Female Hearts Research Articles

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with β-alanine buildup. Raising β-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Heart Failure, Female Sex, and Atrial Fibrillation Are the Main Drivers of Human Atrial Cardiomyopathy: Results From the CATCH ME Consortium.

Atrial cardiomyopathy (atCM) is an emerging prognostic factor in cardiovascular disease. Fibrotic remodeling, cardiomyocyte hypertrophy, and capillary density are hallmarks of atCM. The contribution of etiological factors and atrial fibrillation (AF) to the development of differential atCM phenotypes has not been quantified. This study aimed to evaluate the association between histological features of atCM and the clinical phenotype. We examined left atrial (LA, n=95) and right atrial (RA, n=76) appendages from a European cohort of patients undergoing cardiac surgery. Quantification of histological atCM features was performed following wheat germ agglutinin/CD31/vimentin staining. The contributions of AF, heart failure, sex, and age to histological characteristics were determined with multiple linear regression models. Persistent AF was associated with increased endomysial fibrosis (LA: +1.13±0.47 μm, P=0.038; RA: +0.94±0.38 μm, P=0.041), whereas total extracellular matrix content was not. Men had larger cardiomyocytes (LA: +1.92±0.72 μm, P<0.001), while women had more endomysial fibrosis (LA: +0.99±0.56 μm, P=0.003). Patients with heart failure showed more endomysial fibrosis (LA: +1.85±0.48 μm, P<0.001) and extracellular matrix content (LA: +3.07±1.29%, P=0.016), and a higher capillary density (LA: +0.13±0.06, P=0.007) and size (LA: +0.46±0.22 μm, P=0.044). Fuzzy k-means clustering of histological features identified 2 subtypes of atCM: 1 characterized by enhanced endomysial fibrosis (LA: +3.17 μm, P<0.001; RA: +2.86 μm, P<0.001), extracellular matrix content (LA: +3.53%, P<0.001; RA: +6.40%, P<0.001) and fibroblast density (LA: +4.38%, P<0.001), and 1 characterized by cardiomyocyte hypertrophy (LA: +1.16 μm, P=0.008; RA: +2.58 μm, P<0.001). Patients with fibrotic atCM were more frequently female (LA: odds ratio [OR], 1.33, P=0.002; RA: OR, 1.54, P=0.004), with persistent AF (LA: OR, 1.22, P=0.036) or heart failure (LA: OR, 1.62, P<0.001). Hypertrophic features were more common in men (LA: OR=1.33, P=0.002; RA: OR, 1.54, P=0.004). Fibrotic atCM is associated with female sex, persistent AF, and heart failure, while hypertrophic features are more common in men.

Nicotinamide Riboside, an NAD + Precursor, Protects Against Cardiac Mitochondrial Dysfunction in Fetal Guinea Pigs Exposed to Gestational Hypoxia.

Gestational hypoxia inhibits mitochondrial function in the fetal heart and placenta contributing to fetal growth restriction and organ dysfunction. NAD + deficiency may contribute to a metabolic deficit by inhibiting oxidative phosphorylation and ATP synthesis. We tested the effects of nicotinamide riboside (NR), an NAD + precursor, as a treatment for reversing known mitochondrial dysfunction in hypoxic fetal hearts. Pregnant guinea pigs were housed in room air (normoxia) or placed in a hypoxic chamber (10.5%O2) for the last 14days of gestation (term = 65days) and administered either water or NR (1.6mg/ml) in the drinking bottle. Fetuses were excised at term, and NAD + levels of maternal liver, placenta, and fetal heart ventricles were measured. Indices of mitochondrial function (complex IV activity, sirtuin 3 activity, protein acetylation) and ATP synthesis were measured in fetal heart ventricles of NR-treated/untreated normoxic and hypoxic animals. Hypoxia reduced fetal body weight in both sexes (p = 0.01), which was prevented by NR. Hypoxia had no effect on maternal liver NAD + levels but decreased (p = 0.04) placenta NAD + levels, the latter normalized with NR treatment. Hypoxia had no effect on fetal heart NAD + but decreased (p < 0.05) mitochondrial complex IV and sirtuin 3 activities, ATP content, and increased mitochondrial acetylation, which were all normalized with maternal NR. Hypoxia increased (p < 0.05) mitochondrial acetylation in female fetal hearts but had no effect on other mitochondrial indices. We conclude that maternal NR is an effective treatment for normalizing mitochondrial dysfunction and ATP synthesis in the hypoxic fetal heart.

Sex Differences in the Development of Anthracycline-Associated Heart Failure

Female sex is frequently cited as a risk factor for anthracycline cardiotoxicity based on pediatric data, but the role of sex in the development of cardiotoxicity has not been clearly established in adults. To assess the effect of female sex on the development of incident heart failure (HF) in adult patients treated with anthracyclines. This was a retrospective cohort study of 1525 adult patients with no prior history of HF or cardiomyopathy who were treated with anthracyclines between 1992 and 2019. The primary outcome was new HF within 5 years of the first dose of anthracyclines. The effect of sex was assessed using Cox proportional hazards and competing risk models. Over a median (IQR) follow-up of 1.02 (0.30-3.01) years, 4.78% of patients developed HF (44 men and 29 women). Female sex was not associated with the primary outcome in a multivariable Cox proportional hazards model (HR 0.87; 95% CI 0.53-1.43; P = 0.58). Similar results were observed in a multivariable model accounting for the competing risk of death (HR 0.94; 95% CI 0.39-2.25; P = 0.88). Age, coronary artery disease and hematopoietic stem cell transplant were associated with the primary outcome in a multivariable Cox proportional hazards model. Age and body mass index were associated with the primary outcome in a multivariable competing risk model. In this large, single-center, retrospective cohort study, female sex was not associated with incident HF in adult patients treated with anthracyclines. Female sex is frequently cited as a risk factor for anthracycline cardiotoxicity based on pediatric data, but the role of sex in the development of cardiotoxicity has not been clearly established in adults. In this retrospective cohort study, we assessed the effect of female sex on the development of incident heart failure in adult patients treated with anthracyclines. Using Cox proportional hazards and competing risk regression models, we found that there was no association between female sex and heart failure after treatment with anthracyclines.

Cardioprotective O-GlcNAc signaling is elevated in murine female hearts via enhanced O-GlcNAc transferase activity

The post-translational modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc-cycling in response to I/R injury have not been comprehensively assessed. The cycling of O-GlcNAc is regulated by the collective efforts of two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and hydrolysis of O-GlcNAc respectively. It has previously been shown that baseline heart physiology and pathophysiology are impacted by sex. Here, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation both basally and in ischemic hearts. To address this question, we subjected male and female wild type murine hearts to ex vivo ischemia or ischemia-reperfusion injury. We assessed hearts for protein O-GlcNAcylation, abundance of OGT, OGA, and glutamine:fructose-6-phosphate aminotransferase (GFAT2), activity of OGT and OGA, and UDP-GlcNAc levels. Our data demonstrate elevated O-GlcNAcylation in female hearts both basally and during ischemia. We show OGT activity was enhanced in female hearts in all treatments, suggesting a mechanism for these observations. Furthermore, we found ischemia led to reduced O-GlcNAcylation and OGT specific activity. Our findings provide a foundation for understanding molecular mechanisms that regulate O-GlcNAcylation in the heart and highlight the importance of sex as a significant factor when assessing key regulatory events that control O-GlcNAc-cycling. These data suggest the intriguing possibility that elevated O-GlcNAcylation in females contributes to reduced ischemic susceptibility.

Abstract 19080: The Ketogenic Diet Aggravates the Ischemic Response in Mouse Hearts

Ketone bodies, particularly beta-hydroxybutyrate (B-OHB), have been identified as an important fuel source during physiological and pathological stress. Although controversial, consumption of a ketogenic diet (KD) may be used as a method to increase B-OHB availability. Since the KD is a high-fat, low carbohydrate diet, the diet may have harmful effects on the heart especially during ischemic injury. Therefore, the objective of this study was to investigate the effects of short- term consumption of the KD on cardiac ischemia-reperfusion injury. Male (n=14) and female (n=13) were randomly assigned to KD (90% calories from fat) or normal chow diet. Serum B-OHB and cholesterol were significantly higher in both male and females fed the KD. KD did not increase body weight in male and female mice but significantly increased adipose tissue mass by 2.7 and 1.9 fold, respectively. KD increased hepatic triglyceride content in both males and females and increased cardiac triglycerides only in male mice. Cardiac function was assessed in isolated perfused hearts on a mixed substrate buffer consisting of 0.4mM fatty acids, 5.5mM glucose, 50uU/ml insulin, 1.2mM lactate, and 0.5mM B-OHB. Baseline function was monitored for 15 minutes followed by 18 minutes of global ischemia and 36 minutes of reperfusion. Baseline LV developed pressure (LVDevP) and heart rate (HR) were not affected by the KD in either male or female mice. During ischemia, the end diastolic pressure (EDP) was significantly higher in both male and female KD hearts. Time to peak contracture was accelerated in male and female KD hearts (P&lt;0.05 vs chow), with a delay in female hearts (13.12 ± 1.06 vs. 9.27 ± 0.80 min, P&lt;0.05 vs KD male). At the end of reperfusion, rate pressure product (RPP, the product of LVDevP and HR) was lower in male KD vs male chow (P&lt;0.05) whereas RPP was similar in female KD and female chow. Moreover, RPP in female KD was significantly higher than male KD (6190 ± 929 vs 2133 ± 533 mmHg/min). These findings suggest that the KD may increase vulnerability of the heart to ischemia and may reduce recovery from ischemia, particularly in males. Overall, this study highlights potential sex differences and negative consequences of short-term consumption of the KD.

Abstract 17153: Mitochondrial Cx43 and Gja1-20k Contribute to Sex-Specific Differences of Mitochondrial Response in Septic Cardiomyopathy

Introduction: Cardiac dysfunction contributes to morbidity and mortality of severe sepsis. Preserving mitochondrial (mito) activity is key to maintaining cardiac function. While epidemiological studies consistently report that male sex is an important risk factor for severe sepsis, it is unknown if male heart mito are susceptible to septic injury. We have shown that male cardiomyocyte mito were more vulnerable to TNFα- or H 2 O 2 -induced stress than females. We have also demonstrated that mito connexin 43 (Cx43 or Gja1) and its smaller isoform - Gja1-20k are implicated in sex-related heart response following myocardial ischemia. Hypothesis : In this study, we hypothesize that during sepsis, female (F) hearts exhibit preserved mito Cx43 and Gja1-20k to confer mitochondrial protection, thus improving cardiac function compared to male (M) myocardium. Methods: Age-matched adult M &amp; F C57BL/6 mice received LPS (7.5 mg/kg BW, i.p.) or PBS (n &gt; 6 mice/group). Cardiac function was detected 20h post LPS by 2D echocardiography. Cx43, Gja1-20k, OXPHOS, &amp; PGC-1 were evaluated in M &amp; F mouse heart mito 24h after LPS by Western Blot (n&gt;3 hearts/group). Mito Cx43 was also detected by Immuno-electron microscopy (EM). Mito membrane potential (ΔΨm) was analyzed in cardiomyocytes (from adult M &amp; F mice, n&gt;3 hearts/group) +/- 1h LPS (1 μg/ml) using JC1 fluorescent probe. Mito respiration capacity was further assessed in H9c2 cells transfected with Gja1 siRNA upon LPS exposure by Seahorse XF Mito Stress test. Results: Decreased cardiac function was observed in M mice after LPS injection, but not in F. Higher mito levels of Cx43, Gja1-20k, OXPHOS, &amp; PGC1 were noticed in F mouse hearts compared to M after LPS. Despite no difference in total Cx43 particles between M &amp; F hearts by EM, more gold Cx43 particles were observed in F heart mito compared to M mito following LPS. LPS reduced ΔΨm in M cardiomyocytes, but not in F. Cx43 knockdown decreased mito maximal respiration capacity &amp; ATP production in H9c2 cells with LPS stress. Conclusions: The mitochondria-derived protective mechanism is preserved in F mice, leading to better cardiac function than M mice following LPS-induced sepsis. F cardiac mito are more resistant to LPS challenge than M mito, partially due to preserved mito Cx43 and/or Gja1-20k.

Abstract 16891: High Molecular Weight Hyaluronan Inhibits Macrophage Phagocytosis

Background: After a myocardial infarction (MI), macrophages are recruited to the site of injury to begin the process of wound healing. Macrophages are important because they clear cellular debris via phagocytosis. This period of acute inflammation in the heart coincides with extracellular matrix (ECM) remodeling. Hyaluronan (HA), one of the most abundant ECM components, accumulates in a high molecular weight form (HA HMW ). The relationship between HA HMW accumulation and macrophage function in is not fully understood. Goal: To determine the extent to which hyaluronan impacts phagocytosis in polarized macrophages. Methods and Results: Tissue sections were collected from mice one-week post-MI and stained for hyaluronan binding protein (HABP) to assess HA accumulation. A significant increase in HA was seen in both male (n=4, p=0.0031) and female (n=4, p=0.0210) MI hearts compared to sham hearts. Separately, bone marrow was isolated from naïve mice and cells were treated in culture with macrophage colony stimulating factor (M-CSF) for 7-10 days. The resultant macrophages were then polarized into a pro-inflammatory (M1) phenotype using LPS and INF-γ, or pro-reparative phenotype (M2) using IL-4 and IL-13. Macrophage polarization was confirmed via qRT-PCR. In additional experiments, macrophages were polarized for 24 hours and then incubated for 30 min with FITC-labeled, IgG-coated latex beads in the presence or absence of 1 μM HA HMW . Flow cytometry was used to assess phagocytosis of the beads. HA HMW treatment resulted in a significant reduction in the percent of FITC positive cells in M1 female (n=6, p=0.005), M2 female (n=5, p=0.0406), and M2 male (n=5, p=0.0314) macrophage subsets indicating a decrease in phagocytosis. No significant changes were seen in the M0 and M1 male macrophage subsets. Additionally, the mean fluorescence intensity (MFI) measured in the FITC channel was significantly decreased in all the female subsets as well as the M2 male macrophages (n=5-6, p&lt;0.05). No significant changes were seen in the M0 and M1 male subsets. Conclusion: HA HMW impairs opsonin-induced phagocytosis in macrophages. Given that HA HMW accumulates post-MI, these findings may partially explain the incomplete immune response in the failing heart.

Abstract 12479: Cardiovascular Stroke Nursing Best Abstract Award: Sex Differences in Relationships Between Inflammation and Symptoms in Persons With Heart Failure

Background: Heart failure (HF) is a complex syndrome involving interplay of pathologic processes, such as inflammation and myocardial remodeling. Sex differences in cardiac remodeling and function are linked to differences in inflammation. Female hearts show a pro-inflammatory shift and increased cytokine expression with aging not seen in male hearts. Yet, sex is not often considered in HF interventions. Previous studies suggest sex may moderate the relationship between inflammation and symptoms in cardiometabolic disease, but the relationship in HF is unknown. Purpose: To examine sex differences in inflammation and symptoms in Black adults living with HF. Methods: Black adults living with HF (N=41) were enrolled in this pilot study. Physical symptoms were measured with PROMIS Dyspnea, Heart Failure Somatic Perception Scale (HFSPS), and Multidimensional Fatigue Inventory (MFI). Mood symptoms were measured with Center for Epidemiological Studies Depression Scale (CESD), State-Trait Anxiety Inventory (STAI), and Perceived Stress Scale (PSS). Inflammatory biomarkers were measured via multiplex immunoassay. Xanthine oxidase (XO) activity, an indicator of oxidative stress, was measured via ELISA. Pearson correlations were stratified by sex. Results: The mean age was 57 ± 11 years, and 66% were female. Dyspnea was positively associated with XO activity in both groups. Among females, dyspnea was positively associated with matrix metalloproteinase (MMP)-12 (r=.519, p=.033), interleukin (IL)-6 (p=.564, p=.018), [C-C motif chemokine ligand(CCL)-3, (r=.509, p=.037), and CCL-11 (r=.520, p=.032). HFSPS was positively associated with XO activity among females only (r=.445, p=.014). CCL-11 was positively associated with MFI (r=.616, p=.008), PSS (p=.0537, r=.032), STAI (r=.714, p=.001), and CESD (r=.635, p=.008) symptoms among females but not males. Conclusions: While physical symptoms were associated with similar inflammatory pathways, fatigue and mood symptoms were associated with inflammation among females only. Women may be more vulnerable to inflammation-induced mood changes and fatigue. Further work is needed to better understand how sex modulates the relationship between inflammation and symptoms in persons with HF.

Sex-Specific Limitation of Cardiac Capacity During the Adult Life Span: Return to Fundamental Structure and Function.

Physiology underlying reduced cardiac pumping capacity in women compared with men and its interaction with aging remains unresolved. Herein, the pressure gradient (PG) driving venous return was manipulated to evidence whether cardiac structure and/or function explain sex differences in cardiac capacity. Healthy women/men matched by age and physical activity were included within young (n = 40, age = 25 ± 4 years) and older (n = 55, age = 60 ± 8 years) groups. Cardiac volumes/output (Q) were assessed up-to-peak exercise under 2 hemodynamic conditions ("low"/"high" PG between lower/upper body). Main outcomes included sex differences in delta ("high" - "low" PG) left ventricular (LV) end-diastolic volume (∆LVEDV), stroke volume (∆SV), and Q (∆Q). In young individuals, "high"-PG increased exercise LVEDV and SV in men (p ≤ .002), but not in women (p ≥ .562), relative to "low"-PG (control condition). Accordingly, peak ∆LVEDV, ∆SV, and ∆Q were enhanced in young men versus young women (p ≤ .019). Notwithstanding, right/left atrial volumes during exercise were similarly increased by "high"-PG in both young sexes (p ≤ .007). "High"-PG exclusively prolonged moderate exercise LV filling time in young men (p ≤ .036). In older individuals, "high"-PG did not modify exercise cardiac volumes and reduced LV diastolic function (p ≤ .049). In conclusion, the female young heart is unrestrained by venous return or structural factors external to the myocardium. As determined during moderate exercise, impaired LV filling time lengthening limits female-specific cardiac capacity. With older age, cardiac chambers are not distended and LV relaxation is impaired with increased PG in both sexes. During early but not late adulthood, a functional LV limitation may explain sex differences in cardiac capacity.

Asian Female Athlete's Heart: The CHIEF Heart Study.

The cardiac characteristics of Asian female endurance athletes and strength athletes have rarely been investigated. This study included 177 Taiwanese young women undergoing military training. Cardiac features were assessed by electrocardiography (ECG) and echocardiography. Then, all participants completed a 3000-meter run to assess endurance capacity, and 89 participants completed a 2-minute push-up test to assess muscular strength. Athletes were those whose exercise performance fell one standard deviation above the mean, and the remaining participants were defined as controls. Multiple logistic regression analysis was used to determine the predictors of the cardiac characteristics of female athletes. Compared to the female controls, female endurance athletes had a greater QRS duration (ms) (92.12 ± 10.35 vs. 87.26 ± 9.89, p = 0.01) and a higher prevalence of right axis deviation (RAD) (34.9% vs. 11.1%, p < 0.001). There were no differences in any echocardiographic parameters. Greater QRS duration and RAD and lower systolic blood pressure were independent predictors of female endurance athletes [odds ratios (OR) and 95% confidence intervals: 1.05 (1.01-1.09), 2.91 (1.12-7.59) and 0.93 (0.88-0.98), respectively]. Female strength athletes had a greater right ventricular outflow tract (RVOT) (mm) (28.06 ± 3.57 vs. 25.38 ± 3.61, p = 0.007) but revealed no differences in ECG variables. Greater RVOT was the only predictor of female strength athletes [OR: 1.26 (1.05-1.50)]. In Asian military women, a wider QRS duration and the presence of RAD in ECG rather than heart structure and function were found to characterize endurance athletes, whereas a wider RVOT but no ECG features were found to characterize strength athletes.

Risk factors for long COVID in children and adolescents: a systematic review and meta-analysis.

The long-term sequelae of COVID-19 in children and adolescents remain poorly understood and characterized. This systematic review and meta-analysis sought to summarize the risk factors for long COVID in the pediatric population. We searched six databases from January 2020 to May 2023 for observational studies reporting on risk factors for long COVID or persistent symptoms those were present 12 or more weeks post-infection using multivariable regression analyses. Trial registries, reference lists of included studies, and preprint servers were hand-searched for relevant studies. Random-effects meta-analyses were conducted to pool odds ratios for each risk factor. Individual study risk of bias was rated using QUIPS, and the GRADE framework was used to assess the certainty of evidence for each unique factor. Sixteen observational studies (N = 46,262) were included, and 19 risk factors were amenable to meta-analysis. With moderate certainty in the evidence, age (per 2-year increase), allergic rhinitis, obesity, previous respiratory diseases, hospitalization, severe acute COVID-19, and symptomatic acute COVID-19 are probably associated with an increased risk of long COVID. Female sex, asthma, comorbidity, and heart diseases may be associated with an increased risk of long COVID, and Asian and Black races may be associated with a decreased risk of long COVID. We did not observe any credible subgroup effects for any risk factor. The current body of literature presents several compelling risk factors for the development of long COVID in the pediatric population. Further research is necessary to elucidate the pathophysiology of long COVID.

Sex differences and estrogen effects in cardiac mitochondria in human aortic stenosis and in the mouse heart.

Sex differences in the adaptation to pressure overload have been described in humans, as well as animal models, and have been related to sex-specific expression of mitochondrial genes. We therefore tested whether sex differences in cardiac mitochondrial respiration exist in humans with aortic stenosis (AS). We also examined whether these potential differences may be at least partially due to sex hormones by testing if mitochondrial respiration is affected by estrogen (17ß-estradiol (E2)). Consecutive patients undergoing transapical aortic valve implantation (TAVI) (women, n = 7; men, n = 10) were included. Cardiac biopsies were obtained during TAVI and used directly for mitochondrial function measurements. Male and female C57BL/6J mice (n = 8/group) underwent sham surgery or gonadectomy (GDX) at the age of 2 months. After 14 days, mice were treated once with intraperitoneally injected vehicle (placebo), 17ß-estradiol (E2), estrogen receptor alpha (ERα) agonist [propyl pyrazole triol (PPT)], or ER beta (ERβ) agonist (BAY-1214257). Thereafter, mitochondrial measurements were performed directly in cardiac skinned fibers from isolated left ventricles and musculus solei. Mitochondrial State-3 respiration was higher in female than that in male human heart biopsies (15.0 ± 2.30 vs. 10.3 ± 2.05 nmol/mL/min/mg, p< 0.05). In the mouse model, mitochondrial State-3 respiration decreased significantly after GDX in female (27.6 ± 1.55 vs. 21.4 ± 1.71 nmol/mL/min/mg; p< 0.05) and male hearts (30.7 ± 1,48 vs. 23.7 ± 2,23 nmol/mL/min/mg; p< 0.05). In ovariectomized female mice, E2 and ERβ-agonist treatment restored the State-3 respiration to intact placebo level, whereas ERα-agonist treatment did not modulate State-3 respiration. The treatment with E2, ERα-, or ERβ-agonist did not modulate the State-3 respiration in GDX male mice. We identified sex differences in mitochondrial respiration in the diseased human heart. This is in alignment with known sex differences in the gene expression and proteome level at the functional level. E2 and ERβ affect cardiac mitochondrial function in the mouse model, suggesting that they may also contribute to the sex differences in the human heart. Their roles should be further investigated.

Major Cardiovascular Events after Chemoradiotherapy with or without Durvalumab in Patients with Stage III Non-Small Cell Lung Cancer: Supplementary Analysis

RTOG 0617 showed that cardiac events are relatively common after high-dose thoracic radiotherapy. The aim of this study was to investigate the incidence and risk of major cardiovascular events (MACE) after concurrent chemoradiotherapy (CCRT) with or without durvalumab in patients with stage III non-small cell lung cancer (NSCLC) using the data from a multi-institutional study in Japan. Patients who received CCRT for stage III NSCLC between July 2018 and July 2019 were enrolled in a multi-institutional study in Japan. MACE was defined as follows: symptomatic pericardial effusion, acute coronary syndrome, pericarditis, significant arrhythmia, and heart failure. The cumulative incidence of MACE, accounting for death as a competing risk, was calculated. Pre-existing coronary heart disease (CHD) included coronary artery disease, congestive heart failure, peripheral vascular disease, stroke, and extensive coronary artery calcification. The association between patient/treatment-related factors and MACE was assessed by multivariate analysis. Among 178 patients with a median follow-up period of 42.5 months, 13 patients developed MACEs. The 3-year cumulative incidence of MACE was 6.9% (95% confidence interval [CI], 4.0-11.9%). Univariate analysis showed that female sex and mean heart dose (MHD) were marginally associated (3-year cumulative incidence, male 5.6% vs. female 12.1%; P = 0.12; MHD ≥ 6.3 Gy 4.8% vs. < 6.3 Gy 9.1%; P = 0.13), and pre-existing CHD was significantly associated with an increased risk of MACE (no CHD 4.3% vs. CHD 16.8%; P = 0.026). Consolidation durvalumab was not associated with an increased risk of MACE (no durvalumab 5.2% vs. durvalumab 7.4%; P = 0.89). Multivariate analysis showed that pre-existing CHD was significantly associated with MACE (hazard ratio, 4.22; 95% CI, 1.30-13.7; P = 0.016). The incidence of MACE based on the real-world data in Japan was lower than previously reported. Pre-existing CHD was associated with an increased risk of MACE after CCRT in patients with stage III NSCLC, whereas the administration of consolidation durvalumab was not associated with an increased risk of MACE.

Interleukin-36 is vasculoprotective in both sexes despite sex-specific changes in the coronary microcirculation response to IR injury.

Risks and outcomes of myocardial infarction (MI) are different between men and women and some studies have demonstrated that the latter have a higher risk of mortality. Whilst there are many reasons for this, it may also partially be linked to stronger innate and adaptive immune responses mounted by females compared to males. However, little is known about how sex impacts the coronary microvessels, the site where inflammatory processes take place, after an MI. Intravital and laser speckle microscopy was used to image coronary microvessels and ventricular perfusion in vivo in response to myocardial ischaemia-reperfusion (IR) injury in male and female mice. Interleukin-36 (IL-36) is the latest addition to the IL-1 superfamily of pro-inflammatory cytokines and has recently been shown to mediate inflammation in a number of non-cardiovascular diseases. Its role in mediating potential sex-related microcirculatiory pertubations in the heart are unknown. Therefore, the vasculoprotective efficacy of an IL-36 receptor antagonist (IL-36Ra) was also investigated. Immunostaining and flow cytometry demonstrated higher expression of IL-36 and its receptor in female hearts, an observation confirmed in human samples. Intravital imaging of the anaesthetised mouse beating heart identified significantly greater neutrophil recruitment in female hearts, but a greater burden of thrombotic disease in male hearts. Male mice had reduced functional capillary density and were unable to restore perfusion to baseline values as effectively as females. However, female mice had significantly larger infarcts. Interestingly, IL-36Ra decreased inflammation, improved perfusion, and reduced infarct size in both sexes despite increasing platelet presence in male hearts. Mechanistically, this was explained by IL-36Ra attenuating endothelial oxidative damage and VCAM-1 expression. Importantly, IL-36Ra administration during ischaemia was critical for vasculoprotection to be realised. This novel study identified notable sex-related differences in the coronary microcirculatory response to myocardial IR injury which may explain why some studies have noted poorer outcomes in women after MI. Whilst contemporary MI treatment focuses on anti-platelet strategies, the heightened presence of neutrophils in female IR injured coronary microvessels necessitates the development of an effective anti-inflammatory approach for treating female patients. We also emphasise the importance of early intervention during the ischaemic period in order to maximise therapeutic effectiveness.

Estrogen-mediated mitigation of cardiac oxidative stress in ovariectomized rats is associated with upregulated cardiac circadian clock Per2 and heart-specific miRNAs

AimEstrogen (E2) confers cardioprotection in premenopausal women and in models of menopause and its effects, mostly studied in female reproductive organs, vary on a circadian rhythm basis in relation to the circadian clock genes. However, it remains unknown if a similar circadian pattern exists in the female heart in a manner that explains, at least partly, the cardioprotective effect of E2. The aim of the present investigation was to determine if upregulation of the circadian clock Per2 and its regulated heart-specific miRNAs, and redox enzymes contribute to the E2-mediated cardioprotection in ovariectomized rats. Main methodsRats were subjected to ovariectomy (OVX) 2-weeks prior to a 2-week E2 treatment. On the last treatment day, hearts were collected every 4 h. for ex-vivo biochemical measurements. In parallel studies, telemetric mean arterial pressure (MAP) was obtained at the tissue collection times. Key findingsOVX + E2 rats exhibited lower body weight during daytime and MAP during day and night times, and their hearts exhibited: (1) higher Per2 protein abundance, cardioprotective miRNAs (miRNA1, miRNA133a, miRNA208a, miRNA499), mALDH2, and catalase; (2) lower reactive oxygen species, cardio-detrimental miRNA652, carbonyl, MDA and HO-1 levels. The reciprocal Per2/HO-1 relationship was more evident during the daytime and correlated with the upregulated cardioprotective miRNAs in OVX + E2 rats. Finally, cardiac Per2, heart-specific miRNAs and reactive oxygen species levels and redox enzymes activities were similar in normal female and OVX + E2 rats. SignificanceEnhancement of cardiac Per2, redox enzymes and heart-specific miRNAs likely contribute to E2-mediated mitigation of cardiac oxidative stress in OVX rats.

Abstract P3206: Sex-specific Differences In Proteasome Activity And Mitochondrial Function In Mouse Hearts Lacking Natriuretic Peptide Receptor A

Objective: Atrial natriuretic peptide (ANP) is a cardiac hormone that plays an important role in cardiovascular function and energy metabolism by signaling through natriuretic peptide receptor A (NPRA). Genetic ablation of NPRA in mice resulted in hypertension, cardiac hypertrophy, and fibrosis. The cardiovascular phenotypes were reported to be more severe in male NPRA-knockout (KO) mice. It is known that reductions in proteasomal activities can lead to mitochondrial dysfunction. Similarly, an imbalance between mitochondrial fission and fusion events can impair mitochondrial function. We hypothesized that alterations in proteasomal function and mitochondrial dynamics might form the basis for phenotype differences reported for NPRA-KO mice. Method/Results: Non-invasive echocardiography, RT-QPCR, Western blotting, and proteasomal assays were performed to evaluate cardiac functions. Preliminary results revealed significant decreases in the activities of two proteasome subunits (β1 and β5) in male and female NPRA-KO hearts compared to sex-matched wild-type (WT) hearts. Notably, the percent-activity reductions were much higher in male NPRA-KO hearts. Western blotting analysis revealed that the expression levels of mitochondrial fusion proteins (Mfn1, Mfn2, and Opa1) were significantly increased in both male and female NPRA-KO hearts compared to WT controls. However, the expression of fission proteins (Drp1 &amp; MFF) was significantly increased only in male NPRA-KO hearts. Consistent with a more imbalance in mitochondrial dynamics in male NPRA-KO hearts, the expression levels of enzymes that promote fatty acid oxidation (ECHS1 and phospho-PDH) were unfavorably altered in male but not in female NPRA-KO hearts compared to controls. In addition, various echocardiographic and electrocardiographic parameters related to the cardiac function such as cardiac output, diastolic volume, left ventricular wall thickness, QRS duration, and stroke volume, were altered significantly more in male than in female NPRA-KO hearts. Conclusion: Our findings suggest that more pronounced sex-specific alterations in proteasome/mitochondrial functions in male NPRA-KO hearts can lead to changes in cardiac metabolism and a severe disease phenotype.

Abstract P3106: Sex-specific Limitation Of Cardiac Capacity: Return To Fundamental Structure And Function.

Objective: The physiology underlying the sex-specific limitation of cardiac pumping capacity remains unresolved. This study experimentally manipulated the pressure gradient (PG) driving venous return to evidence whether cardiac structure and/or function explain sex differences in cardiac capacity in young and older individuals. Methods: Healthy women and men matched by age and physical activity were included within young ( n =40, age=25±4 yr) and older ( n =55, age=60±8 yr) age groups. Cardiac volumes and output (Q) were assessed via transthoracic echocardiography from moderate to peak exercise under two hemodynamic conditions (‘low’ and ‘high’ PG between lower and upper body) generated via opposite PG identically delivered in a lower body pressure chamber. Main outcomes included sex differences in delta (‘high’ ‘low’ PG) left ventricular (LV) end-diastolic volume (ΔLVEDV), stroke volume (ΔSV) and Q (ΔQ). Results: In young individuals, ‘high’ PG increased exercise LVEDV and SV in men ( P ≤0.002), but not in women ( P ≥0.562), relative to ‘low’ PG. Accordingly, ΔLVEDV, ΔSV and ΔQ were enhanced in young men compared with young women, specifically at the highest exercise intensities ( P ≤0.019). In contrast, right and left atrial volumes were similarly increased by ‘high’ PG in both sexes ( P ≤0.007). ‘High’ PG exclusively prolonged LV filling time during exercise in young men ( P ≤ 0.036). In older women and men, ‘high’ PG did not modify exercise cardiac volumes ( P ≥0.124) and impaired established markers of LV diastolic function ( P ≤0.049). Conclusions: The female young heart is not constrained by venous return or structural factors external to the myocardium. An impaired ability to lengthen LV filling time with augmented venous return may limit female-specific cardiac capacity. With older age, cardiac chambers are not distended and LV diastolic function is impaired with increased PG in both sexes.

Abstract P3124: Chronic Infusion Of Angiotensin II Promotes Ventricular Fibrosis And Diastolic Dysfunction In Aging Male But Not Female Mouse Hearts

Heart failure prevalence increases with age and affects men and women differently. Chronic activation of the renin-angiotensin system (RAS) in heart failure increases circulating angiotensin II (AII) levels, which elevates blood pressure (BP), promotes fibrosis and leads to adverse remodelling of the heart. Whether maladaptive responses to AII differ between the sexes at older ages is unclear, as most studies use young male animal models. We investigated whether adverse cardiac remodeling induced by chronic exposure to AII differed between the sexes in older C57BL/6 mice. Osmotic minipumps with pressor doses of AII (3 mg/kg/day) or saline were implanted into older mice (≈16 mos) of both sexes for 6 weeks. BP (tail cuff) and ventricular structure/function (echocardiography) were measured at baseline, midpoint, and endpoint. Ventricular fibrosis markers (transforming growth factor-ß (TGF-ß), fibronectin collagen I, collagen III) were quantified with qPCR. Mean arterial pressure increased similarly in both sexes (76.3±4.2 vs. 136.1±4.6 mmHg in males and 73.9±2.9 vs. 116.1±8.7 mmHg in females; p&lt;0.05; n=6-8/group). AII infusion also increased LV mass and reduced ejection fraction in males and females. By contrast, AII increased signs of diastolic dysfunction in males but had little impact on diastolic function in females. For example, isovolumic relaxation time increased over 6 weeks of AII infusion in males (11.7±1.0 vs. 19.0±1.4 ms; p&lt;0.05) but not in females (14.6±0.9 vs. 16.4±1.2 ms). Similar results were seen for E/A ratios. Molecular studies showed a marked increase in mRNA abundance for TGF-ß in male ventricles (1.02±0.13 vs. 1.50±0.16; p&lt;0.05) but not in females (1.01±0.08 vs. 1.25±0.10). Similar male-female differences were seen for collagens I and III (fibronectin was similar in both sexes, and not affected by AII). As TGF-ß signalling increases the production of profibrotic mediators like collagens I and III, these findings suggest that chronic RAS activation increases fibrosis and disrupts diastolic function in aging male hearts. By contrast, older female hearts are resistant to these deleterious effects of RAS activation. These sex-specific differences may modify heart failure expression and responses to treatment in older men and women.

Assessment of coronary microcirculation alterations in a porcine model of no-reflow using ultrasound localization microscopy: a proof of concept study.

Coronary microvascular obstruction also known as no-reflow phenomenon is a major issue during myocardial infarction that bears important prognostic implications. Alterations of the microvascular network remains however challenging to assess as there is no imaging modality in the clinics that can image directly the coronary microvascular vessels. Ultrasound Localization Microscopy (ULM) imaging was recently introduced to map microvascular flows at high spatial resolution (∼10μm). In this study, we developed an approach to image alterations of the microvascular coronary flow in exvivo perfused swine hearts. A porcine model of myocardial ischemia-reperfusion was used to obtain microvascular coronary alterations and no-reflow. Four female hearts with myocardial infarction in addition to 6 controls were explanted and placed immediately in a dedicated preservation and perfusion box manufactured for ultrasound imaging. Microbubbles (MB) were injected into the vasculature to perform Ultrasound Localization Microscopy (ULM) imaging and a linear ultrasound probe mounted on a motorized device was used to scan the heart on multiple slices. The coronary microvascular anatomy and flow velocity was reconstructed using dedicated ULM algorithms and analyzed quantitatively. We were able to image the coronary microcirculation of exvivo swine hearts at a resolution of tens of microns and measure flow velocities ranging from 10mm/s in arterioles up to more than 200mm/s in epicardial arteries. Under different aortic perfusion pressures, we measured in large arteries of a subset of control hearts an increase of flow velocity from 31±11mm/s at 87mmHg to 47±17mm/s at 132mmHg (N=3 hearts, P<0.05). This increase was compared with a control measurement with a flowmeter in the aorta. We also compared 6 control hearts to 4 hearts in which no-reflow was induced by the occlusion and reperfusion of a coronary artery. Using average MB velocity and average density of MB per unit of surface as two ULM quantitative markers of perfusion, we were able to detect areas of coronary no-reflow in good agreement with a control anatomical pathology analysis of the cardiac tissue. In the no-reflow zone, we measured an average perfusion of 204±305MB/mm2 compared to 3182±1302MB/mm2 in the surrounding re-perfused area. We demonstrated this approach can directly image and quantify coronary microvascular obstruction and no-reflow on large mammal perfused hearts. This is a first step for noninvasive, quantitative and affordable assessment of the coronary microcirculation function and particularly coronary microvascular anatomy in the infarcted heart. This approach has the potential to be extended to other clinical situations characterized by microvascular dysfunction. This study was supported by the French National Research Agency (ANR) under ANR-21-CE19-0002 grant agreement.