Association of epicardial adipose tissue volume with heart weight in post-mortem cases.

Epicardial adipose tissue (EAT) deposition has been long associated with heart weight. However, recent research has failed to replicate this association. We aimed to determine the association of EAT volume with heart weight in post-mortem cases and identify potential confounding variables. EAT volume derived from post-mortem computed tomography (PMCT) and heart weight were measured in post-mortem cases (N = 87, age: 56 ± 16 years, 28% female). Cases with hypertrophied heart weights (N = 44) were determined from reference tables. Univariable associations were tested using Spearman correlation and simple linear regression. Independence was determined with stepwise regression. In the total cohort, EAT volume (median 66 ± 45 cm3) was positively associated with heart weight (median 435 ± 132 g) at the univariable level (r = 0.6, P < 0.0001) and after adjustment for age, female sex, and various body size metrics (R2 adjusted = 0.41-0.57). Median EAT volume was 1.9-fold greater in cases with hypertrophic hearts (P < 0.0001) but with considerably greater variability, especially in cases with extreme EAT volume or heart weight. As such, EAT volume was not associated with heart weight in hypertrophic cases, while a robust independent association was found in non-hypertrophic cases (R2 adjusted = 0.62-0.86). EAT mass estimated from EAT volume found that EAT comprised approximately 13% of overall heart mass in the total cases. This was significantly greater in cases with hypertrophy (median 15.5%; range, 3.6-36.6%) relative to non-hypertrophied cases (12.5%, 3.3-24.3%) (P = 0.04). EAT volume is independently and positively associated with heart weight in post-mortem cases. Excessive heart weight significantly confounded this association.

Cardiac effects of myoregulin in ischemia-reperfusion.

Myoregulin is a recently discovered micropeptide that controls calcium levels by inhibiting the intracellular calcium pump sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). Keeping calcium levels balanced in the heart is essential for normal heart functioning, thus myoregulin has the potential to be a crucial regulator of cardiac muscle performance by reducing the rate of intracellular Ca2+ uptake. We provide the first report of myoregulin mRNA expression in human heart tissue, absence of expression in human plasma, and the effects of myoregulin on cardiac hemodynamics in an ex vivo Langendorff isolated rat heart model of ischemia/reperfusion. In this preliminary study, myoregulin provided a cardio-protective effect, as assessed by preservation of left ventricular contractility and relaxation, during ischemia/reperfusion. This study provides the foundation for future research in this area.

An arrhythmogenic metabolite in atrial fibrillation.

BACKGROUND: Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its in vitro effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting. METHODS AND RESULTS: Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF. CONCLUSION: Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF.

Identifying sex differences in predictors of epicardial fat cell morphology.

Predictors of overall epicardial adipose tissue deposition have been found to vary between males and females. Whether similar sex differences exist in epicardial fat cell morphology is currently unknown. This study aimed to determine whether epicardial fat cell size is associated with different clinical measurements in males and females. Fat cell sizes were measured from epicardial, paracardial, and appendix adipose tissues of post-mortem cases (N= 118 total, 37 females). Epicardial, extra-pericardial, and visceral fat volumes were measured by computed tomography from a subset of cases (N= 70, 22 females). Correlation analyses and stepwise linear regression were performed to identify predictors of fat cell size in males and females. Median fat cell sizes in all depots did not differ between males and females. Body mass index (BMI) and age were independently predictive of epicardial, paracardial, and appendix fat cell sizes in males, but not in females. Epicardial and appendix fat cell sizes were associated with epicardial and visceral fat volumes, respectively, in males only. In females, paracardial fat cell size was associated with extra-pericardial fat volume, while appendix fat cell size was associated with BMI only. No predictors were associated with epicardial fat cell size in females at the univariable or multivariable levels. To conclude, no clinical measurements were useful surrogates of epicardial fat cell size in females, while BMI, age, and epicardial fat volume were independent, albeit weak, predictors in males only.

Estimating heart mass from heart volume as measured from post-mortem computed tomography.

Heart mass can be predicted from heart volume as measured from post-mortem computed tomography (PMCT), but with limited accuracy. Although related to heart mass, age, sex, and body dimensions have not been included in previous studies using heart volume to estimate heart mass. This study aimed to determine whether heart mass estimation can be improved when age, sex, and body dimensions are used as well as heart volume. Eighty-seven (24 female) adult post-mortem cases were investigated. Univariable predictors of heart mass were determined by Spearman correlation and simple linear regression. Stepwise linear regression was used to generate heart mass prediction equations. Heart mass estimate performance was tested using median mass comparison, linear regression, and Bland-Altman plots. Median heart mass (P = 0.0008) and heart volume (P = 0.008) were significantly greater in male relative to female cases. Alongside female sex and body surface area (BSA), heart mass was univariably associated with heart volume in all cases (R2 = 0.72) and in male (R2 = 0.70) and female cases (R2 = 0.64) when segregated. In multivariable regression, heart mass was independently associated with age and BSA (R2 adjusted = 0.46-0.54). Addition of heart volume improved multivariable heart mass prediction in the total cohort (R2 adjusted = 0.78), and in male (R2 adjusted = 0.74) and female (R2 adjusted = 0.74) cases. Heart mass estimated from multivariable models incorporating heart volume, age, sex, and BSA was more predictive of actual heart mass (R2 = 0.75-0.79) than models incorporating either age, sex, and BSA only (R2 = 0.48-0.57) or heart volume only (R2 = 0.64-0.73). Heart mass can be more accurately predicted from heart volume measured from PMCT when combined with the classical predictors, age, sex, and BSA.

Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G.

Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca2+ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 µM) to activate endogenous PKG. In cells transfected with luminal Ca2+ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca2+ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca2+ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 µM) also reduced the threshold for spontaneous Ca2+ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca2+ release propensity or luminal Ca2+ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca2+ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca2+ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.

Long-chain acylcarnitine 18:1 acutely increases human atrial myocardial contractility and arrhythmia susceptibility.

Long-chain acylcarnitines (LCACs) are known to directly alter cardiac contractility and electrophysiology. However, the acute effect of LCACs on human cardiac function is unknown. We aimed to determine the effect of LCAC 18:1, which has been associated with cardiovascular disease, on the contractility and arrhythmia susceptibility of human atrial myocardium. Additionally, we aimed to assess how LCAC 18:1 alters Ca2+ influx and spontaneous Ca2+ release in vitro. Human right atrial trabeculae (n = 32) stimulated at 1 Hz were treated with LCAC 18:1 at a range of concentrations (1-25 µM) for a 45-min period. Exposure to the LCAC induced a dose-dependent positive inotropic effect on myocardial contractility (maximal 1.5-fold increase vs. control). At the 25 µM dose (n = 8), this was paralleled by an enhanced propensity for spontaneous contractions (50% increase). Furthermore, all LCAC 18:1 effects on myocardial function were reversed following LCAC 18:1 washout. In fluo-4-AM-loaded HEK293 cells, LCAC 18:1 dose dependently increased cytosolic Ca2+ influx relative to vehicle controls and the short-chain acylcarnitine C3. In HEK293 cells expressing ryanodine receptor (RyR2), this increased Ca2+ influx was linked to an increased propensity for RyR2-mediated spontaneous Ca2+ release events. Our study is the first to show that LCAC 18:1 directly and acutely alters human myocardial function and in vitro Ca2+ handling. The metabolite promotes proarrhythmic muscle contractions and increases contractility. The exploratory findings in vitro suggest that LCAC 18:1 increases proarrhythmic RyR2-mediated spontaneous Ca2+ release propensity. The direct effects of metabolites on human myocardial function are essential to understand cardiometabolic dysfunction.NEW & NOTEWORTHY For the first time, the fatty acid metabolite, long-chain acylcarnitine 18:1, is shown to acutely increase the arrhythmia susceptibility and contractility of human atrial myocardium. In vitro, this was linked to an influx of Ca2+ and an enhanced propensity for spontaneous RyR2-mediated Ca2+ release.

Human Atrial Fibrillation Is Not Associated With Remodeling of Ryanodine Receptor Clusters.

The release of Ca2+ by ryanodine receptor (RyR2) channels is critical for cardiac function. However, abnormal RyR2 activity has been linked to the development of arrhythmias, including increased spontaneous Ca2+ release in human atrial fibrillation (AF). Clustering properties of RyR2 have been suggested to alter the activity of the channel, with remodeling of RyR2 clusters identified in pre-clinical models of AF and heart failure. Whether such remodeling occurs in human cardiac disease remains unclear. This study aimed to investigate the nanoscale organization of RyR2 clusters in AF patients - the first known study to examine this potential remodeling in diseased human cardiomyocytes. Right atrial appendage from cardiac surgery patients with paroxysmal or persistent AF, or without AF (non-AF) were examined using super-resolution (dSTORM) imaging. Significant atrial dilation and cardiomyocyte hypertrophy was observed in persistent AF patients compared to non-AF, with these two parameters significantly correlated. Interestingly, the clustering properties of RyR2 were remarkably unaltered in the AF patients. No significant differences were identified in cluster size (mean ∼18 RyR2 channels), density or channel packing within clusters between patient groups. The spatial organization of clusters throughout the cardiomyocyte was also unchanged across the groups. RyR2 clustering properties did not significantly correlate with patient characteristics. In this first study to examine nanoscale RyR2 organization in human cardiac disease, these findings indicate that RyR2 cluster remodeling is not an underlying mechanism contributing to altered channel function and subsequent arrhythmogenesis in human AF.

Long-Chain Acylcarnitines and Cardiac Excitation-Contraction Coupling: Links to Arrhythmias.

A growing number of metabolomic studies have associated high circulating levels of the amphiphilic fatty acid metabolites, long-chain acylcarnitines (LCACs), with cardiovascular disease (CVD) risk. These studies show that plasma LCAC levels can be correlated with the stage and severity of CVD and with indices of cardiac hypertrophy and ventricular function. Complementing these recent clinical associations is an extensive body of basic research that stems mostly from the twentieth century. These works, performed in cardiomyocyte and multicellular preparations from animal and cell models, highlight stereotypical derangements in cardiac electrophysiology induced by exogenous LCAC treatment that promote arrhythmic muscle behavior. In many cases, this is coupled with acute inotropic modulation; however, whether LCACs increase or decrease contractility is inconclusive. Linked to the electromechanical alterations induced by LCAC exposure is an array of effects on cardiac excitation-contraction coupling mechanisms that overload the cardiomyocyte cytosol with Na+ and Ca2+ ions. The aim of this review is to revisit this age-old literature and collate it with recent findings to provide a pathophysiological context for the growing body of metabolomic association studies that link circulating LCACs with CVD.

Inotropic and lusitropic, but not arrhythmogenic, effects of adipocytokine resistin on human atrial myocardium.

The adipocytokine resistin is released from epicardial adipose tissue (EAT). Plasma resistin and EAT deposition are independently associated with atrial fibrillation. The EAT secretome enhances arrhythmia susceptibility and inotropy of human myocardium. Therefore, we aimed to determine the effect of resistin on the function of human myocardium and how resistin contributes to the proarrhythmic effect of EAT. EAT biopsies were obtained from 25 cardiac surgery patients. Resistin levels were measured by ELISA in 24-h EAT culture media (n = 8). The secretome resistin concentrations increased over the culture period to a maximal level of 5.9 ± 1.2 ng/mL. Coculture with ß-adrenergic agonists isoproterenol (n = 4) and BRL37344 (n = 13) had no effect on EAT resistin release. Addition of resistin (7, 12, 20 ng/mL) did not significantly increase the spontaneous contraction propensity of human atrial trabeculae (n = 10) when given alone or in combination with isoproterenol. Resistin dose-dependently increased trabecula-developed force (maximal 2.9-fold increase, P < 0.0001), as well as the maximal rates of contraction (2.6-fold increase, P = 0.002) and relaxation (1.8-fold increase, P = 0.007). Additionally, the postrest potentiation capacity of human trabeculae was reduced at all resistin doses, suggesting that the inotropic effect induced by resistin might be due to altered sarcoplasmic reticulum Ca2+ handling. EAT resistin release is not modulated by common arrhythmia triggers. Furthermore, exogenous resistin does not promote arrhythmic behavior in human atrial trabeculae. Resistin does, however, induce an acute dose-dependent positive inotropic and lusitropic effect.

Correlation between epicardial adipose tissue and body mass index in New Zealand ethnic populations.

AIM: We aimed to investigate the correlation between epicardial adipose tissue (EAT) and body mass index (BMI) in different ethnic groups in New Zealand. METHODS: The study included 205 individuals undergoing open heart surgery. Maori and Pacific groups were combined to increase statistical power. EAT was measured using 2D echocardiography. RESULTS: There were 164 New Zealand Europeans (NZE) and 41 Maori/Pacific participants. The mean (SD) age of the study group was 67.9 (10.1) years, 69.1 (9.5) for NZE and 63.5 (11.4) for Maori/Pacific. BMI was 29.6 (5.5) kg/m2 for NZE and 31.8 (6.2) for Maori/Pacific. EAT thickness was 6.2 (2.2) mm and 6.0 (1.8) mm for NZE and Maori/Pacific, respectively. Using univariate linear regression, BMI showed moderate correlation with EAT in NZE (R2=0.26, p<0.001); however, there was no significant correlation between BMI and EAT in Maori/Pacific patients (R2=0.05, p=0.17). Using multivariate analysis, BMI remained a significant predictor of EAT thickness in NZE (R2 =0.27, p<0.001). CONCLUSIONS: BMI was associated with EAT thickness in NZE patients, but not in Maori/Pacific patients. The same level of BMI can carry different connotations of risk in different ethnic groups, with BMI likely being an inconsistent measure of obesity in in Maori/Pacific patients.

Acute interaction between human epicardial adipose tissue and human atrial myocardium induces arrhythmic susceptibility.

Epicardial adipose tissue (EAT) deposition has a strong clinical association with atrial arrhythmias; however, whether a direct functional interaction exists between EAT and the myocardium to induce atrial arrhythmias is unknown. Therefore, we aimed to determine whether human EAT can be an acute trigger for arrhythmias in human atrial myocardium. Human trabeculae were obtained from right atrial appendages of patients who have had cardiac surgery (n = 89). The propensity of spontaneous contractions (SCs) in the trabeculae (proxy for arrhythmias) was determined under physiological conditions and during known triggers of SCs (high Ca2+, ß-adrenergic stimulation). To determine whether EAT could trigger SCs, trabeculae were exposed to superfusate of fresh human EAT, and medium of 24 h-cultured human EAT treated with ß1/2 (isoproterenol) or ß3 (BRL37344) adrenergic agonists. Without exposure to EAT, high Ca2+ and ß1/2-adrenergic stimulation acutely triggered SCs in, respectively, 47% and 55% of the trabeculae that previously were not spontaneously active. Acute ß3-adrenergic stimulation did not trigger SCs. Exposure of trabeculae to either superfusate of fresh human EAT or untreated medium of 24 h-cultured human EAT did not induce SCs; however, specific ß3-adrenergic stimulation of EAT did trigger SCs in the trabeculae, either when applied to fresh (31%) or cultured (50%) EAT. Additionally, fresh EAT increased trabecular contraction and relaxation, whereas media of cultured EAT only increased function when treated with the ß3-adrenergic agonist. An acute functional interaction between human EAT and human atrial myocardium exists that increases the propensity for atrial arrhythmias, which depends on ß3-adrenergic rather than ß1/2-adrenergic stimulation of EAT.

Relationship between epicardial adipose tissue thickness and epicardial adipocyte size with increasing body mass index.

Macroscopic deposition of epicardial adipose tissue (EAT) has been strongly associated with numerous indices of obesity and cardiovascular disease risk. In contrast, the morphology of EAT adipocytes has rarely been investigated. We aimed to determine whether obesity-driven adipocyte hypertrophy, which is characteristic of other visceral fat depots, is found within EAT adipocytes. EAT samples were collected from cardiac surgery patients (n = 49), stained with haematoxylin & eosin, and analysed for mean adipocyte size and non-adipocyte area. EAT thickness was measured using echocardiography. A significant positive relationship was found between EAT thickness and body mass index (BMI). When stratified into standardized BMI categories, EAT thickness was 58.7% greater (p = 0.003) in patients from the obese (7.3 ± 1.8 mm) compared to normal (4.6 ± 0.9 mm) category. BMI as a continuous variable significantly correlated with EAT thickness (r = 0.56, p < 0.0001). Conversely, no correlation was observed between adipocyte size and either BMI or EAT thickness. No difference in the non-adipocyte area was found between BMI groups. Our results suggest that the increased macroscopic EAT deposition associated with obesity is not caused by adipocyte hypertrophy. Rather, alternative remodelling via adipocyte proliferation might be responsible for the observed EAT expansion.

Epicardial adipocyte size does not correlate with body mass index.

BACKGROUND: Epicardial adipose tissue (EAT) deposition has a strong association with aspects of metabolic dysfunction, including obesity. The size of the EAT adipocytes in relation to obesity, however, has rarely been researched. Therefore, to contextualise EAT within the broader framework of pathophysiological adipocyte size changes in obesity, we aimed to determine whether EAT adipocyte size is associated with body mass index (BMI). METHODS: During routine post-mortem examination, adipose tissue biopsies were obtained from four depots of 43 cases, including EAT, as well as pericardial (PAT), appendix mesenteric (AAT), and clavicular subcutaneous (SAT) adipose tissues. Tissues were fixed, sectioned, and stained using haematoxylin and eosin. The size (measured as area) of each adipocyte imaged from the depots was analysed in relation to BMI. RESULTS: Mean size of EAT adipocytes was significantly smaller than that from SAT and AAT depots, while not differing from PAT adipocytes. BMI positively correlated with the size of adipocytes isolated from SAT (r=0.5893, P<.0001), PAT (r=0.5854, P<.0001), and AAT (r=0.5829, P<.0001) depots, but not from EAT (r=0.1242, P=.4274), even after multivariate adjustment for age and sex. CONCLUSIONS: EAT adipocyte size is not associated with increased BMI despite significant associations within adipocytes from other adipose depots.