Calpain Activation Contributes to Hyperglycaemia-Induced Apoptosis in Cardiomyocytes
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Cardiovascular Research (2009) 84, 100–110 doi:10.1093/cvr/cvp189 Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes Ying Li1,2, Yanwen Li3, Qingping Feng1,2,4, Malcolm Arnold1,2,4, and Tianqing Peng1,2,5* 1Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, VRL 6th Floor, A6-140, 800 Commissioners Road, London, ON, Canada N6A 4G5; 2Department of Medicine, University of Western Ontario, London, ON, Canada N6A 4G5; 3Department of Microbiology, Imperial College London, Flowers Building, Armstrong Road, London SW7 2AZ, UK; 4Department of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada N6A 4G5; and 5Department of Pathology, University of Western Ontario, London, ON, Canada N6A 4G5 Received 22 June 2008; revised 11 May 2009; accepted 29 May 2009; online publish-ahead-of-print 8 June 2009 Time for primary review: 23 days KEYWORDS Aims Cardiomyocyte apoptosis contributes to cardiac complications of diabetes. The aim of this study Cardiomyocyte; was to investigate the role of calpain in cardiomyocyte apoptosis induced by hyperglycaemia. Apoptosis; Methods and results In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased Calpain; calpain activity and induced apoptosis, concomitant with the impairment of Naþ/Kþ ATPase activity. NADPH oxidase; These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain þ þ Na /K ATPase; inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a Downloaded from High glucose specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Naþ/Kþ ATPase. Furthermore, blocking gp91phox-NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced dia- by guest on August 10, 2015 betes, administration of different calpain inhibitors blocked calpain activation, increased the Naþ/Kþ ATPase activity, and decreased apoptosis in the heart. Conclusion Calpain-1 activation induces apoptosis through down-regulation of the Naþ/Kþ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucose- induced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain acti- vation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases. 1. Introduction the diabetic cardiomyopathy.11,12 As such, suppression of car- diomyocyte apoptosis results in a significant prevention of the Cardiovascular disease is one of the major complications of development of diabetic heart diseases.7 However,the mech- diabetes mellitus and has become the main cause of mortality 1 anisms by which hyperglycaemia induces apoptosis in cardio- in the diabetic population. Diabetic patients have a worse myocytes have not been fully understood. outcome after myocardial infarction, with higher risk of Calpains belong to a family of calcium-dependent thiol- heart failure.2,3 Hyperglycaemia is considered the main 4 proteases. Two major isoforms, calpain-1 and calpain-2, are cause of chronic diabetic complications. Studies also ubiquitously expressed, whereas the remaining isoforms suggest that hyperglycaemia is associated with significant have more limited tissue distribution. All 15 gene products higher mortality rates in patients hospitalized with acute cor- 5 of the calpain family reported in mammals are present in onary syndromes. One important consequence of hypergly- the cytosol as inactive proenzymes. Calcium (Ca2þ)is caemia in the heart is the induction of cardiomyocyte required for calpain-1 and calpain-2 activation. Binding of apoptosis. An increase in cardiomyocyte apoptosis has been Ca2þ to calpain-1 and calpain-2 induces the release of con- reported in diabetic animal models and patients, and straints imposed by domain interactions and results in the causes a loss of contractile tissue that initiates cardiac remo- 6–10 release of a regulatory subunit and then the rearrangement delling. The loss of ventricular cardiomyocytes and hyper- of the active site cleft in a catalytic subunit.13 Both trophy of the remaining viable cardiomyocytes characterize calpain-1 and calpain-2 are specifically countered by the endogenous inhibitor, calpastatin. Over-expression of calpas- Corresponding author. Tel: þ1 519 685 8300; fax: þ1 519 685 8341. tatin has been shown to inhibit calpain-1 and calpain-2 in in * 14,15 E-mail address: [email protected] vitro and in vivo transgenic mice. Calpain is involved in Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009. For permissions please email: [email protected]. Role of calpain in apoptosis 101 a variety of biological functions such as cell cycle, cell manufacturer’s protocol as described in our previous migration, differentiation, and apoptosis.13 Both calpain-1 studies with modifications.21,22 Briefly, we mixed 3 mL and calpain-2 are expressed in the heart.16 Activation of Enhancer, 84 mL Buffer ECR, 4.5 mL siRNA (10 mM), 6 mL calpain is implicated in ischaemia/reperfusion-induced apop- TransMessenger, and 220 mL MEM without serum/antibiotics tosis in the heart, and plays a role in TNF-a-mediated apopto- for each well (12-well plates). Four hours after incubation sis in cardiomyocytes.17–19 These studies suggest that calpain with the siRNA mixture, the medium was changed with a activation may contribute to the progression of heart failure. fresh normal culture medium. The cells were then incubated This was indeed supported by a recent study which demon- for 24 h before treatment with high glucose. strated that cardiac over-expression of calpain-1 was suffi- cient to cause heart failure in transgenic mice.16 However, 2.5 Adenoviral infection of cultured adult the role of calpain in cardiac complications of diabetes has rat cardiomyocytes not been demonstrated. Cardiomyocytes were infected with adenoviral vectors con- In the present study, we employed an in vitro model of taining either the gene for rat calpastatin (Ad-CAST, Univer- adult rat cardiomyocyte death induced by high glucose and sity of Buffalo, USA) or beta-gal (Ad-gal, Vector Biolabs) as a a mouse model of streptozotocin (STZ)-induced type-1 dia- control at a multiplicity of infection of 10 PFU/cell. betes to examine the role of calpain in cardiomyocyte Adenovirus-mediated gene transfer was implemented as apoptosis. described previously.20 2. Experimental procedures 2.6 Calpain activity 2.1 Animals and adult rat cardiomyocytes culture Calpain activity was determined by using a fluorescence sub- strate N-succinyl-LLVY-AMC (Cedarlane Laboratories) as This investigation conforms with the Guide for the Care and 20 Use of Laboratory Animals published by the US National described previously. The calpain inhibitor PD150606 was Institutes of Health (NIH Publication No. 85-23). All exper- used to determine the specificity of the assay. imental procedures were approved by the Animal Use Sub- 1 1 committee at the University of Western Ontario, Canada. 2.7 Na /K ATPase activity assay Downloaded from Breeding pairs of C57BL/6 mice were purchased from the Cultured cardiomyocytes or heart tissues were homogenized Jackson Laboratory. Adult male rats (Sprague Dawley, with a lysis buffer containing 10 mM Tris, pH 7.4, 2 mM EDTA, 200 g body weight) were purchased from Charles River and 250 mM sucrose. After four freeze–thaw cycles, the by guest on August 10, 2015 Labs. Adult rat ventricular cardiomyocytes were isolated ouabain-sensitive Naþ/Kþ ATPase activity in homogenates 20 and cultured as described in our recent study. was determined by measuring 3-O-methylfluorescein phos- phatase (3-O-MFPase) activity as described previously.23 2.2 Reagents The fluorescence intensity of cleaved 3-O-MFP was quanti- Streptozotocin, diphenyleneiodonium (DPI), N-acetylcysteine fied with a multilabel reader (excitation, 475 nm; emission, þ (NAC), verapamil, dantrolene, D-glucose, and mannitol 515 nm). The K -stimulated 3-O-MFPase activity was deter- were purchased from Sigma. Ouabain, PD150606, calpain mined as the difference between the slopes generated from inhibitor-I, and calpain inhibitor-III were from Calbiochem. the same sample with or without the addition of KCl, which Annexin-V conjugated with FITC and Hoechst 33324 were was ouabain inhibitable. from Invitrogen. Peptides gp91ds-tat and scramble-tat were synthesized by ProImmune Ltd (UK). 2.8 Active caspase-3 As described in detail previously,24 caspase-3 activity in 2.3 STZ hyperglycaemic mice myocardial tissues and cardiomyocytes was measured by Adult male mice (25–30 g body weight) were intrapenitone- using a caspase-3 fluorescent assay kit (BIOMOL Research ally (i.p.) injected with a single dose of STZ at 150 mg/kg Laboratories). body weight, dissolved in 10 mM sodium citrate buffer (pH 4.5), a well-established agent that destroys pancreatic 2.9 Cell death measurement cells. On Day 3 after STZ treatment, whole-blood was Cardiomyocyte death was measured by annexin-V and obtained from the mouse tail-vein, and the blood glucose Hoechst 33342 staining as described in our recent study.20 levels