Left Ventricular Fractional Shortening Research Articles

Protective effect of naringin against radiation-induced heart disease in rats via Sirt1/NF-κB signaling pathway and endoplasmic reticulum stress.

This study was designed to explore the protective effect and mechanism of naringin (NG) on radiation-induced heart disease (RIHD) in rats. Rats were divided into four x-ray (XR) irradiation groups with different absorbed doses (0/10/15/20 Gy), or into three groups (control, XR, and XR + NG groups). Subsequently, the ultrasonic diagnostic apparatus was adopted to assess and compare the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diameter at end diastole (LVIDd), and left ventricular internal diameter at end systole (LVIDs) in rats. Hematoxylin-eosin (H&E) staining and Masson staining were applied to detect the pathological damage and fibrosis of heart tissue. Western blot was used to measure the expression levels of myocardial fibrosis-related proteins, endoplasmic reticulum stress-related proteins, and Sirt1 (silent information regulator 1)/NF-κB (nuclear factor kappa-B) signaling pathway-related proteins in cardiac tissues. Additionally, enzyme-linked immunosorbent assay was utilized to detect the activities of pro-inflammatory cytokines, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in cardiac tissue. The results showed that NG treatment significantly attenuated the 20 Gy XR-induced decline of LVEF and LVFS and the elevation of LVIDs. Cardiac tissue damage and fibrosis caused by 20 Gy XR were significant improved after NG treatment. Meanwhile, in rats irradiated by XR, marked downregulation was identified in the expressions of fibrosis-related proteins (Col I, collagen type I; α-SMA, α-smooth muscle actin; and TGF-β1, transforming growth factor-beta 1) and endoplasmic reticulum stress-related proteins (GRP78, glucose regulatory protein 78; CHOP, C/EBP homologous protein; ATF6, activating transcription factor 6; and caspase 12) after NG treatment. Moreover, NG treatment also inhibited the production of pro-inflammatory cytokines [interleukin-6, interleukin-1β, and monocyte chemoattractant protein-1 (MCP-1)], reduced the expression of MDA, and promoted the activities of SOD and CAT. Also, NG treatment promoted Sirt1 expression and inhibited p65 phosphorylation. Collectively, XR irradiation induced cardiac injury in rats in a dose-dependent manner. NG could improve the cardiac injury induced by XR irradiation by inhibiting endoplasmic reticulum stress and activating Sirt1/NF-κB signaling pathway.

Effects of aquatic and land high intensity interval training on hemodynamics and vascular function of middle-aged men.

Objective: To investigate the effects of 8-week aquatic and land high intensity interval training (HIIT) on hemodynamics and vascular function in middle-aged men. Methods: Thirty middle-aged men with low physical activity were selected and divided into 15 men (52.43 ± 4.11) in aquatic group and 15 men (52.74 ± 5.62) in land group by random number table. They performed HIIT exercise in aquatic and land 3 times a week for 8weeks. Pre-test, inter-test and post-test respectively measure hemodynamics and blood vessel function. Results: (1) Body composition: After 8weeks of exercise, weight, body mass index (BMI) and body fat rate (BF) were lower than before exercise (aquatic group: p < 0.01, land group: p < 0.05). The improvement of BF in the aquatic group was better than that in the land group (p < 0.05); (2) Cardiac function: After 8weeks of exercise, stroke volume (SV), left ventricular end-diastolic volume (EDV), cardiac output (CO), and left ventricular fractional shortening (FS), were higher than before exercise (aquatic group: p < 0.01, land group: p < 0.05), heart rate (HR) and left ventricular end-systolic volume (ESV) were lower than before exercise (aquatic group: p < 0.01, land group: p < 0.05). The improvement of SV, HR, EDV, ESV, CO and FS in the aquatic group was better than that in the land group (p < 0.05); (3) Hemodynamics: After 8weeks of exercise, systolic blood pressure (SBP), diastolic blood pressure (DBP) were lower than before exercise (aquatic group: p < 0.01, land group: p < 0.05), wall shear stress (WSS) and peak systolic velocity (PSV) were higher than before exercise (aquatic group: p < 0.01, land group: p < 0.05). The improvement of SBP, WSS and PSV in the aquatic group was better than that in the land group (p < 0.05); (4) Vascular function: basal diameter and brachial artery flow-mediated dilatation (FMD) level in aquatic group and land group was higher than before exercise, pulse wave velocity (PWV) level was lower than before exercise (aquatic and land group: p < 0.05). The improvement of FMD in the aquatic group was better than that in the land group. Conclusion: The body composition, hemodynamics and vascular function of middle-aged men were improved by 8-week aquatic and land HIIT. Aquatic HIIT has better effect on body fat rate, hemodynamics and vascular endothelial function in middle-aged men due to the effect of aquatic pressure and temperature.

Effects and mechanisms of Salvia miltiorrhiza Bunge extract on myocardial cell apoptosis in rat heart failure model.

This work aimed to investigate the effects of Tanshinone IIA (Tan IIA) on myocardial cell (MC) apoptosis in a rat model of heart failure (HF). Tan IIA was extracted from Salvia miltiorrhiza Bunge (SMB) using an ethanol reflux method. Fifty rats were randomly divided into five groups: sham (no treatment), mod (HF model establishment), low dose (LD: 0.1 mL/kg Tan IIA), medium dose (MD: 0.3 mL/kg Tan IIA), and high dose (HD: 0.5 mL/kg Tan IIA), with 10 rats in each group. The effects of different doses of Tan IIA on cardiac function, MC apoptosis, and the levels of proteins associated with the PI3K/Akt/mTOR signaling pathway were compared. Mod group showed a significant decrease in systolic arterial pressure, mean arterial pressure, heart rate, left ventricular systolic pressure, left ventricular ejection fraction, left ventricular fractional shortening, and the levels of p-PI3K, p-Akt, and p-mTOR proteins versus sham group (p < 0.05). Additionally, the left ventricular end-diastolic diameter (LVIDd), end-systolic diameter, diastolic pressure, and MC apoptosis were significantly increased (p < 0.05). LD, MD, and HD groups exhibited significant improvements across various indicators of cardiac function and MC apoptosis versus mod group (p < 0.05). Tan IIA may improve cardiac function and inhibit MC apoptosis in rats with HF by modulating the PI3K/Akt/mTOR signaling pathway.

Echocardiographic Evaluation of Infants Born to Mothers with Gestational Diabetes Mellitus: A Cross-sectional Analytical Study

Introduction: Gestational Diabetes Mellitus (GDM) is a chronic and progressive disease characterised by changes in the metabolism of carbohydrates, lipids, and proteins, leading to glucose intolerance. The foetal heart in GDM is affected throughout the gestational period, which may result in hyperplasia and hypertrophy of myocardial cells. Aim: To determine the cardiac function and structural changes in infants of gestational diabetic mothers using Dopplerechocardiographic data. Materials and Methods: A cross-sectional analytical study was conducted on 30 infants of mothers with GDM and 30 infants of non diabetic mothers at the Department of Physiology, R.G. Kar Medical College and Hospital, in collaboration with the Departments of Paediatric Medicine and Cardiology from January 2023 to August 2023. Neonatal screening echocardiography was performed by a cardiologist using a Philips Echocardiograph machine, Model: EPIQ 7C, Software Version: 4.0.2, with a linear convex probe of S8-3 MHz frequency to assess cardiac structure and function using 2D, M mode, and Conventional Doppler. The parameters studied included aortic root diameter (mm), left atrial diameter (mm), Interventricular Septal (IVS) in diastole (mm), left ventricular posterior wall thickness in diastole (mm), left ventricular internal diameter in diastole (mm), left ventricular internal diameter in end systole (mm), left ventricular fractional shortening (%), and left ventricular ejection fraction (%). Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version-20.0 with an Independent Student’s t-test. Results: The mean gestational age for the cases was 38.50±2.57 weeks and 39.30±1.97 weeks for controls. The mean maternal age for cases was 25±4.37 years and for controls was 27±5.03 years. The left atrial diameter in cases (12.83±2.14 mm) was significantly smaller than that of the control (14.20±0.55 mm). The left ventricular posterior wall diameter in diastole was significantly increased in cases (4.53±0.50 mm) compared to controls (3.87±0.35 mm). Significant differences were observed in the interventricular septum in diastole {cases: 4.73±0.45 mm, control: 3.97±0.32 mm} and in the left ventricular internal diameter in end systole {cases: 12.30±1.84 mm, control: 13.83±1.02 mm} between cases and controls. Aortic root diameter was increased in 13.3% of cases. The percentage of left ventricular fractional shortening (cases: 35.47±3.74 %, Control: 38.53±2.43 %), Left Ventricular Ejection fraction (cases: 67.63±4.52 %, control: 72.17±4.89 %) were significantly decreased in cases compared to control. Conclusion: The present study demonstrated the development of ventricular hypertrophy and compromised myocardial contractility in infants of diabetic mothers.

Chinese Medicine Supplementing Qi and Activating Blood Circulation Relieves the Progression of Diabetic Cardiomyopathy

Diabetic cardiomyopathy (DCM) is the leading cause of diabetic death as the final occurrence of heart failure and arrhythmia. Traditional Chinese medicine is usually used to treat various diseases including diabetes. This study sought to investigate the effects of Traditional Chinese medicine supplementing Qi and activating blood circulation (SAC) in DCM. After the construction of the DCM model by streptozotocin (STZ) injection and high glucose/fat diet feeding, rats were administered intragastrically with SAC. Then, cardiac systolic/diastolic function was evaluated by detecting left ventricular systolic pressure (LVSP), maximal rate of left ventricular pressure rise (+LVdp/dtmax), and fall (-LVdp/dtmax), heart rate (HR), left ventricular ejection fraction (EF), LV fractional shortening (FS) and left ventricular end-diastolic pressure (LVEDP). Masson’s and TUNEL staining were used to assess fibrosis and cardiomyocyte apoptosis. DCM rats exhibited impaired cardiac systolic/diastolic function manifested by decreasing LVSP, + LVdp/dtmax, -LVdp/dtmax, HR, EF and FS, and increasing LVEDP. Intriguingly, traditional Chinese medicine SAC alleviated the above-mentioned symptoms, indicating a potential role in improving cardiac function. Masson’s staining substantiated that SAC antagonized the increased collagen deposition and interstitial fibrosis area and the elevations in protein expression of fibrosis-related collagen I and fibronectin in heart tissues of DCM rats. Furthermore, TUNEL staining confirmed that traditional Chinese medicine SAC also attenuated cardiomyocyte apoptosis in DCM rats. Mechanically, DCM rats showed the aberrant activation of the TGF-β/Smad signaling, which was inhibited after SAC. SAC may exert cardiac protective efficacy in DCM rats via the TGF-β/Smad signaling, indicating a new promising therapeutic approach for DCM.

Lack of cardiac benefit after intramyocardial or intravenous injection of mesenchymal stem cell-derived extracellular vesicles supports the need for optimized cardiac delivery.

To determine the differences in improvement in cardiac function by intramyocardial (IM) vs. intravenous (IV) injection of human bone mesenchymal stem cell-derived extracellular vesicles (HBMSC-EV) after acute MI. FVB mice underwent acute MI via left anterior descending coronary artery ligation and subsequent injection of: (1) IM saline control; (2) IM HBMSC-EV; (3) IV saline control; and (4) IV HBMSC-EV. Cardiac function was evaluated with weekly postoperative echocardiography. On postoperative day 28, the mice were euthanized, and the heart, lungs, liver, spleen, and kidneys were harvested. Given previous studies showing HBMSC-EV hepatic uptake after IV injection, the liver was evaluated for changes in inflammation, fibrosis, and proliferation. On postoperative day 28, there were no significant differences in left ventricular ejection fraction (P = 0.6151), fractional shortening (P = 0.1135), or anterior border zone fibrosis (P = 0.6333) in any of the experimental groups. Interestingly, there was a strong trend demonstrating improvement in infarct size on fibrosis staining, which did not reach significance (P = 0.05620). There were no differences in hepatic inflammation, fibrosis, and proliferation. Although there was a trend in the improvement in infarct size, a single-dose administration of neither IM nor IV injection of HBMSC-EV resulted in significant improvement in post-MI cardiac function. A major limitation of this study is the lack of trials determining the optimal dose of HBMSC-EV needed in this model. However, the current study demonstrates that future studies are required to either optimize administration or bioengineer HBMSC-EV with cardiac-homing properties.

Shenfu Injection improves chronic heart failure by regulating pyroptosis based on NLRP3/caspase-1 pathway

This study investigated the mechanisms and targets of Shenfu Injection in the intervention in chronic heart failure(CHF) through the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/caspase-1 signaling pathway. A CHF model was induced in rats by subcutaneous injection of isoproterenol. Model rats were randomly divided into a model group, a Shenfu Injection group, and a MCC950(NLRP3 inhibitor) group, and a blank group was also set up as a control. After 15 days of treatment, echocardiography was performed to measure cardiac function parameters [left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS)]. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), interleukin(IL)-1β, and IL-18. Hematoxylin-eosin(HE) and Masson staining were used to observe morphological changes in myocardial tissues, and Western blot was used to measure the expression levels of NLRP3/caspase-1 pathway-related proteins [NLRP3, caspase-1, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), IL-1β, and IL-18]. The study found that isoproterenol-induced CHF in rats resulted in decreased cardiac function, worsened myocardial fibrosis, increased expression levels of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 in myocardial tissues, elevated serum inflammatory factors, and induced myocardial cell pyroptosis. Following Shenfu Injection intervention, the Shenfu Injection group showed significantly improved LVEF and LVFS, a significant decrease in NT-proBNP, a marked downregulation of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 protein expression levels, reduced serum inflammatory factors IL-1β and IL-18 expression in CHF rats, and a decrease in the rate of TUNEL-positive cells. Shenfu Injection can significantly improve cardiac function in CHF, inhibit myocardial fibrosis, and alleviate the progression of myocardial cell pyroptosis through the inhibition of the NLRP3/caspase-1 pathway.

Longitudinal echocardiographic parameters before and after pacemaker placement in congenital complete heart block

BackgroundCongenital complete heart block (CCHB) is seen in 1:15,000–1:20,000 live births, with risk of left ventricular (LV) dysfunction or dilated cardiomyopathy in 7%–23% of subjects. ObjectiveThe purpose of this study was to investigate serial changes in LV size and systolic function in paced CCHB subjects to examine the effect of time from pacemaker on echocardiographic parameters. MethodsSingle-center retrospective cohort analysis of paced CCHB subjects was performed. Echocardiographic data were collected before and after pacemaker placement. Linear mixed effect regression of left ventricular end-diastolic dimension (LVEDD) z-score, left ventricular shortening fraction (LVSF), and left ventricular ejection fraction (LVEF) was performed, with slopes compared before and after pacemaker placement. ResultsOf 114 CCHB subjects, 52 had echocardiographic data before and after pacemaker placement. Median age at CCHB diagnosis was 0.6 [interquartile range 0.0–3.5] years; age at pacemaker placement 3.4 [0.5–9.0] years; and pacing duration 10.8 [5.2–13.7] years. Estimated LVEDD z-score was 1.4 at pacemaker placement and decreased –0.08 per year (95% confidence interval [CI] –0.12 to –0.04; P = .002) to 0.2 (95% CI –0.3 to +0.3) 15 years postplacement. Estimated LVSF decreased –1.1% per year (95% CI –1.7% to –0.6%; P <.001) from 6 months prepacemaker placement to 34% (95% CI 32%–37%) 4 years postplacement. There was no significant change in LVSF between 4 and 15 years postplacement. Estimated LVEF did not change significantly after pacemaker placement, with estimated LVEF 59% (95% CI 55%–62%) 15 years postplacement. ConclusionIn 52 paced CCHB subjects, estimated LVEDD z-score decreased significantly after pacemaker placement, and estimated LVSF and LVEF remained within normal limits at 15 years postpacemaker placement.

Investigate the role of neutrophil extracellular traps in immune checkpoint inhibitor-associated myocarditis with programmed death protein-1 inhibitors involvement

Objective: To investigate the role of neutrophil extracellular traps (NETs) in immune checkpoint inhibitor-associated myocarditis (ICIAM) with programmed death protein-1 (PD-1) inhibitors involvement, and to explore the therapeutic potential of targeting NETs in the treatment of ICIAM. Methods: Thirty 6-week-old male BALB/c mice were randomly divided into control group (n=10), myocarditis group (n=10), and treatment group (n=10). Apart from the control group, each mouse was subcutaneously injected with 100 μl of complete Freund's adjuvant containing 250 μg of mouse cardiac troponin I peptide on the 1st and 7th day. Starting on the 8th day, PD-1 inhibitor (15 μg/per mouse) was intraperitoneally injected every other day for a total of 5 times. Since 1 day before the beginning of PD-1+TnI injection, the treatment group was injected with PF-1355 (50 mg·kg-1·d-1) for 16 consecutive days. The mice's general state was observed during the whole process. Real-time fluorescence quantitative PCR (RTFQ-PCR) was carried out to evaluate the transcriptional regulation of neutrophil related chemokines, NETs, pyronecrosis related factors and proinflammatory cytokines. Immunohistochemistry, immunofluorescence and western blot were applied to determine the changes of pyrosis related molecules. Echocardiography showed the differences of main cardiac indexes while cardiac pathology compared the degree of inflammatory infiltration in 3 gruops. Results: The immunofluorescence intensity of myocardial NETs in the myocarditis group was significantly increased compared to the control group mice (2.49±0.08 and 0.99±0.26, P<0.001). The protein expression levels of pyroptosis-related NLRP3, cleaved-Caspase 1, Caspase 1, cleaved-GSDMD, GSDMD, IL-1β and IL-18 in myocardial tissue of the model group were higher than those of the control group (all P<0.05). After treatment with PF-1355, compared to the myocarditis group, the left ventricular ejection fraction (LVEF) (73.58%±5.31% and 58.12%±3.19%, P<0.001) and left ventricular fraction shortening (LVFS) (39.78%±4.31% and 33.89%±2.19%, P<0.001) increased. H-E staining showed a reduction in inflammatory infiltration area in the treatment group compared to the myocarditis group (30.12%±3.57% and 14.92%±2.46%, P<0.001). The immunofluorescence intensity of NETs decreased in the treatment group compared to the myocarditis group (2.52±0.04 and 1.03±0.05, P<0.001). The levels of NLRP3 and other pyroptosis-related molecules were downregulated in the treatment group compared to the myocarditis group (all P<0.05). Conclusions: NETs lead to myocardial cell pyroptosis by activating the NLRP3 inflammasome in PD-1 inhibitor-associated myocarditis. The specific MPO inhibitor PF-1355 shows a therapeutic potential by regulating the formation of NETs, decreasing NLRP3 level and relieving myocardial pyroptosis, thus reducing myocardial damage.

Treatment with recombinant Sirt1 rewires the cardiac lipidome and rescues diabetes-related metabolic cardiomyopathy.

Metabolic cardiomyopathy (MCM), characterized by intramyocardial lipid accumulation, drives the progression to heart failure with preserved ejection fraction (HFpEF). Although evidence suggests that the mammalian silent information regulator 1 (Sirt1) orchestrates myocardial lipid metabolism, it is unknown whether its exogenous administration could avoid MCM onset. We investigated whether chronic treatment with recombinant Sirt1 (rSirt1) could halt MCM progression. db/db mice, an established model of MCM, were supplemented with intraperitoneal rSirt1 or vehicle for 4weeks and compared with their db/ + heterozygous littermates. At the end of treatment, cardiac function was assessed by cardiac ultrasound and left ventricular samples were collected and processed for molecular analysis. Transcriptional changes were evaluated using a custom PCR array. Lipidomic analysis was performed by mass spectrometry. H9c2 cardiomyocytes exposed to hyperglycaemia and treated with rSirt1 were used as in vitro model of MCM to investigate the ability of rSirt1 to directly target cardiomyocytes and modulate malondialdehyde levels and caspase 3 activity. Myocardial samples from diabetic and nondiabetic patients were analysed to explore Sirt1 expression levels and signaling pathways. rSirt1 treatment restored cardiac Sirt1 levels and preserved cardiac performance by improving left ventricular ejection fraction, fractional shortening and diastolic function (E/A ratio). In left ventricular samples from rSirt1-treated db/db mice, rSirt1 modulated the cardiac lipidome: medium and long-chain triacylglycerols, long-chain triacylglycerols, and triacylglycerols containing only saturated fatty acids were reduced, while those containing docosahexaenoic acid were increased. Mechanistically, several genes involved in lipid trafficking, metabolism and inflammation, such as Cd36, Acox3, Pparg, Ncoa3, and Ppara were downregulated by rSirt1 both in vitro and in vivo. In humans, reduced cardiac expression levels of Sirt1 were associated with higher intramyocardial triacylglycerols and PPARG-related genes. In the db/db mouse model of MCM, chronic exogenous rSirt1 supplementation rescued cardiac function. This was associated with a modulation of the myocardial lipidome and a downregulation of genes involved in lipid metabolism, trafficking, inflammation, and PPARG signaling. These findings were confirmed in the human diabetic myocardium. Treatments that increase Sirt1 levels may represent a promising strategy to prevent myocardial lipid abnormalities and MCM development.

Tristetraprolin in cardiomyocytes has a protective role in pressure-overloaded hearts

Abstract Background Elevated circulating levels of TNFα, IL-6 and the soluble TNF receptors predict adverse outcomes in patients with heart failure. However, the targeted anti TNF approaches resulted in worsening heart failure or death. The entire scenario of how the expression levels of inflammatory cytokines are maintained in failing hearts must be elucidated to develop novel and effective treatments for heart failure. Although transcriptional control is a determinant of the kinetics of proinflammatory cytokine gene expression, the stability of the mRNA also has a key function in coordinating immune responses. Tristetraprolin (TTP) has been shown to control mRNA decay by associating with the 3’- untranslated region and critical for the decay of the mRNAs for cytokines. The role of TTP in nonimmune cells such as cardiomyocytes remains to be elucidated. Purpose The purpose of this study is to examine the in vivo role of proinflammatory cytokine degradation by TTP in cardiomyocytes during cardiac remodelling under pressure overload. Methods Cardiomyocyte-specific TTP–deficient mice were generated. The mice were subjected to pressure overload by means of transverse aortic constriction (TAC) to induce heart failure. Cardiac remodelling was assessed by echocardiography as well as histological and molecular analyses 4 weeks after operation. Cytokine mRNA levels were examined by quantitative PCR. Results Cardiomyocyte-specific TTP–deficient mice showed no cardiac phenotypes under baseline conditions. However, the mice exhibited severe systolic dysfunction, indicated by left ventricular fractional shortening, after 4 weeks of pressure overload by means of TAC compared with control littermates (fractional shortening, control littermates 35.8% versus cardiomyocyte-specific TTP-deficient mice 19.9%, p = 0.000). In addition, those hearts showed increases in collagen deposition and the mRNA levels of Col1a2 and Nppb (1.4 times (p = 0.023), 1.5 times (p = 0.033), and 2.0 times (p = 0.020), respectively) compared to corresponding control hearts. Four weeks after TAC, the Il6 mRNA level was upregulated 2.7 times (p = 0.000), but not other cytokine mRNAs, including Tnfα, in cardiomyocyte-specific TTP–deficient hearts compared to controls. Conclusions The degradation of cytokine mRNA by TTP in cardiomyocytes plays a protective role in failing hearts and the TTP–mediated pathway in cardiomyocytes might be a therapeutic target to treat patients with heart failure.

FUDNC1 ameliorates doxorubicin-induced cardiotoxicity by restoring MERCs structure and blocked autophagic flux

Abstract Background Doxorubicin (DOX)-induced cardiotoxicity (DIC) is frequently observed in clinical practice. Autophagy dysregulation is known to be a mechanism of DIC. Mitochondrial-Endoplasmic Reticulum Contact sites (MERCs) is where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a mitochondria outer membrane protein that serves as a mitophagy receptor as well as a tethering protein of MERCs. Purpose The aim of our study was to investigate the effect of DOX on MERCs and explore whether it is involved in the dysregulation of autophagy. Our hypothesis is that FUNDC1 overexpression ameliorates DIC by restoring MERCs structure and dysregulated autophagic flux. Method We use confocal microscopy and transmission electron microscopy to assess MERCs structure. Overall autophagic flux was analyzed by RFP-EGFP-LC3B fluorescence assay and western blotting for LC3BII. A total dose of 15 mg/kg of doxorubicin was administrated in C57BL/6J mice to construct a DIC model in vivo. Adeno-associated virus (AAV) was used to cardiac-specifically overexpress FUNDC1. Cardiac remodeling was evaluated by Masson’s trichrome staining. Cardiac function (left ventricle ejection fraction, LVEF; left ventricle fraction shortening, LVFS) was measured by echocardiography. Results DOX treatment led to a significant downregulation of multiple tethering proteins (including FUNDC1) of MERCs and inhibition of MERCs formation, as demonstrated by decreased colocalization coefficients of mitochondria and endoplasmic reticulum in cardiomyocytes. The overexpression of FUNDC1 abolished DOX-induced inhibition on MERCs formation. In addition, DOX treatment blocked the normal autophagic flux, as demonstrated by the decreased autophagosome synthesis and impaired autophagosome turnover. Maintaining the MERCs structure by FUNDC1 overexpression was able to restore the blocked autophagic flux mentioned above. Moreover, FUNDC1 overexpression restored the blocked autophagic flux in vivo, as demonstrated by the increased LC3B turnover compared to the DOX treatment group. Importantly, global heart function of FUNDC1 overexpressed mice was significantly improved post DOX treatment compared to the Wildtype DOX treatment group, as demonstrated by the increased LVEF and LVFS. In addition, LV fibrosis level was found to be significantly decreased in FUNDC1 overexpressed mice treated with DOX compared to the Wildtype DOX treatment group. Conclusion We reported for the first time that the impaired MERCs formation in DOX-treated cardiomyocytes and revealed that restoring the disrupted MERCs structure by FUNDC1 overexpression was able to revert the blocked autophagic flux induced by DOX treatment. Furthermore, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. These findings may shed light on the prevention and treatment of DIC.Figure

Protective effects of nicotinamide mononucleotide on radiation-related cardiac injury

Abstract Background Radiation therapy for cancer treatment can cause damage to the heart. There are numerous possible mechanisms related to radiation-related cardiac dysfunction, among which increased oxidative stress is an important factor. However, the detailed mechanisms remain unclear and effective treatments on radiation-related cardiac dysfunction are still lacking. Nicotinamide mononucleotide (NMN), the most direct precursor of nicotinamide adenine dinucleotide (NAD+), can effectively reduce reactive oxygen species (ROS) production and thus could be a candidate to alleviate radiation-induced damage. However, the protective effects and the underlying mechanisms of NMN on radiation-related cardiac injury remain to be elucidated. Therefore, this study was designed to identify the mechanisms of radiation-related cardiac injury and investigate the protective actions of NMN. Methods Wild-type c57 mice were divided into radiation exposure (7.2Gy radiation) and non-exposure groups. Wild-type C57BL6/J mice supplied with NMN for 12 months before radiation. Echocardiography to detect mice cardiac function. Epicardial electrical mapping technique to detect electrical conduction in the heart. Non-targeted metabolomics was used to detect cardiac different metabolites. In parallel, we observed the effect of radiation on survival and mitochondrial function (ROS production and ATP content) of H9C2 cells with or without NMN. Results In the radiation exposure group, echocardiography showed that the left ventricular ejection fraction (LVEF) and fractional shortening (FS) were reduced, whilst epicardial mapping demonstrated slowed conduction velocity and change in the direction of propagation. Non-targeted metabolomics showed the different metabolites between the radiation group and the control group were mainly in nucleotide and amino acid metabolism. Metabolic pathway enrichment analysis showed that different metabolites were implicated in the metabolism of pyrimidine, folate, arginine and proline, and tryptophan. Mice with NMN before radiation showed reduced mechanical and electrical dysfunction, with non-targeted metabolomic results showed NMN rescued the radiation-induced down-regulation of 1-(4-aminobutyl)urea, 4-aminobenzoic acid, 3-hydroxyanthranilic acid, which ultimately exerted cardioprotective effects. We also found that radiation can reduce the survival, increase ROS production, and reduce ATP content of H9C2 cells in vitro, and these harmful effects can be partially reversed by NMN. Conclusion The mechanical and electrical dysfunction caused by radiation were associated with altered nucleotide and amino acid metabolism in the myocardium, and these harmful effects could be partially mitigated by NMN supplement.

OpiCa1-PEG-PLGA nanomicelles antagonize acute heart failure induced by the cocktail of epinephrine and caffeine

BackgroundReducing Ca2+ content in the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) by calcin is a potential intervention strategy for the SR Ca2+ overload triggered by β-adrenergic stress in acute heart diseases. MethodsOpiCal-PEG-PLGA nanomicelles were prepared by thin film dispersion, of which the antagonistic effects were observed using an acute heart failure model induced by epinephrine and caffeine in mice. In addition, cardiac targeting, self-stability as well as biotoxicity were determined. ResultsThe synthesized OpiCa1-PEG-PLGA nanomicelles were elliptical with a particle size of 72.26 nm, a PDI value of 0.3, and a molecular weight of 10.39 kDa. The nanomicelles showed a significant antagonistic effect with 100 % survival rate to the death induced by epinephrine and caffeine, which was supported by echocardiography with significantly recovered heart rate, ejection fraction and left ventricular fractional shortening rate. The FITC labeled nanomicelles had a strong membrance penetrating capacity within 2 h and cardiac targeting within 12 h that was further confirmed by immunohistochemistry with a self-prepared OpiCa1 polyclonal antibody. Meanwhile, the nanomicelles can keep better stability and dispersibility in vitro at 4 °C rather than 20 °C or 37 °C, while maintain a low but stable plasma OpiCa1 concentration in vivo within 72 h. Finally, no obvious biotoxicities were observed by CCK-8, flow cytometry, H&E staining and blood biochemical examinations. ConclusionOur study also provide a novel nanodelivery pathway for targeting RyRs and antagonizing the SR Ca2+ disordered heart diseases by actively releasing SR Ca2+ through RyRs with calcin.

Abstract 16402: Pitx2c-Conditional Knockout Mice May Become a Useful Model to Elucidate the Pathogenesis of Post-Capillary Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is an intractable disease that causes cardiopulmonary dysfunction due to elevated pulmonary arterial pressure. The pathogenesis of PH varies greatly depending on whether the primary lesion is upstream or downstream of the pulmonary capillaries, and the latter, post-capillary PH (pc-PH) caused by pulmonary vein stenosis/obstruction or left heart disease, currently has not an appropriate animal models to study its pathophysiology and pharmacological effects. Hypothesis: We hypothesized that a tissue-specific deletion of a gene involved in the formation of pulmonary veins could create a pathological mouse model of pc-PH. Methods: Focusing on the homeobox transcription factor Pitx2c, which has been reported to be indispensable for the formation of pulmonary veins, we generated Pitx2c-conditional knockout (cKO) mice specific for pulmonary vein and atrial myocardium by crossing Pitx2c-floxed mouse lines with Cre mouse lines for sarcolipin, a sarcoplasmic reticulum membrane protein. Then, we examined the cardiac morphology and hemodynamics of the Pitx2c-cKO mice. Results: Tissue weights of 8-week-old Pitx2c-cKO mice showed that the relative weights of both atria (especially the right atrium), right ventricle and lungs were significantly increased in Pitx2c-cKO mice, while the relative weight of the left ventricle was decreased. Furthermore, echocardiography performed on 3-month-old Pitx2c-cKO mice revealed that the right ventricle was markedly enlarged in Pitx2c-cKO mice, with enlarged pulmonary artery annulus diameter and increased velocity time integral (VTI) of blood flow through the right ventricular outflow tract. Pitx2c-cKO mice also had significantly decreased left ventricular end-diastolic diameter (LVDd), end-systolic diameter (LVDs), and mass (LVM). On the other hand, there was no difference in left ventricular fractional shortening (%FS) or tricuspid annular plane systolic excursion (TAPSE), indicating no cardiac dysfunction in Pitx2c-cKO mice. Cardiac catheterization also revealed elevated right ventricular pressure in some Pitx2c-cKO mice. Conclusion: These primary findings suggest that the Pitx2c-cKO mice may be a model for understanding the pathogenesis of pc-PH.

Abstract 13361: The Effect of Electronic Cigarette Exposure on Flow Mediated Vasodilation and LV Remodeling in a Chronic Myocardial Infarction Model

Introduction: There is little information about the effect of electronic cigarette (e-C) exposure on the cardiovascular system in the setting of myocardial infarction (MI) and whether e-C’s could alter vascular or cardiac function during the healing phase of MI. We determine the effect of e-C on endothelial function, cardiac remodeling and function during the healing phase of MI. Methods: Sprague Dawley rats (both sexes) were anesthetized and subjected to a proximal left coronary artery occlusion to induce a large anterior wall MI. At one week, rats were randomized to receive either 12 weeks of exposure to purified air (n=37) or e-cig vapor plus 15 mg/ml of nicotine (e-Cn=32). At 12 weeks endothelial function was tested by assessing femoral artery diameter and blood flow following a 5- minute femoral artery occlusion and reperfusion (ultrasound and flow probe). Cardiac function was assessed by echocardiography; remodeling was also assessed by quantitative histologic analysis. Results: Baseline blood flow in the femoral artery did not differ between groups, but peak reperfusion blood flow was blunted in the e-Cn group (1.59 ± 0.15 ml/min) vs. the air group (2.11 ± 0.18 ml/min; p=0.034). Femoral artery diameter after reperfusion was narrower in the e-Cn group (0.54 ± 0.02mm) compared to the air group (0.60 ± 0.02mm; p=0.023). Postmortem left ventricular (LV) volumes were similar in the e-Cn (0.69 ± 0.04 ml) and air groups (0.73 ± 0.04 ml; p=NS); and myocardial infarct expansion index did not differ between groups (1.4 ± 0.1 in e-Cn group versus 1.3 ± 0.1 in air group; p=NS). Gross pathology LV wall thickness was greater in the e-Cn group (1.38 ± 0.12 mm) versus the air group (1.04 ± 0.10 mm) despite no increase in blood pressure at 12 weeks in the e-CN group. LV fractional shortening by echo did not differ between groups at 12 weeks (e-Cn at 29 ± 2% and air at 27 ± 1%; p=NS). Conclusion: Exposure to e-Cn during the healing phase of MI was associated with altered endothelial function with reduced femoral artery blood flow and diameter at reperfusion, but not with worsened LV dilation or worsened cardiac function. E-Cn did increase gross pathologic LV wall thickness. E-Cn exposure during the healing phase of MI has deleterious effects on the CV system but does not dilate the LV.

Abstract 17015: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

Introduction: Protein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes. Mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L), induce protein aggregates with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. The functional relevance of this pathway in myocardial proteostasis is unknown. Hypothesis: We hypothesize that mitophagy removes cytosolic protein aggregates in cardiac myocytes. Methods: Mice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assayed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Transgenic mice expressing R120G CRYAB (R120G TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. Results: Poly-ubiquitinated protein-rich aggregates accumulate in the cytosol and within mitochondria with desmin mislocalization in TRAF2icKO myocardium. CRYAB R120G and BAG3 P209L mutant proteins are taken up by isolated mitochondria. CRYAB R120G protein aggregates co-localize with damaged mitochondria and lysosomes by Coupa imaging, and accumulate in the mitochondrial fraction in R120G TG myocardium with upregulation of TRAF2 and induction of mitophagy. Adult-onset haplo-insufficiency of Traf2 accelerated mortality (P=0.003), impaired left ventricular (LV) fractional shortening (P&lt;0.001) and worsened mitochondrial morphology in R120G TG mice. AAV9-cTnT-TRAF2 transduction delayed mortality (P=0.027), rescued LV fractional shortening (P=0.039), reduced mitochondrial protein aggregates and improved desmin localization in R120G TG mice. Conclusions: Mitophagy facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

Abstract 17881: A Predictive Algorithm to Identify Late Gadolinium Enhancement Using Echocardiography

Background: Late gadolinium enhancement (LGE) on cardiac MRI (CMR) correlates with fibrosis and arrhythmic risk, but cannot be used in several clinical settings compared the availability of echocardiography (TTE). Aim: To develop and evaluate a machine learning (ML) model that uses standard clinical and echocardiography (TTE) variables to identify LGE on paired CMR. Methods: Amongst 725 patients (mean age 56.9 ± 16.5 years, 40.3% female) with TTE and CMR within 3 months at our institution, 2018-2023, we derived and tested several ML models (recursive partitioning, random forest, boosted tree, neural networks, LASSO/Elastic Net) to identify LGE on CMR using age and 24 TTE features. Ten-fold cross validation was used for internal validation. Results: A total of 165 (23%) had the presence of LGE (40% ischemic, 60% nonischemic pattern). Of tested models, a boosted neural network performed best (validation area under the curve [AUC] = 0.98; Figure ). In the validation cohort, the algorithm had a sensitivity and specificity of 93.8% and 94.6% for identifying presence of LGE. Accuracy remained high regardless of gender (M vs. F, AUC, 0.987 vs. 0.988) or race (blacks vs. whites, AUC, 0.987 vs. 0.984) and was not related to time between studies (r = -0.06, p = 0.09). Accuracy was better for ischemic LGE (AUC = 0.94) than nonischemic LGE (AUC = 0.90). Left ventricular size, age, fractional shortening, and wall thickness, were amongst the top predictors of LGE presence in the model. Conclusions: In this single center study, a boosted neural network using TTE features identified LGE on paired CMR with high sensitivity and specificity. If confirmed in external data, this suggests possible utility of ML for improving the diagnostic value of TTE for LGE assessment.

Abstract 16213: No Cardiac Benefit After Single Dose of Intramyocardial or Intravenous Mesenchymal Stem Cell-Derived Extracellular Vesicles Post-Myocardial Infarction Supports Need for Bioengineered Extracellular Vesicles With Non-Invasive Cardiac Uptake

Introduction: In pre-clinical models, mesenchymal stem cell-derived extracellular vesicles (MSC-EV) have benefits in ischemic CVD. We previously showed that only intramyocardial (IM) injection of MSC-EV, an invasive method of administration, and not intravenous (IV) injection, resulted in reliable cardiac uptake after acute MI. No prior studies have compared the functional outcome of IM versus IV injection after acute MI. Hypothesis: IM injection of MSC-EV will result in improved cardiac outcomes whereas IV injection will not. Aim: To demonstrate the long-term outcomes of IM versus IV MSC-EV after acute MI. Methods: MSC-EV were isolated and characterized. FVB mice underwent left anterior descending coronary artery ligation and subsequent injection of the following: 1) IM saline control (IM-C; n = 8), 2) IM 2*10 9 MSC-EV particles (IM-EV; n = 9), 3) IV saline control (IV-C; n = 8), and 4) IV 2*10 9 MSC-EV particles (IV-EV; n = 8). The mice underwent pre-operative, and post-operative weekly echocardiograms. On post-operative day 28, the mice were euthanized, and the hearts were harvested. Results: Preliminary data showed no significant differences in left ventricular ejection fraction (p = 0.6151) or fractional shortening (p = 0.1135) in the IM-EV and IV-EV groups compared to the controls IM-C and IV-C, respectively, at post-operative day 28 (Figure 1A). There were also no significant differences in infarct size (p = 0.05620) (Figure 1B) or anterior border zone fibrosis (p = 0.6333) (Figure 1C) on Masson-Trichrome staining. Conclusions: Preliminary findings demonstrate that a one-time IM or IV dose of MSC-EV given immediately after acute MI results in no observable effects. Additional MSC-EV doses after MI may produce beneficial effects. Results from our ongoing studies on the creation of stem cells bioengineered to produce MSC-EV with surface cardiac targeting peptides and immune-evasion properties will be presented in August 2023.

Abstract 19126: Discovery of TMEM65 as a Positive Regulator of Mitochondrial Calcium Efflux

Introduction: The balance between mitochondrial calcium ( m Ca 2+ ) uptake and efflux regulates ATP production, but if perturbed causes m Ca 2+ depletion and energy starvation or m Ca 2+ overload and cell death. Disrupted m Ca 2+ homeostasis is implicated in ischemia-reperfusion injury, heart failure, and neurodegeneration. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of m Ca 2+ efflux in excitable tissues, supporting NCLX as a promising therapeutic target to limit pathogenic m Ca 2+ overload in organs like the heart and brain. However, a critical barrier to translation is that little is known about the mechanisms controlling NCLX activity. Goals: We used proximity biotinylation screening to identify the NCLX interactome and proteins that may regulate NCLX function. We identified TMEM65, a mitochondrial inner membrane protein of unknown function, as an NCLX-proximal protein. Hypothesis: Null mutation of TMEM65 causes mitochondrial encephalomyopathy and phenocopies genetic NCLX disruption. Therefore, we hypothesized that TMEM65 modulates m Ca 2+ efflux through NCLX. Approach: Here, we used measurements of m Ca 2+ exchange in AC16 cardiomyocytes with TMEM65 overexpression (OE) or CRISPR/Cas9 genetic disruption of TMEM65 to test TMEM65’s effect on m Ca 2+ flux and its functional dependence on NCLX. We used AAV-mediated Tmem65 knockdown (KD) and TMEM65 OE in mice to assess the effects of gain- or loss of TMEM65 expression on cardiac function. Results: TMEM65 OE potently enhanced m Ca 2+ efflux in vitro , and NCLX inhibition with CGP-37157 mitigated this effect. Knockout of TMEM65 attenuated m Ca 2+ efflux. In vivo Tmem65 KD reduced left ventricular fractional shortening and reduced S293 phosphorylation of pyruvate dehydrogenase in the heart, indicative of increased m Ca 2+ loading. In vivo TMEM65 OE did not alter cardiac function. Conclusions: We conclude that TMEM65 has an essential function to promote m Ca 2+ efflux, likely via positive regulation of NCLX, and is critical to limiting deleterious m Ca 2+ overload in the intact heart. Ongoing work is defining the functional interactions between TMEM65 and NCLX, and establishing the utility of targeting TMEM65 to restore m Ca 2+ homeostasis in heart failure and other excitable tissue diseases.