dynamics in the beneficial effects of fenofibrate, support its repurposing for the treatment of cardiac hypertrophy and provide new potential targets for its pharmacological function.

Ethanol-induced lung and cardiac right ventricular inflammation and remodeling underlie progression to pulmonary arterial hypertension.

Current research on ethanol-induced cardiovascular anomalies has focused on left ventricular (LV) function and blood pressure. To extend this area of research, we sought to determine whether ethanol-induced alterations in the structure and function of the right cardiac ventricle (RV) and pulmonary artery (PA) lead to pulmonary arterial hypertension (PAH). Two groups of male Sprague-Dawley rats received a balanced liquid diet containing 5% ethanol (w/v) or a pair-fed isocaloric liquid diet for 8 weeks. Weekly echocardiography was conducted to evaluate cardiopulmonary function, and lung and RV tissues were collected for exvivo histological and molecular studies. The ethanol-treated rats exhibited: (1) Elevated mean pulmonary arterial pressure and decreased pulmonary artery acceleration time/ejection time; (2) Pulmonary vascular remodeling comprising intrapulmonary artery medial layer thickening; and (3) RV hypertrophy along with increased RV/LV + septum, RV diameter, RV cardiomyocyte cross-sectional area, and LV mass/body weight ratio. These responses were associated with increased lung and RV pro-inflammatory markers, endothelin-1 (ET-1), TNF-α, and IL-6 levels and higher ET-1,ET-1 type A/B receptor ratio, and downregulation of the cytoprotective protein, bone morphogenetic protein receptor 2 (BMPR2), in the lungs. These findings show that moderate ethanol-induced cardiopulmonary changes underlie progression to PAH via an upregulated proinflammatory ET1-TNFα-IL6 pathway and suppression of the anti-inflammatory BMPR2.

CXCL10 regulates pressure overload-induced heart failure

Rationale: Heart failure involves tissue inflammation with elevated serum levels of CXCL9 and CXCL10, which are CXCR3 receptor ligands. Both chemokines are expressed by cardiac fibroblasts and myeloid cells in mice subjected to pressure overload induced by transverse aortic constriction (TAC) surgery. Although it is known that CXCR3−/− animals are protected from cardiac remodeling induced by TAC, the role of CXCL10 is unknown. The present study was undertaken to test the hypothesis that CXCL10 is crucial in the cardiac remodeling response to pressure overload induced by TAC in mice. Methods: We subjected wild-type and CXCL10−/− mice to TAC surgery, resulting in 4 experimental groups: WT Sham, WT TAC, CXCL10−/− Sham and CXCL10−/− TAC. WGA was used to analyze the cross-sectional area of cardiomyocytes. To analyze total collagen deposition, PicroSirius Red was used. Immunohistochemistry was performed to measure the number of CD64, CD3g and CD45 positive cells. Echocardiography was used to analyze cardiac function. Results are presented as the mean and standard error of the mean. Comparisons between means were made using two-way variance analysis (ANOVA), as well as Tukey's post hoc test for parametric comparisons, or Kruskal-Wallis and Dunn‘s post hoc test for non-parametric data. Differences with p<0.05 were considered significant. Results: After 1 week, the CXCL10−/− mice showed the same level of cardiac hypertrophy and similar changes in echocardiography parameters, when compared to wild-type mice. However, after 6 weeks, we observed that the CXCL10−/− mice showed an attenuation of heart weight with a reduction in the total collagen deposition compared with wild-type TAC mice. At the cellular level, the genetic deletion of CXCL10 prevented the TAC-induced increase in the cross-sectional area of cardiomyocytes. Moreover, we observed that TAC increased the number of CD3+ T-cells in the heart quantified from stained cross sections of both wild type and CXCL10−/− mice, while no differences were observed for the total number of macrophages. Importantly, echocardiographic changes, including dilation and cardiac dysfunction, induced by pressure overload in wild animals were blunted in CXCL10−/− mice. Corroborating this, the genetic deletion of CXCL10 prevented the increase in lung weight induced by TAC. Conclusion: CXCL10 regulates cardiac hypertrophy and fibrosis induced by pressure overload in mice, as well as the functional changes that characterize heart failure. We are currently unraveling the mechanism through which CXCL10 can accelerate the development of heart failure in response to cardiac pressure overload. Interaction between T cells and macrophages are likely mediating these effects. R01HL155993. 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.

Left Ventricular Dilation Resulting from the Absence of Either Rgs2 or Rgs5 Is Associated with Increased Immune Cell Infiltration in the Myocardium

Sustained sympathoexcitation is a hallmark of heart failure, with equally sustained activation of downstream signaling pathways involving Gs- and Gi/o-coupled β-adrenergic receptors (βAR) stimulating cAMP production and PKA activation. A critical mechanism controlling G protein-mediated βAR signaling is the activity of regulator of G protein signaling (RGS) proteins, including members of the B/R4 subfamily, which accelerate the termination of Gq/11 and Gi/o protein signaling. Previous studies by us and others reported that moderation of Gi/o activity separately by RGS2 and RGS5 of the R4 subfamily is key to maintaining normal ventricular rhythm. The loss of one or both R4 RGS proteins also results in left ventricular (LV) dilation and reduced ejection fraction. Here, we tested the hypothesis that the LV structural remodeling is due to the disruption of the coordinated activity of RGS2 and 5 resulting in sensitization of βAR signaling. To test this hypothesis, we challenged wild type (WT), Rgs2 KO ,Rgs5 KO, and Rgs2/5 double knockout (dbKO) adult male mice with sub-chronic infusion of isoproterenol (ISO, 30 mg/kg/day, 3 days) via a peritoneal osmotic minipump implantation. Rgs2 KO and Rgs5 KO mice showed increased LV chamber size at baseline, which was unchanged by ISO administration. In saline-treated animals, the loss of Rgs2 increased Rgs5 mRNA expression, while Rgs5 absence increased Rgs4 mRNA expression. ISO infusion induced concentric cardiac hypertrophy in WT mice, as evidenced by increased heart weight/tibia length (HW/TL) ratio and cardiomyocyte cross sectional area. While ISO infusion increased interstitial fibrosis in midventricular tissue and HW/TL ratio in dbKO mice, it did not affect cardiomyocyte cross sectional area or exacerbate LV dilation, despite increased expression of the cardiac fetal gene Nppa as well as the sarcomeric protein α-actinin and Ca2+/calmodulin-dependent kinase II (CaMKII). Cardiact tissue from saline-treated Rgs2 KO and Rgs5 KO mice had markedly increased CD45+ cells vs WT, while tissue from Rgs5 KO mice showed increased CD68+ cells, as revealed by immunohistochemistry. Taken together, these results indicate that ventricular remodeling seen in Rgs2 KO, Rgs5 KO, and Rgs2/5 dbKO mice is insensitive to sustained βAR stimulation but may be due to increased cardiac resident immune cell population. This work was supported by NIH R56 DK132859-01A1 and R01 HL139754, and Craig H. Neilsen Foundation Grant 382566 to Patrick Osei-Owusu. 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.

Effect of GLP-1 Receptor Agonist on Ischemia Reperfusion Injury in Rats with Metabolic Syndrome.

Metabolic syndrome (MetS) represents an important factor that increases the risk of myocardial infarction, and more severe complications. Glucagon Like Peptide-1 Receptor Agonists (GLP-1RAs) exhibit cardioprotective potential, but their efficacy in MetS-related myocardial dysfunction has not been fully explored. Therefore, we aimed to assess the effects of exenatide and dulaglutide on heart function and redox balance in MetS-induced rats. Twenty-four Wistar albino rats with induced MetS were divided into three groups: MetS, exenatide-treated (5 µg/kg), dulaglutide-treated (0.6 mg/kg). After 6 weeks of treatment, in vivo heart function was assessed via echocardiography, while ex vivo function was evaluated using a Langendorff apparatus to simulate ischemia-reperfusion injury. Heart tissue samples were analyzed histologically, and oxidative stress biomarkers were measured spectrophotometrically from the coronary venous effluent. Both exenatide and dulaglutide significantly improved the ejection fraction by 3% and 7%, respectively, compared to the MetS group. Histological analyses corroborated these findings, revealing a reduction in the cross-sectional area of cardiomyocytes by 11% in the exenatide and 18% in the dulaglutide group, indicating reduced myocardial damage in GLP-1RA-treated rats. Our findings suggest strong cardioprotective potential of GLP-1RAs in MetS, with dulaglutide showing a slight advantage. Thus, both exenatide and dulaglutide are potentially promising targets for cardioprotection and reducing mortality in MetS patients.

The Cardiometabolic Impact of Rebaudioside A Exposure during the Reproductive Stage.

The consumption of non-sugar sweeteners (NSS) has increased during pregnancy. The European Food Safety Agency suggested that steviol glycosides, such as Rebaudioside A (RebA), the major sweetener component of stevia, are safe for humans up to a dose of 4 mg/kg body weight/day. However, the World Health Organization recommended in 2023 the restraint of using NSS, including stevia, at any life stage, highlighting the need to study NSS safety in early periods of development. We aimed to study the mitochondrial and cardiometabolic effects of long-term RebA consumption during the reproductive stage of the life cycle. Female rats were exposed to RebA (4 mg steviol equivalents/kg body weight/day) in the drinking water from 4 weeks before mating until weaning. Morphometry, food and water consumption, glucose and lipid homeostasis, heart structure, function, and mitochondrial function were assessed. RebA showed an atrophic effect in the heart, decreasing cardiomyocyte cross-sectional area and myocardial fibrosis without repercussions on cardiac function. Mitochondrial and myofilamentary functions were not altered. Glucose tolerance and insulin sensitivity were not affected, but fasting glycemia and total plasma cholesterol decreased. This work suggests that this RebA dose is safe for female consumption during the reproductive stage, from a cardiometabolic perspective. However, studies on the effects of RebA exposure on the offspring are mandatory.