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Experimental Physiology – Research Paper
Myocardial overexpression of adenine nucleotide translocase 1 ameliorates diabetic cardiomyopathy in mice ∗
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Yong Wang1,2 , Linda Ebermann1 , Anja Sterner-Kock3 , Sylwia Wika1 , Heinz-Peter Schultheiss1 , Andrea D¨orner1 and Thomas Walther1,2 1
Department of Cardiology, Charit´e-Universit¨atsmedizin Berlin, Campus Benjamin Franklin (CBF), Berlin, Germany Centre for Biomedical Research, Hull York Medical School, University of Hull, Hull, UK 3 Institute for Veterinary Pathology, Freie Universit¨at, Berlin, Germany 2
Mitochondrial dysfunction is implicated in the pathogenesis of diabetic cardiomyopathy, a common complication of diabetes. Adenosine nucleotide translocase (ANT) translocates ADP/ATP across the inner mitochondrial membrane. Our study aimed to test the hypothesis that overexpression of ANT1 in cardiomyocytes has cardioprotective effects in diabetic cardiomyopathy induced by streptozotocin (STZ). Mice specifically overexpressing murine ANT1 in the heart were generated using α-myosin heavy chain promoter. Expression of ANT1 mRNA and protein in hearts was characterized by real-time polymerase chain reaction and Western blot analysis. Five- to 6-month-old male transgenic mice and their age-matched wild-type littermates were subjected to type 1 diabetes induced by STZ. Six weeks later, haemodynamic measurement was performed to assess cardiac function. Ventricular mRNA expression of atrial natriuretic peptide, a molecular marker of heart failure, was characterized by RNase-protection assay. Both ANT1 mRNA and ANT1 protein were specifically overexpressed in the heart of transgenic mice. Heart weight was decreased and cardiac function was dramatically impaired in wild-type mice 6 weeks after induction of diabetes, but ANT1 overexpression prevented these significant changes. The mRNA expression level of atrial natriuretic peptide confirmed the haemodynamic findings, being upregulated in wild-type mice receiving STZ, but showing no statistical differences in ANT1 transgenic mice. Cardiomyocyterestricted overexpression of ANT1 prevents the development of diabetic cardiomyopathy; therefore, accelerated ADP/ATP exchange could be a new promising target to treat diabetic cardiomyopathy. (Received 1 September 2008; accepted after revision 22 October 2008; first published online 22 October 2008) Corresponding author T. Walther: Centre for Biomedical Research, Hull York Medical School, University of Hull, Hull, East Yorkshire HU6 7RX, UK. Email:
[email protected]
Accumulated evidence indicates that heart failure in diabetes is due, at least in part, to a specific form of cardiomyopathy, referred to as diabetic cardiomyopathy, which is independent of hypertension, coronary artery disease or any other known cardiac diseases. Although the pathogenesis of diabetic cardiomyopathy is poorly understood, a variety of mechanisms contribute to its development, including microangiopathy, myocardial fibrosis, perturbations in cardiac energy metabolism, oxidative damage and cell death (Fiordaliso et al. 2000;
∗
Y. Wang and L. Ebermann contributed equally to this work.
DOI: 10.1113/expphysiol.2008.044800
Cai & Kang, 2001; Monkemann et al. 2002; Feuvray, 2004; Tschope et al. 2004). Mitochondria are the primary source of energy generation within the cell. Electrons from various substrates are obtained from fatty acid β-oxidation, glycolysis and the tricarboxylic acid cycle and feed the electron transport chain, where ATP is ultimately generated within the mitochondria. The adenine nucleotide translocase (ANT) is the carrier that facilitates the transport of ADP and ATP across the inner mitochondrial membrane. The dimeric ANT complex of rodents is nuclear encoded by three distinct genes (ANT1, ANT2 and ANT4) that are coexpressed in tissue-specific C 2008 The Authors. Journal compilation C 2009 The Physiological Society
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patterns (Dorner et al. 1999; Rodic et al. 2005). In the heart ANT1 is the predominant isoform. In addition to its role in linking mitochondrial energy production and cytosolic energy consumption, ANT1 is a binding partner of pro-apoptotic and anti-apoptotic proteins (Belzacq et al. 2003), transcription factors (Bottero et al. 2001) and cell-signalling proteins (Sharer et al. 2002) and thus is also involved in the regulation of apoptosis and intracellular communication. A deficit of ANT has been implicated in the pathogenesis of myocardial remodelling and cardiac insufficiency (Portman, 2002). ANT1 knockout mice have biochemical, histological, metabolic and physiological characteristics of cardiomyopathy, resulting from a severe cardiac energy deficit (Graham et al. 1997). Inhibition of mitochondrial ANT has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing reactive oxygen species and adenosine (Ciapaite et al. 2006). Defects in structure and function of mitochondria were also observed in type 1 diabetic hearts (Shen et al. 2004). In diabetes mellitus, the restricted glucose uptake and glycolysis lead to a switch of energy source from multiple substrates to fatty acids only. The altered metabolism is currently considered as a factor in the development of diabetic cardiomyopathy (An & Rodrigues, 2006). Our recent studies showed that myocardial ANT1 overexpression protected against hypertension-induced cardiac pathology in rats (Walther et al. 2007). Thus, improvement of mitochondrial function may be a basic principle for new strategies in treating heart diseases. To test the hypothesis that ANT1 overexpression in mitochondria also has a cardioprotective role in diabetic cardiomyopathy, we generated transgenic (TG) mice selectively overexpressing ANT1 in cardiomyocytes. These TG mice and their age-matched wild-type (WT) littermates were subjected to type 1 diabetes induced by streptozotocin (STZ), and haemodynamic characteristics were investigated. Methods
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mice according to our standard protocol (Wang et al. 2007) by a commercial provider (RCC Ltd, F¨ullinsdorf, Switzerland). Southern blot
Southern blot was used to screen potential founder lines and to determine the integrated copy number in founder lines. To detect copy number of the integrated cDNA, 5, 10 and 20 μg of genomic DNA were digested by BamHI or BamHI/SspI to give similar sized fragments of endogenous and transgene ANT1, respectively. The ANT1 PCR product was labelled and used as a southern probe as described before (Walther et al. 1998). Reverse transcriptase-polymerase chain reaction (RT-PCR)
Semi-quantitative RT-PCR was used to detect the mRNA abundance of αMHC–ANT1 (forward primer 5 -GTGGTGCCTCGTTCCAGCTG-3 , reverse primer 5 CAGTTTGACCCTCTCGATCGG-3 ) in different tissues. The mRNA was normalized to β-actin mRNA levels (forward primer 5 -AGGGAAATCGTGCGTGACAT-3 , reverse primer 5 -CATCTGCTGGAAGGTGGACA-3 ). Amplification was performed in a thermal cycler at 94◦ C for 30 s, at 55◦ C for 30 s and at 72◦ C for 1 min for 36 cycles. Real-time PCR
Quantitative real-time PCR was performed using the Light Cycler system (Roche, Mannheim, Germany) to quantify ANT1 and ANT2 mRNA expression. Total RNA was isolated from myocardium, and cDNA was generated. R Gene The ANT1 mRNA was quantified using TaqMan Expression Assay from Applied Biosystems (Darmstadt, Germany). Gene expression was normalized in relation to the expression of the endogenous housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1), also R Gene Expression Assay from Applied using TaqMan Biosystems (Foster City, CA, USA).
Generation of transgenic mice
All animal studies were performed according to the guidelines of the Federal Law on the Use of Experimental Animals in Germany and were approved by local authorities. Mouse ANT1 coding sequence was amplified by polymerase chain reaction (PCR) using reversetranscribed cardiac complementary DNA (cDNA) as template and subcloned into a vector containing rat α-myosin heavy chain (αMHC) gene promoter and the bovine growth hormone (BGH) polyadenylation signal. Linearized DNA fragment was microinjected into the pronuclei of fertilized oocytes isolated from C57BL/6 C 2008 The Authors. Journal compilation C 2009 The Physiological Society
Mitochondrial isolation
A mitochondria-enriched fraction was isolated from the hearts according to the method of Smith (1967). Mitochondrial protein concentration was determined by bicinchoninic acid test (Pierce, Bonn, Germany). Western blot analysis
Mitochondrial protein (3 μg) was separated by electrophoresis on a 12% polyacrylamide gel and blotted
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onto a polyvinylidene difluoride membrane. Western blots were performed using a standard protocol with a specific antibody raised in rabbits against ANT1 (1:750 dilution; Dorner et al. 2006). As secondary antibody, horseradish peroxidase-conjugated goat antirabbit immunoglobulin G (Dako, Glostrup, Denmark) was used at a dilution of 1:10 000. Induction of diabetes
Diabetes mellitus was induced in 5- to 6-month-old male transgenic mice and their wild-type littermates by daily low-dose intraperitoneal injections of STZ (50 mg STZ in 0.1 M citrate buffer, pH 4.5, per kg body weight) for 5 days. The sham group received citrate buffer injection only. One and 6 weeks later, a one-drop blood sample was obtained from all mice from the tip of the tail for the determination of blood glucose concentration by using a reflectance meter (Accu-Chek, Roche Diagnostics GmbH, Mannheim, Germany).
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ANP (290 nucleotides) antisense probe complementary to target mRNA. The RNA complementary to 127 nucleotides of rL32 mRNA was used as a positive control. Ten micrograms of each RNA sample was hybridized with approximately 40 000 c.p.m. of ANP- and 40 000 c.p.m. of rL32-radiolabelled antisense probe in the same reaction. The hybridized fragments, protected from RNase A + T1 digestion, were separated by electrophoresis on a denaturing gel (5% w/v polyacrylamide, 8 M urea) and analysed using a FUJIX BAS 2000 Phospho-Imager system (Raytest GmbH, Straubenhardt, Germany). Quantitative analyses were performed by measuring the intensity of the target bands normalized to the intensity of rL32. Statistical analysis
All data are expressed as means ± S.E.M. Differences between two groups were determined using unpaired Student’s t test. Multiple-parameter comparisons were performed by two-way ANOVA followed by Bonferroni’s post hoc test. A value of P < 0.05 was considered statistically significant.
Haemodynamic characterization
Six weeks after injecting STZ or vehicle, general anaesthesia was induced by 4% isoflurane and then mice were intubated and artificially ventilated with a mixture of 70% room air and 30% oxygen using a small animal ventilator (TSE Systems, Bad Homburg, Germany), to which 2% isoflurane was added for anaesthesia, as described previously (Wang et al. 2007). A 1.4 F-microtipped pressure transducer catheter (Millar Instruments, Houston, TX, USA) was inserted into the left ventricular (LV) lumen via the right carotid artery to measure arterial and LV pressure. Subsequently, baseline recordings were obtained for systolic blood pressure (SBP), heart rate (HR), LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP). In addition, we measured the LV contractility parameter LV dP/dt max and relaxation parameter LV dP/dt min . After the experiment, mice were killed by excision of the heart under general anaesthesia; heart, lung and kidneys were weighed and snap-frozen in liquid nitrogen for further analysis. RNase-protection assay (RPA) R Total RNA was isolated from tissues using TRIzol reagent (Invitrogen GmbH, Karlsruhe, Germany) with subsequent chloroform–isopropanol extraction according to the manufacturer’s instructions. Atrial natriuretic peptide (ANP) mRNA expression was identified by RPA using the Ambion RPA II kit (Ambion [Europe] Ltd, Huntingdon, UK) as described elsewhere (Walther et al. 2007). In brief, SP6-RNA polymerase transcribed radioactive
Results Generation and characterization of ANT1-transgenic mice
Transgenic mice selectively overexpressing ANT1 in cardiomyocytes, which was controlled by α-MHC promoter, were generated. Integration of ANT1 transgene into chromosomal DNA was confirmed by southern blot, and different lines were detected according to the distinct pattern of integration (data not shown). Two lines, 17 and 20, have been chosen for further basal characterization. The copy number of the transgenic construct in both lines was comparable, with approximately 10–12 copies (Fig. 1A), and the latter line was selected for the present studies. Both male and female mice from line 20 were vital and fertile, and generated normal litter size. Body weight did not differ from their non-transgenic control littermates after weaning until the defined end-point of 12 months. There was also no difference in 1 year survival rate until this end-point. To exclude ectopic transgene expression, we detected ANT1 mRNA expression in different organs from TG mice and their WT littermates. As expected, two different splicing variants with or without 5 untranslated region of ANT1 mRNA (522 and 175 bp, respectively, estimated ratio 1:2) were specifically detected in the transgenic heart, but not in any of the other investigated organs of transgenic mice (Fig. 1B). To investigate ontogenetic regulation, the mRNA expression level of ANT1 was evaluated in mice aged from 1 day to 20 months. At all investigated C 2008 The Authors. Journal compilation C 2009 The Physiological Society
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time points, ANT1 expression was significantly higher compared with age-matched WT control mice (Fig. 1C). Consequently, ANT1 mRNA overexpression resulted in significantly more ANT1 protein in transgenic mitochondria (Fig. 1D). Importantly, overexpression of ANT1 in cardiomyocytes did not affect the copy number of mitochondrial DNA (data not shown).
Overexpression of ANT1 prevents significant deterioration of cardiac function
Haemodynamic measurement was performed to assess cardiac function 6 weeks after injection of STZ or citrate buffer. Under baseline conditions, all investigated parameters showed no significant differences between WT and TG mice. Although the blood glucose concentrations were comparable in diabetic wild-type and transgenic mice, the extent of cardiac function impairment induced by hyperglycaemia was different compared with the non-diabetic control mice. An indicator of left ventricular contraction function, LVSP, was significantly reduced in diabetic wild-type mice 6 weeks after induction of diabetes (Fig. 3A). However, overexpression of ANT1 in cardiomyocytes prevented this significant deterioration (Fig. 3A). This improvement was also seen in the measurements of systolic blood pressure. A high blood glucose level led to a significant decrease of SBP in WT mice, but no change was observed in transgenic mice (Fig. 3B). The LV contraction parameter LV dP/dt max (Fig. 3C) and LV relaxation parameter LV dP/dt min (Fig. 3D) were also significantly impaired in diabetic WT mice, while there was no significant difference in diabetic mice overexpressing ANT1 in the heart compared with their normoglycaemic control mice.
Basic characteristics of diabetic mice
Throughout the 6 week study period, both WT and ANT1-TG mice that received STZ developed severe hyperglycaemia (Fig. 2A), loss of body weight (Fig. 2B) and an increase in the ratio of kidney weight to body weight (Fig. 2C), with no significant differences between the WT and TG lines. The ventricular mass as an absolute value was significantly decreased in the wild-type STZ group compared with the corresponding non-diabetic control mice (data not shown). This loss of cardiac weight was still significant if calculated as heart-to-body weight ratio (Fig. 2D). However, hyperglycaemia did not alter the ratio of heart weight to body weight (Fig. 2D) or heart weight in transgenic mice specifically overexpressing ANT1 in cardiomyocytes.
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Figure 1. Selective overexpression of ANT1 in transgenic mouse hearts A, integrated DNA copy numbers in transgenic lines 17 and 20 were determined by southern blot. Different concentrations of genomic DNA were digested by BamHI or BamHI/SspI to give similar sized fragments of endogenous and transgene ANT1, respectively. B, specific overexpression of ANT1 mRNA in TG mice hearts was confirmed by semi-quantitative RT-PCR. C, ontogenetic regulation of ANT1 mRNA in wild-type (open bars) and transgenic hearts (filled bars) from 1-day-old to 20-month-old mice was investigated by real-time PCR. The ANT1 expression was normalized to that of the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1). n = 3–4. D, Western blot was used to determine mitochondrial ANT1 protein levels in 3- and 24-month-old mice, and data were expressed as x−fold to WT, n = 4–6. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. WT. C 2008 The Authors. Journal compilation C 2009 The Physiological Society
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Figure 2. Basic characteristics of diabetic and non-diabetic mice A, hyperglycaemia in STZ-treated WT and ANT1-TG mice was confirmed by blood glucose measurement. B, body weight changes are shown in diabetic and non-diabetic WT and ANT1-TG mice 6 weeks after induction of diabetes. C, ratios of kidney to body weight are shown in WT and TG mice 6 weeks after injection of STZ or citrate buffer. D, ratio of heart weight to body weight is shown for diabetic and non-diabetic mice. n = 11 (WT control), 14 (WT STZ), 10 (TG control) and 13 (TG STZ). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. corresponding control column.
To further characterize effects of diabetes induced by STZ on ANT expression, both ANT1 and ANT2 mRNA expression was quantitatively analysed by real-time PCR. In transgenic mice, ANT1 mRNA was significantly
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increased, as already described. However, although ANT1 is the main isoform in the heart, diabetes did not alter its expression in either wild-type or transgenic mice (Fig. 4A). In sham conditions, cardiac overexpression of ANT1 did
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Figure 3. Haemodynamic parameters in diabetic and non-diabetic mice Cardiac function parameters LVSP (A), SBP (B), LV contractility parameter LV dP/dt max (C) and LV relaxation parameter LV dP/dt min (D) were measured in both WT and ANT1-TG mice receiving citrate buffer (open bars) or STZ (filled bars). n = 7 (WT control), 11 (WT STZ), 7 (TG control) and 10 (TG STZ). ∗ P < 0.05 vs. corresponding control column. C 2008 The Authors. Journal compilation C 2009 The Physiological Society
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not alter ANT2 expression (WT, 100 ± 2.7% vs. TG, 92.7 ± 5.2%; P = 0.22; n = 5–6; Fig. 4B). Nevertheless, ANT2 expression was significantly induced by STZ in both groups (Fig. 4B). In accordance with the haemodynamic parameters, the mRNA of ANP, a well-known molecular marker of diabetic cardiomyopathy (Howarth et al. 2005), was upregulated 6 weeks after induction of diabetes in WT mice, but no significant difference was detected in diabetic ANT1-transgenic mice (Fig. 4C and D). Although the rise in cardiac ANP mRNA is more pronounced in diabetes than the increase in mRNA of B-type natriuretic peptide (BNP), a more common marker of heart failure, BNP mRNA was also significantly increased in diabetic wildtype mice but not in hyperglycaemic transgenic mice (data not shown).
phenotype of diabetic cardiomyopathy (An & Rodrigues, 2006). This suggestion is strongly supported by a series of studies using genetically modified animal models, increasing fatty acid uptake (Yagyu et al. 2003), transport (Chiu et al. 2005) and oxidation (Chiu et al. 2001; Finck et al. 2002), leading to a similar cardiac phenotype to that seen in diabetic cardiomyopathy. Mitochondrial structural damage and metabolic dysfunction have also been implicated in diabetic complications (Shen et al. 2004; Ciapaite et al. 2006). Therefore, it is reasonable to postulate mitochondria as an important target of diabetes in hearts. Our recent studies demonstrated that accelerating ADP/ATP transport by overexpression ANT1 in rat hearts significantly improved hypertension-induced heart disease. Therefore, we hypothesized that accelerating ADP/ATP transport across the inner mitochondrial membrane may also ameliorate diabetic cardiomyopathy. Thus, we generated transgenic mice selectively overexpressing ANT1 in cardiomyocytes, with mitochondrial ANT1 increased by approximately 40%, and treated them with STZ to induce diabetes. Although no differences were observed in the level of hyperglycaemia and the body weight loss between wild-type and transgenic mice 6 weeks after injection of STZ, the overexpression of ANT1 prevented the significant decrease in ventricular weight observed in diabetic WT mice. In agreement with previous reports in STZ-induced diabetic animal models (Nielsen et al. 2002; Pacher et al. 2002), both systolic and diastolic function were significantly impaired in WT mice receiving
Discussion Although diabetic cardiomyopathy is a common complication of diabetes and can also lead to heart failure, its pathogenesis is still not fully understood. Among the postulated mechanisms, disturbed energy metabolism is thought to play an important role in the development of diabetic cardiomyopathy (Carvajal & Moreno-Sanchez, 2003; An & Rodrigues, 2006). Cardiac function depends on sufficient supply of ATP generated in cellular mitochondria. In diabetes, cardiac metabolism rapidly switches from glucose to fatty acid, and high fatty acid uptake and metabolism may contribute to the
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Figure 4. Molecular analysis of diabetic cardiomyopathy Quantification of ANT1 (A) and ANT2 (B) by real-time PCR was expressed as a percentage of the values in WT control mice. C, RNase-protection assay showing ANP expression in ventricles of WT and ANT1-TG mice 6 weeks after injection of STZ or citrate buffer. D, quantification of ANP mRNA expression in hearts normalized to rL32 mRNA levels. ∗ P < 0.05 and ∗∗ P < 0.01 vs. corresponding control column; and ###P < 0.001 vs. corresponding WT control. C 2008 The Authors. Journal compilation C 2009 The Physiological Society
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STZ in comparison with their normoglycaemic control mice; however, there was no significant deterioration detected in hyperglycaemic mice overexpressing ANT1 in cardiomyocytes. Furthermore, this cardioprotective effect of ANT1 overexpression was further supported by a lack in elevation of ANP mRNA, which is an mRNA marker of cardiac failure. Thus, this is the first report to describe beneficial effects of ANT1 on diabetic cardiomyopathy using a transgenic animal model. Although the long-term effects of ANT1 overexpression on diabetic cardiomyopathy need to be addressed in further research, our data clearly support the assumption that stimulation of ANT1 expression could be a basic principle for the treatment of diabetic cardiomyopathy through improving mitochondrial function. While an insufficient ANT function contributes to cardiac damage, a rise in ANT expression has currently been discussed as part of a cardioprotective programme that occurs during ischaemic and hypothermic preconditioning (Ning et al. 1998; McLeod et al. 2004). Gene expression of mitochondrial proteins, such as cytochrome oxidase, cytochrome c, β-F(1) ATPase and ANT1, which were seen to be downregulated in failing hearts, is stabilized by ischaemic preconditioning (Ning et al. 1998; McLeod et al. 2004). In addition, Bcl-2, an anti-apoptotic member of the Bcl-2 protein family, increases ANT transport activity (Belzacq et al. 2003), and overexpression of Bcl-2 improves the energy state of ischaemic hearts (Imahashi et al. 2004). Thus, stressed myocardium is able to run a cardioprotective gene programme, including increased ANT1 expression, with the final intention of stabilizing mitochondrial integrity and thereby securing the essential energy supply. However, in the present study, quantification of Bcl-2 and Bcl-2 associated X protein (Bax) showed no difference between WT and TG mice (data not shown). A considerable amount of evidence suggests that disturbed energy metabolism can induce apoptosis of cardiac myocytes, which may also contribute to the progressive development of diabetic cardiomyopathy (Ghosh & Rodrigues, 2006). Fiordaliso et al. (2004) reported that type 1 diabetes reduced myocyte numbers by 29%, and the number of dead and apoptotic myocytes increased 5.0- and 6.7-fold, respectively, in rats 3 months after injection of STZ. Consistent with their findings, in the present study, heart weight was also significantly decreased in wild-type diabetic mice in comparison with their control littermates. Since comparable hyperglycaemia did not change heart weight in ANT1-transgenic mice, our data imply that overexpression of ANT1 in mitochondria may be a potential inhibitor of diabetesinduced cardiomyocyte apoptosis or necrosis. Whether or not ANT1 overexpression can also inhibit the reduction of cardiomyocyte size in diabetic mice needs further investigation.
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In conclusion, the present study provides direct in vivo evidence that increased expression of ANT1 in mitochondria can ameliorate significant deterioration of cardiac function 6 weeks after induction of type 1 diabetes in mice. The authors cannot completely exclude the possibility that the long-term overexpression of ANT1 in cardiomyocytes may up- or downregulate other proteins with impact on cardiac function under hyperglycaemic conditions. Thus, follow-up research should focus on the development of pharmaceutical approaches to test our hypothesis and to develop new pharmaceutical strategies to activate cardiac ANT1 in patients with diabetes. Further work is needed to investigate the molecular mechanisms responsible for the significant cardioprotective effect of overexpressed ANT1 to build a bridge between our experimental findings and new clinical therapies. However, the illustrated beneficial effects of ANT1 overexpression in the heart on diabetic cardiomyopathy could provide new insights into therapeutical strategies for management of diabetic complications.
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Acknowledgements L.E. was supported by a PhD scholarship from the Freie Universit¨at, Berlin, Germany.