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

Correspondence: Rajesh Katare ([email protected]) 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-β receptor-1 (TGFβR1) 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 TGFβR1 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

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

Abstract 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 β-myosin heavy chain (MHC) and thyroid hormone receptor-α (TR-α), the negative regulator of β-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 β-MHC and downregulation of TR-α. Interestingly, upregulation of miR-208a preceded the switch of α-/β-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 β-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

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.</p

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]

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]

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