565629 research-article2015
DVR0010.1177/1479164114565629Diabetes & Vascular Disease ResearchJoshi et al.
Original Article
Role of mitochondrial dysfunction in hyperglycaemia-induced coronary microvascular dysfunction: Protective role of resveratrol
Diabetes & Vascular Disease Research 2015, Vol. 12(3) 208–216 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1479164114565629 dvr.sagepub.com
Mandar S Joshi1,2,3, David Williams1, Duncan Horlock1, Thilini Samarasinghe2, Karen L Andrews4, Ann-Maree Jefferis4, Philip J Berger2, Jaye P Chin-Dusting4 and David M Kaye1,5
Abstract Microvascular complications are now recognized to play a major role in diabetic complications, and understanding the mechanisms is critical. Endothelial dysfunction occurs early in the course of the development of complications; the precise mechanisms remain poorly understood. Mitochondrial dysfunction may occur in a diabetic rat heart and may act as a source of the oxidative stress. However, the role of endothelial cell-specific mitochondrial dysfunction in diabetic vascular complications is poorly studied. Here, we studied the role of diabetes-induced abnormal endothelial mitochondrial function and the resultant endothelial dysfunction. Understanding the role of endothelial mitochondrial dysfunction in diabetic vasculature is critical in order to develop new therapies. We demonstrate that hyperglycaemia leads to mitochondrial dysfunction in microvascular endothelial cells, and that mitochondrial inhibition induces endothelial dysfunction. Additionally, we show that resveratrol acts as a protective agent; resveratrol-mediated mitochondrial protection may be used to prevent long-term diabetic cardiovascular complications. Keywords Diabetes, microvascular, mitochondria, resveratrol, cardiac dysfunction
Introduction Cardiovascular (CV) complications account for a substantial proportion of the morbidity and mortality that occurs in patients living with diabetes. In particular, the development of endothelial dysfunction is thought to play a major role in the pathogenesis of diabetes-associated cardiovascular disease (CVD) in addition to other pathology including retinopathy and nephropathy.1,2 The prevention and early identification of vascular complications are a central issue in the care of patients with diabetes. Recent data suggest that endothelial dysfunction occurs very early in the course of the development of vascular pathology. For example, these features have been reported in preadolescent children with type I diabetes,3 and other studies have also reported the increased presence of markers of endothelial activation and perturbation along with carotid intima–media thickening,4 in early type I diabetes. Thus, understanding the mechanisms of early endothelial dysfunction in diabetes is critical for prevention of long-term complications. Several crucial factors, such as alterations in oxidative stress, lipid metabolism, insulin resistance and inflammation,
have been suggested to play a role in the causation of endothelial dysfunction. The precise source of the oxidative stress remains controversial, and potential sources include nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and mitochondria. Interestingly in the context of inflammation, mitochondrial sources of reactive oxygen species (ROS) have been proposed to be particularly important.5 Consistent with 1Heart
Failure Research Group, Cardiology Division, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia 2The Ritchie Centre, Monash University, Melbourne, VIC, Australia 3Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, USA 4Vascular Pharmacology, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia 5Heart Failure Unit, Alfred Hospital, Melbourne, VIC, Australia Corresponding author: David M Kaye, Heart Failure Research Group, Cardiology Division, Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, VIC 3150, Australia. Email: [email protected]
Downloaded from dvr.sagepub.com at TEXAS A&M UNIV TEXARKANA on August 30, 2015
209
Joshi et al. this concept, mitochondrial function has been shown to be impaired in the rat heart, providing a possible explanation for the pathogenesis of diabetic cardiomyopathy,6 although the possibility that abnormal endothelial mitochondrial function is a specific contributor has not been examined in detail. In this study, we hypothesized that diabetes induces abnormal endothelial mitochondrial function, and that this contributes to endothelial dysfunction in diabetes. Given the relatively low abundance of mitochondria in endothelial cells (ECs), it had been assumed that they were unlikely to contribute significantly to cellular function. However, it is now apparent that in addition to adenosine triphosphate (ATP) generation, mitochondria also play a role in EC homeostasis via modulation of nitric oxide (NO), ROS and Ca2+.7 Stimulation of mitochondrial Ca2+ uptake can activate NO production that in turn can modulate mitochondrial Ca2+ uptake and efflux, demonstrating a negative feedback regulation.8 Thus, understanding the role of EC-specific mitochondrial dysfunction in diabetic vasculature is critical in order to develop new therapies. Our data show that coronary perfusion is reduced early in the course of experimental diabetes. To understand the underlying cause of reduced microvascular function in diabetic heart, we examined the role of mitochondrial dysfunction as a cause for endothelial dysfunction. We demonstrate that hyperglycaemia leads to mitochondrial dysfunction in microvascular ECs, and that mitochondrial dysfunction induces endothelial dysfunction that manifests as impaired pharmacologic and regenerative capacity. Finally, we showed that resveratrol exerts protective actions in the setting of diabetes-associated endothelial dysfunction.
Methods Animal model All experiments were carried out in accordance with the guidelines set forth by the institutional animal ethics committee. Hyperglycaemia was induced in pathogen-free male Sprague-Dawley rats (200–250 g) with a single dose of streptozotocin [STZ, 65 mg/kg intraperitoneal (i.p.) prepared fresh in citrate buffer pH 4.5]. Control animals received equal volume of the citrate buffer. Blood glucose level was determined at each time point using Optium Omega (Abbott Diabetes Care, Alameda, CA) clinical blood glucose monitor (Figure 8; supplementary data available online). Animals with blood glucose level of
DVR0010.1177/1479164114565629Diabetes & Vascular Disease ResearchJoshi et al.
Original Article
Role of mitochondrial dysfunction in hyperglycaemia-induced coronary microvascular dysfunction: Protective role of resveratrol
Diabetes & Vascular Disease Research 2015, Vol. 12(3) 208–216 © The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1479164114565629 dvr.sagepub.com
Mandar S Joshi1,2,3, David Williams1, Duncan Horlock1, Thilini Samarasinghe2, Karen L Andrews4, Ann-Maree Jefferis4, Philip J Berger2, Jaye P Chin-Dusting4 and David M Kaye1,5
Abstract Microvascular complications are now recognized to play a major role in diabetic complications, and understanding the mechanisms is critical. Endothelial dysfunction occurs early in the course of the development of complications; the precise mechanisms remain poorly understood. Mitochondrial dysfunction may occur in a diabetic rat heart and may act as a source of the oxidative stress. However, the role of endothelial cell-specific mitochondrial dysfunction in diabetic vascular complications is poorly studied. Here, we studied the role of diabetes-induced abnormal endothelial mitochondrial function and the resultant endothelial dysfunction. Understanding the role of endothelial mitochondrial dysfunction in diabetic vasculature is critical in order to develop new therapies. We demonstrate that hyperglycaemia leads to mitochondrial dysfunction in microvascular endothelial cells, and that mitochondrial inhibition induces endothelial dysfunction. Additionally, we show that resveratrol acts as a protective agent; resveratrol-mediated mitochondrial protection may be used to prevent long-term diabetic cardiovascular complications. Keywords Diabetes, microvascular, mitochondria, resveratrol, cardiac dysfunction
Introduction Cardiovascular (CV) complications account for a substantial proportion of the morbidity and mortality that occurs in patients living with diabetes. In particular, the development of endothelial dysfunction is thought to play a major role in the pathogenesis of diabetes-associated cardiovascular disease (CVD) in addition to other pathology including retinopathy and nephropathy.1,2 The prevention and early identification of vascular complications are a central issue in the care of patients with diabetes. Recent data suggest that endothelial dysfunction occurs very early in the course of the development of vascular pathology. For example, these features have been reported in preadolescent children with type I diabetes,3 and other studies have also reported the increased presence of markers of endothelial activation and perturbation along with carotid intima–media thickening,4 in early type I diabetes. Thus, understanding the mechanisms of early endothelial dysfunction in diabetes is critical for prevention of long-term complications. Several crucial factors, such as alterations in oxidative stress, lipid metabolism, insulin resistance and inflammation,
have been suggested to play a role in the causation of endothelial dysfunction. The precise source of the oxidative stress remains controversial, and potential sources include nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and mitochondria. Interestingly in the context of inflammation, mitochondrial sources of reactive oxygen species (ROS) have been proposed to be particularly important.5 Consistent with 1Heart
Failure Research Group, Cardiology Division, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia 2The Ritchie Centre, Monash University, Melbourne, VIC, Australia 3Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, USA 4Vascular Pharmacology, Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia 5Heart Failure Unit, Alfred Hospital, Melbourne, VIC, Australia Corresponding author: David M Kaye, Heart Failure Research Group, Cardiology Division, Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, VIC 3150, Australia. Email: [email protected]
Downloaded from dvr.sagepub.com at TEXAS A&M UNIV TEXARKANA on August 30, 2015
209
Joshi et al. this concept, mitochondrial function has been shown to be impaired in the rat heart, providing a possible explanation for the pathogenesis of diabetic cardiomyopathy,6 although the possibility that abnormal endothelial mitochondrial function is a specific contributor has not been examined in detail. In this study, we hypothesized that diabetes induces abnormal endothelial mitochondrial function, and that this contributes to endothelial dysfunction in diabetes. Given the relatively low abundance of mitochondria in endothelial cells (ECs), it had been assumed that they were unlikely to contribute significantly to cellular function. However, it is now apparent that in addition to adenosine triphosphate (ATP) generation, mitochondria also play a role in EC homeostasis via modulation of nitric oxide (NO), ROS and Ca2+.7 Stimulation of mitochondrial Ca2+ uptake can activate NO production that in turn can modulate mitochondrial Ca2+ uptake and efflux, demonstrating a negative feedback regulation.8 Thus, understanding the role of EC-specific mitochondrial dysfunction in diabetic vasculature is critical in order to develop new therapies. Our data show that coronary perfusion is reduced early in the course of experimental diabetes. To understand the underlying cause of reduced microvascular function in diabetic heart, we examined the role of mitochondrial dysfunction as a cause for endothelial dysfunction. We demonstrate that hyperglycaemia leads to mitochondrial dysfunction in microvascular ECs, and that mitochondrial dysfunction induces endothelial dysfunction that manifests as impaired pharmacologic and regenerative capacity. Finally, we showed that resveratrol exerts protective actions in the setting of diabetes-associated endothelial dysfunction.
Methods Animal model All experiments were carried out in accordance with the guidelines set forth by the institutional animal ethics committee. Hyperglycaemia was induced in pathogen-free male Sprague-Dawley rats (200–250 g) with a single dose of streptozotocin [STZ, 65 mg/kg intraperitoneal (i.p.) prepared fresh in citrate buffer pH 4.5]. Control animals received equal volume of the citrate buffer. Blood glucose level was determined at each time point using Optium Omega (Abbott Diabetes Care, Alameda, CA) clinical blood glucose monitor (Figure 8; supplementary data available online). Animals with blood glucose level of
