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.

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.

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.

Diabetes induces the activation of pro-ageing miR-34a in the heart, but has differential effects on cardiomyocytes and cardiac progenitor cells.

Increased apoptosis and premature cellular ageing of the diabetic heart underpin the development of diabetic heart disease. The molecular mechanisms underlying these pathologies are still unclear. Here we determined the role of pro-senescence microRNA (miR)-34a in accelerating the ageing of the diabetic heart. RT-PCR analysis showed a significant increase in the level of circulating miR-34a from early stages in asymptomatic type-2 diabetic individuals compared to non-diabetic controls. We also observed significant upregulation of miR-34a in the type-2 human diabetic heart suggesting circulating miR-34a may be cardiac in origin. Moreover, western blot analysis identified marked downregulation of the pro-survival protein sirtuin 1 (SIRT1), a direct target of miR-34a. Analysis of cultured human adult cardiomyocytes exposed to high glucose and cardiac progenitor cells (CPCs) isolated from the diabetic heart confirmed significant upregulation of miR-34a and downregulation of SIRT1, associated with a marked increase in pro-apoptotic caspase-3/7 activity. Although therapeutic inhibition of miR-34a activity restored SIRT1 expression in both cardiomyocytes and CPCs, p53 expression was further upregulated in cardiomyocytes but conversely downregulated in CPCs. In spite of increased p53, miR-34a inhibition significantly reduced high glucose induced apoptotic cell death in cardiomyocytes. However, this effect was not observed in CPCs, which in fact showed reduced proliferation following miR-34a inhibition. Taken together, our results demonstrate upregulation of miR-34a in the diabetic heart and in the circulation from an early stage of the disease. However, inhibition of miR-34a activity has differential effects depending on the cell type, thereby warranting the need to eliminate off-target effects when introducing miR-based therapy.

Down-regulation of miR-15a/b accelerates fibrotic remodelling in the Type 2 diabetic human and mouse heart.

Aim: Myocardial fibrosis is a well-established cause of increased myocardial stiffness and subsequent diastolic dysfunction in the diabetic heart. The molecular regulators that drive the process of fibrotic events in the diabetic heart are still unknown. We determined the role of the microRNA (miR)-15 family in fibrotic remodelling of the diabetic heart.Methods and results: Right atrial appendage (RAA) and left ventricular (LV) biopsy tissues collected from diabetic and non-diabetic (ND) patients undergoing coronary artery bypass graft surgery showed significant down-regulation of miR-15a and -15b. This was associated with marked up-regulation of pro-fibrotic transforming growth factor-ß receptor-1 (TGFßR1) and connective tissue growth factor (CTGF), direct targets for miR-15a/b and pro-senescence p53 protein. Interestingly, down-regulation of miR-15a/b preceded the development of diastolic dysfunction and fibrosis in Type 2 diabetic mouse heart. Therapeutic restoration of miR-15a and -15b in HL-1 cardiomyocytes reduced the activation of pro-fibrotic TGFßR1 and CTGF, and the pro-senescence p53 protein expression, confirming a causal regulation of these fibrotic and senescence mediators by miR-15a/b. Moreover, conditioned medium (CM) collected from cardiomyocytes treated with miR-15a/b markedly diminished the differentiation of diabetic human cardiac fibroblasts.Conclusion: Our results provide first evidence that early down-regulation of miR-15a/b activates fibrotic signalling in diabetic heart, and hence could be a potential target for the treatment/prevention of diabetes-induced fibrotic remodelling of the heart.

The diagnostic sensitivity of circulating cardio-enriched microRNAs is increased after normalization of high-density lipoprotein levels.

BACKGROUND: Emerging evidence suggests that microRNAs (miRs) could be a potential biomarker to identify early molecular alterations in the heart. HDL are the major carriers for miRs into the circulation. This study tested whether changes in the level of HDL could affect the diagnostic sensitivity of miRs. METHODS AND RESULTS: Peripheral blood samples were collected from 20 diabetic and 22 age and gender matched non-diabetic patients undergoing coronary artery bypass graft (CABG) surgery for ischemic heart disease (IHD). Total RNA was extracted from the separated plasma and stored in -80°C. Reverse transcription and amplification using specific primers against cardio-enriched miR-1, -34a, -126, -133, and -499 showed significant correlation between HDL levels and miR-1, -133 and -499. Importantly, normalization of miR levels with HDL showed a significant downregulation of miR-1, -133 and -499 in diabetic plasma, which was not observed before normalization with HDL levels. CONCLUSION: Normalization of circulating miR levels with HDL increases the diagnostic sensitivity of circulating miRs.

Down-regulation of proangiogenic microRNA-126 and microRNA-132 are early modulators of diabetic cardiac microangiopathy.

AIM: Microangiopathy due to endothelial dysfunction is a major contributing factor to the development of diabetes-induced cardiovascular disease (CVD). Dysregulation of endothelial-specific microRNAs (miRs) is correlated with impaired angiogenesis and cell survival. We investigated the profile of two angiomiRs, miR-126, and miR-132, in the plasma of type 2 diabetic individuals without any known history of CVD as well as in the cardiac tissues collected from diabetics undergoing cardiac surgery. METHODS AND RESULTS: The presence of diabetes alone significantly decreased both angiomiRs in the plasma and the myocardium. The down-regulation of angiomiRs was also associated with reduced capillaries and arterioles and increased endothelial cell apoptosis, the hallmark of microangiopathy. Importantly, a time course study in a type 2 diabetic mouse model confirmed that the down-regulation of angiomiRs preceded endothelial apoptosis as well as alterations in the density of the microvasculature. Finally, therapeutic overexpression of both angiomiRs in diabetic aortic rings and human umbilical vein endothelial cells exposed to high glucose (HG) abrogated the deleterious effects of diabetes and HG on cell survival and proliferation and restored their angiogenic potential. CONCLUSIONS: These novel findings demonstrate that the down-regulation of angiomiRs is a major underlying mechanism for the development of microangiopathy in diabetic hearts. Therefore, therapeutic restoration of angiomiRs could become a potential approach to combat the cardiovascular complications of diabetes.

Progenitor cells from atria, ventricle and peripheral blood of the same patients exhibit functional differences associated with cardiac repair.

AIM: Deciding the best cell type for cardiac regeneration remains a big challenge. No studies have directly compared the functional efficacy of cardiac progenitor cells (CPCs) with extra-cardiac stem cells isolated from the same patient. METHODS AND RESULTS: We compared the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CPCs and left ventricular (LV) CPCs isolated from the same patients (n=14). Within the same heart, RAA and LV CPCs exhibited marked differences in surface marker expression, with RAA CPCs exhibiting better expansion potential and migration properties. When subjected to hypoxia and serum starvation to simulate in vivo ischemic environment, RAA and LV CPCs exhibited similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under hypoxia. RT-profiler array showed comparable gene expression pattern in RAA and LV CPCs, while they were differentially expressed in EPCs. Further, treating human umbilical vein endothelial cells with conditioned medium (CM) from LV showed maximum angiogenic potential, while cardiomyocytes treated with CM from RAA showed greatest survival under hypoxic conditions. CONCLUSIONS: Results from this study provide the first evidence that progenitor cells from different regions exhibit functional differences within the same patient.

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.

Kinky circumflex grafts: the left appendage flip maneuver.

The "too-long," or redundant, circumflex graft is notorious for its tendency to form a kink. The acute angle (the kink) typically occurs over the front of the pulmonary trunk. Described herein are (1) the left appendage flip maneuver, a simple solution to correct graft kink, and (2) analysis and explanation of vein graft kink.

Composite venous coronary grafts: construction made simple.

We describe an efficient, safe, and effective method for constructing a satisfactory composite venous conduit from 2 portions of saphenous vein, which by themselves would be inadequate in length to function as independent conduits.