Epithelial Sodium Channel δ Subunit Is Expressed in Human Arteries and Has Potential Association With Hypertension.

BACKGROUND: Elevated expression and increased activity of vascular epithelial sodium channel (ENaC) can result in vascular dysfunction in small animal models. However, there is limited or no knowledge on expression and function of ENaC channels in human vasculature. Hence, this study explored the expression and function of ENaC in human arteries and their association with hypertension. METHODS: Human internal mammary artery (IMA) and aorta were obtained from cardiovascular patients undergoing coronary artery bypass graft surgery. Expression of the ENaC subunit was analyzed by polymerase chain reaction, Western blot, and immunohistochemistry. ENaC function was observed by patch-clamp electrophysiology in endothelial cells isolated from IMA. Levels of ENaC subunit expression levels were compared between arteries from normotensive, uncontrolled hypertensive, and controlled hypertensive patients. RESULTS: For the first time, expression of α, ß, γ, and δ was detected at mRNA and protein levels in human IMA and aorta. Single-channel patch-clamp recordings identified both αßγ- and δßγ-like channel conductance in primary endothelial cells isolated and cultured from IMA. Reduced expression of the δ subunit was observed in controlled hypertensive IMA, whereas reduced expression of γ-ENaC was observed in controlled hypertensive aorta. CONCLUSIONS: These data suggest that functional ENaC channels are expressed in human arteries and their expression levels are associated with hypertension.

Thiamine increases resident endoglin positive cardiac progenitor cells and atrial contractile force in humans: A randomised controlled trial.

BACKGROUND: The heart has an intrinsic ability to regenerate, orchestrated by progenitor or stem cells. However, the relative complexity of non-resident cardiac progenitor cell (CPC) therapy makes modulation of resident CPCs a more attractive treatment target. Thiamine analogues improve resident CPC function in pre-clinical models. In this double blinded randomised controlled trial (identifier: ACTRN12614000755639), we examined whether thiamine would improve CPC function in humans. METHODS AND RESULTS: High dose oral thiamine (one gram twice daily) or matching placebo was administered 3-5 days prior to coronary artery bypass surgery (CABG). Right atrial appendages were collected at the time of CABG, and CPCs isolated. There was no difference in the primary outcome (proliferation ability of CPCs) between treatment groups. Older age was not associated with decreased proliferation ability. In exploratory analyses, isolated CPCs in the thiamine group showed an increase in the proportion of CD34-/CD105+ (endoglin) cells, but no difference in CD34-/CD90+ or CD34+ cells. Thiamine increased maximum force developed by isolated trabeculae, with no difference in relaxation time or beta-adrenergic responsiveness. CONCLUSION: Thiamine does not improve proliferation ability of CPC in patients undergoing CABG, but increases the proportion of CD34-/CD105+ cells. Having not met its primary endpoint, this study provides the impetus to re-examine CPC biology prior to any clinical outcome-based trial examining potential beneficial cardiovascular effects of thiamine.

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.

Elevated myocardial fructose and sorbitol levels are associated with diastolic dysfunction in diabetic patients, and cardiomyocyte lipid inclusions in vitro.

Diabetes is associated with cardiac metabolic disturbances and increased heart failure risk. Plasma fructose levels are elevated in diabetic patients. A direct role for fructose involvement in diabetic heart pathology has not been investigated. The goals of this study were to clinically evaluate links between myocardial fructose and sorbitol (a polyol pathway fructose precursor) levels with evidence of cardiac dysfunction, and to experimentally assess the cardiomyocyte mechanisms involved in mediating the metabolic effects of elevated fructose. Fructose and sorbitol levels were increased in right atrial appendage tissues of type 2 diabetic patients (2.8- and 1.5-fold increase respectively). Elevated cardiac fructose levels were confirmed in type 2 diabetic rats. Diastolic dysfunction (increased E/e', echocardiography) was significantly correlated with cardiac sorbitol levels. Elevated myocardial mRNA expression of the fructose-specific transporter, Glut5 (43% increase), and the key fructose-metabolizing enzyme, Fructokinase-A (50% increase) was observed in type 2 diabetic rats (Zucker diabetic fatty rat). In neonatal rat ventricular myocytes, fructose increased glycolytic capacity and cytosolic lipid inclusions (28% increase in lipid droplets/cell). This study provides the first evidence that elevated myocardial fructose and sorbitol are associated with diastolic dysfunction in diabetic patients. Experimental evidence suggests that fructose promotes the formation of cardiomyocyte cytosolic lipid inclusions, and may contribute to lipotoxicity in the diabetic heart.

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.

Frailty in Elderly Patients Undergoing Cardiac Surgery Increases Hospital Stay and 12-Month Readmission Rate.

BACKGROUND: Cardiac surgery risk scoring systems predict operative mortality but not outcomes related to preoperative frailty. The aim of this study was to assess frailty in a cohort of older cardiac surgery patients as a predictor of postoperative outcomes. METHODS: Prospective data was collected on patients 65 years of age and older undergoing cardiac surgery between September 2015 and October 2016 at Dunedin Hospital. Frailty was assessed with the Edmonton frail scale and five-metre gait speed. The primary endpoint was length of hospital stay. Secondary outcomes included postoperative complications, major adverse events, death and 12-month readmission rate. RESULTS: Among the 96 patients, median age was 74 (interquartile range 10.5) and 65 (68%) were males. Of the sample 64 (67%) were scored as not frail, 22 (23%) as vulnerable, and 10 (10%) as frail. The median (interquartile range) postoperative days' stay were: not frail 6 (2), vulnerable 9.5 (8), and frail 15 (13). Survival analysis adjusting for EuroSCORE II, age, sex and surgery type showed that greater Edmonton Frail Scale scores were independently predictive of longer post-surgery hospital stay with a hazard ratio for discharge of 0.83 (95% confidence interval 0.76-0.91, p<0.001) per point. The Edmonton Frail Scale score was associated with the 12-month post discharge number of readmissions (adjusted incidence rate ratio 1.24 (95% confidence interval 1.13-1.37, p<0.001) per point. CONCLUSIONS: The Edmonton Frail Scale score predicts length of hospital stay post cardiac surgery and 12-month readmission rate in patients older than 65 years of age.

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.

Early dysregulation of cardiac-specific microRNA-208a is linked to maladaptive cardiac remodelling in diabetic myocardium.

BACKGROUND: The diabetic heart undergoes remodelling contributing to an increased incidence of heart failure in individuals with diabetes at a later stage. The molecular regulators that drive this process in the diabetic heart are still unknown. METHODS: Real-time (RT) PCR analysis was performed to determine the expression of cardiac specific microRNA-208a in right atrial appendage (RAA) and left ventricular (LV) biopsy tissues collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery. To determine the time-dependent changes, cardiac tissue were collected from type 2 diabetic mice at different age groups. A western blotting analysis was conducted to determine the expression of contractile proteins α- and ß-myosin heavy chain (MHC) and thyroid hormone receptor-α (TR-α), the negative regulator of ß-MHC. To determine the beneficial effects of therapeutic modulation of miR-208a, high glucose treated adult mouse HL-1 cardiomyocytes were transfected with anti-miR-208a. RESULTS: RT-PCR analysis showed marked upregulation of miR-208a from early stages of diabetes in type 2 diabetic mouse heart, which was associated with a marked increase in the expression of pro-hypertrophic ß-MHC and downregulation of TR-α. Interestingly, upregulation of miR-208a preceded the switch of α-/ß-MHC isoforms and the development of diastolic and systolic dysfunction. We also observed significant upregulation of miR-208a and modulation of miR-208a associated proteins in the type 2 human diabetic heart. Therapeutic inhibition of miR-208a activity in high glucose treated HL-1 cardiomyocytes prevented the activation of ß-MHC and hence the hypertrophic response. CONCLUSION: Our results provide the first evidence that early modulation of miR-208a in the diabetic heart induces alterations in the downstream signaling pathway leading to cardiac remodelling and that therapeutic inhibition of miR-208a may be beneficial in preventing diabetes-induced adverse remodelling of the heart.

Integrated microRNA and messenger RNA analysis in aortic stenosis.

Aortic valve stenosis (AS) is a major cause of morbidity and mortality, with no effective medical therapies. Investigation into the underlying biology of AS in humans is limited by difficulties in obtaining healthy valvular tissue for use as a control group. However, micro-ribonucleic acids (miRNAs) are stable in post-mortem tissue. We compared valve specimens from patients undergoing aortic valve replacement for AS to non-diseased cadaveric valves. We found 106 differentially expressed miRNAs (p < 0.05, adjusted for multiple comparisons) on microarray analysis, with highly correlated expression among up- and down-regulated miRNAs. Integrated miRNA/gene expression analysis validated the microarray results as a whole, while quantitative polymerase chain reaction confirmed downregulation of miR-122-5p, miR-625-5p, miR-30e-5p and upregulation of miR-21-5p and miR-221-3p. Pathway analysis of the integrated miRNA/mRNA network identified pathways predominantly involved in extracellular matrix function. A number of currently available therapies target products of upregulated genes in the integrated miRNA/mRNA network, with these genes being predominantly more peripheral members of the network. The identification of a group of tissue miRNA associated with AS may contribute to the development of new therapeutic approaches to AS. This study highlights the importance of systems biology-based approaches to complex diseases.

Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway.

BACKGROUND: Diabetes promotes progressive loss of cardiac cells, which are replaced by a fibrotic matrix, resulting in the loss of cardiac function. In the current study we sought to identify if excessive autophagy plays a major role in inducing this progressive loss. METHODS AND RESULTS: Immunofluorescence and western blotting analysis of the right atrial appendages collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery showed a marked increase in the level of autophagy in the diabetic heart, as evidenced by increased expression of autophagy marker LC3B-II and its mediator Beclin-1 and decreased expression of p62, which incorporates into autophagosomes to be efficiently degraded. Moreover, a marked activation of pro-apoptotic caspase-3 was observed. Electron microscopy showed increased autophagosomes in the diabetic heart. In vivo measurement of autophagic flux by choloroquine injection resulted in further enhancement of LC3B-II in the diabetic myocardium, confirming increased autophagic activity in the type-2 diabetic heart. Importantly, in-vitro genetic depletion of beclin-1 in high glucose treated adult rat cardiomyocytes markedly inhibited the level of autophagy and subsequent apoptotic cell death. CONCLUSIONS: These findings demonstrate the pathological role of autophagy in the type-2 diabetic heart, opening up a potentially novel therapeutic avenue for the treatment of diabetic heart disease.

Data supporting the activation of autophagy genes in the diabetic heart.

This data article contains full list of autophagy related genes that are altered in diabetic heart. This article also shows data from in vitro cultured cardiomyocytes that are exposed the high glucose treatment to simulate hyperglycemic state in vitro. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in "Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway" (Munasinghe et al., in press) [1].

Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction.

BACKGROUND: Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. METHODS: Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and ß-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of ß1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. RESULTS: In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to ß-adrenergic stimulation, despite no change in ß1-adrenoreceptor expression levels. CONCLUSIONS: Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during ß-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes in an attempt to maintain diastolic function at rest despite impaired relaxation in the diabetic fibrotic atrial myocardium. Our study addresses important aspects of the underlying mechanisms of diabetes-associated diastolic dysfunction, which is crucial to developing new therapeutic treatments.

Rapid onset of cardiomyopathy in STZ-induced female diabetic mice involves the downregulation of pro-survival Pim-1.

BACKGROUND: Diabetic women are five times more likely to develop congestive heart failure compared with two fold for men. The underlying mechanism for this gender difference is not known. Here we investigate the molecular mechanisms responsible for this female disadvantage and attempt safeguarding cardiomyocytes viability and function through restoration of pro-survival Pim-1. METHODS AND RESULTS: Diabetes was induced by injection of streptozotocin in CD1 mice of both genders. Functional and dimensional parameters measurement using echocardiography revealed diastolic dysfunction in female diabetic mice within 8 weeks after STZ-induced diabetes. This was associated with significant downregulation of pro-survival Pim-1 and upregulation of pro-apoptotic Caspase-3, microRNA-1 and microRNA-208a. Male diabetic mice did not show any significant changes at this time point (P < 0.05 vs. female diabetic). Further, the onset of ventricular remodelling was quicker in female diabetic mice showing marked left ventricular dilation, reduced ejection fraction and poor contractility (P < 0.05 vs. male diabetic at 12 and 16 weeks of STZ-induced diabetes). Molecular analysis of samples from human diabetic hearts confirmed the results of pre-clinical studies, showing marked downregulation of Pim-1 in the female diabetic heart (P < 0.05 vs. male diabetic). Finally, in vitro restoration of Pim-1 reversed the female disadvantage in diabetic cardiomyocytes. CONCLUSIONS: We provide novel insights into the molecular mechanisms behind the rapid onset of cardiomyopathy in female diabetics. These results suggest the requirement for the development of gender-specific treatments for diabetic cardiomyopathy.

Combined thyroidectomy and cardiac surgery.

A combination of cardiac surgery and thyroidectomy as a single stage operation has rarely been reported in the literature. We report on the management of a 64-year-old female undergoing a combined aortic valve replacement and excision of a primary (ectopic) intrathoracic goiter. The literature on combined cardiac surgery and thyroidectomy is also reviewed.

Benign metastasizing leiomyoma: a rare metastatic lesion in the right ventricle.

Cardiac tumors require resection for diagnostic purposes and to avoid complications associated with an intracardiac mass. We present the case of a 41-year-old woman with a known uterine leiomyoma who presented 3 months after elective cesarian section and hysterectomy with a right ventricular mass that was confirmed histologically to be a benign leiomyoma of the same pathologic type as the uterine primary. Benign metastasizing leiomyoma is a rare pathologic entity occurring in women with a history of a uterine leiomyoma. This is the second reported case of cardiac metastasis from a benign uterine leiomyoma.

Expanding retrograde saphenous vein graft aneurysm treated with endovascular coiling.

Saphenous vein graft aneurysm is a potentially fatal complication of coronary artery bypass grafting and usually requires surgery. This report describes endovascular coiling of a saphenous vein graft aneurysm that developed after redo coronary artery bypass grafting. The aneurysm occurred in a proximally occluded saphenous vein graft after revascularization of the same target vessel. The procedure required a retrograde approach through a patent left internal mammary and left anterior descending artery to reach and successfully thrombose the aneurysm.