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Mitochondrial transplantation for myocardial protection in diabetic hearts

Mitochondrial transplantation for myocardial protection in diabetic hearts Abstract OBJECTIVES Type 2 diabetes causes mitochondrial dysfunction, which increases myocardial susceptibility to ischaemia–reperfusion injury. We investigated the efficacy of transplantation of mitochondria isolated from diabetic or non-diabetic donors in providing cardioprotection from warm global ischaemia and reperfusion in the diabetic rat heart. METHODS Ex vivo perfused hearts from Zucker diabetic fatty (ZDF fa/fa) rats (n = 6 per group) were subjected to 30 min of warm global ischaemia and 120 min reperfusion. Immediately prior to reperfusion, vehicle alone (VEH) or vehicle containing mitochondria isolated from either ZDF (MTZDF) or non-diabetic Zucker lean (ZL +/?) (MTZL) skeletal muscle were delivered to the coronary arteries via the aortic cannula. RESULTS Following 30-min global ischaemia and 120-min reperfusion, left ventricular developed pressure was significantly increased in MTZDF and MTZL groups compared to VEH group (MTZDF: 92.8 ± 5.2 mmHg vs MTZL: 110.7 ± 2.4 mmHg vs VEH: 44.3 ± 5.9 mmHg; P < 0.01 each); and left ventricular end-diastolic pressure was significantly decreased (MTZDF 12.1 ± 1.3 mmHg vs MTZL 8.6 ± 0.8 mmHg vs VEH: 18.6 ± 1.5 mmHg; P = 0.016 for MTZDF vs VEH and P < 0.01 for MTZL vs VEH). Total tissue ATP content was significantly increased in both MT groups compared to VEH group (MTZDF: 18.9 ± 1.5 mmol/mg protein/mg tissue vs MTZL: 28.1 ± 2.3 mmol/mg protein/mg tissue vs VEH: 13.1 ± 0.5 mmol/mg protein/mg tissue; P = 0.018 for MTZDF vs VEH and P < 0.01 for MTZL vs VEH). Infarct size was significantly decreased in the MT groups (MTZDF: 11.8 ± 0.7% vs MTZL: 9.9 ± 0.5% vs VEH: 52.0 ± 1.4%; P < 0.01 each). CONCLUSIONS Mitochondrial transplantation significantly enhances post-ischaemic myocardial functional recovery and significantly decreases myocellular injury in the diabetic heart. Mitochondrial transplantation , Myocardial ischaemia–reperfusion injury , Diabetes INTRODUCTION Type 2 diabetes (T2D) affects 30 million people in the USA and is increasing by ∼1.5 million per year [1]. There are sufficient data to show that T2D increases the susceptibility of the myocardium to ischaemia–reperfusion injury (IRI) [2]. We and others have demonstrated that mitochondria play a key role in the modulation of IRI in the heart [3–6]. Our studies have shown that IRI detrimentally affects mitochondrial function and cellular energetics. These events occur during ischaemia and persist despite the restoration of coronary blood flow and tissue reperfusion, ultimately negatively impacting post-ischaemic myocardial contractile function and cellular viability [3–6]. These effects are exacerbated by T2D and significantly increase morbidity and mortality in T2D patients requiring cardiac interventions [7]. Recently, we have pioneered a novel therapy, mitochondrial transplantation, that replaces native mitochondria damaged by IRI with viable, structurally intact, respiration competent mitochondria isolated from non-ischaemic tissue obtained from the patient’s own body [3–6, 8–16]. Mitochondrial transplantation has been clinically proven to enhance post-ischaemic cardiac viability and function. We have validated the efficacy of mitochondrial transplantation in vitro and in perfused large animal heart models, and finally in human paediatric cardiac patients [4, 8–11, 14, 15, 17]. Our studies have demonstrated that there is no direct or indirect, acute or chronic alloreactivity, allorecognition or damage-associated molecular pattern reaction to single or serial injections of either syngeneic or allogeneic mitochondria. These studies demonstrated that heterologous mitochondrial transplantation is safe and that there is no immunoreaction between the donor mitochondria and the host [18]. We hypothesized that mitochondrial transplantation will significantly enhance myocardial function, viability and energy production following IRI in the ex vivo isolated perfused diabetic heart. In this study, we investigate the efficacy of mitochondrial transplantation isolated from diabetic or non-diabetic donors in providing cardioprotection from IRI in a model of T2D. MATERIALS AND METHODS Animal care and biosafety This investigation was conducted under the National Institutes of Health’s guidelines on animal care and use and was approved by the Boston Children’s Hospital’s Animal Care and Use Committee (Protocol no. 15-01-2722) [19]. Animal model Zucker diabetic fatty rats (ZDFLeprfa/Crl; ZDF) and non-diabetic Zucker lean rats (Lean fa/+; ZL) were used [20]. All rats were males, aged to 14 weeks old (n = 24), obtained from Charles River Laboratory (Wilmington, MA, USA) and had an average weight of 344 ± 16 g. ZDF rats develop T2D at the age of 12 weeks, which is fully manifested at 14 weeks and lasts up to 24 weeks [20]. ZL rats do not spontaneously develop T2D and maintain normal serum glucose and normal body weight [7, 20]. Experimental model All animals were anaesthetized in an isoflurane chamber (2–4%). Following confirmation of anaesthesia, the chest was opened with an inverted T-shaped incision. Heparin (500 U.I.) was injected into the abdominal aorta and allowed to circulate for 10 s. The heart was removed enbloc with the lungs. Upon harvesting of the heart, the pectoralis major muscle was dissected, and two #6 biopsy punch samples were removed for the isolation of mitochondria [8]. Mitochondrial isolation Tissue samples were obtained from the pectoralis major using a 6-mm biopsy punch. Tissue weight was 0.18 ± 0.04 g (wet weight). Mitochondria were isolated with the use of a commercial tissue dissociator and differential filtration in <30 min as previously described [8]. The isolated mitochondria were suspended in a vehicle [250 mmol/l sucrose, 20 mmol/l K+-HEPES buffer (pH 7.2), 0.5 mmol/l K+-EGTA (pH 8.0)] and were used for mitochondrial transplantation [3, 6]. Langendorff perfusion The heart was excised and placed in a 4°C bath of Krebs-Ringer containing 100 mM NaCl, 4.7 mM KCl, 1.1 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 1.7 mM CaCl2, 11.5 mM glucose, 4.9 mM pyruvic acid and 5.4 mM fumaric acid bubbled with 5% CO2 and 95% O2 (pH 7.4 at 37°C), where spontaneous beating ceased within seconds. The heart was then immediately secured onto a metallic cannula and perfused in the Langendorff mode as previously described [4]. Hearts were perfused at a constant pressure of 60 mmHg. The temperature of the perfusate was maintained at 37°C. Hearts were paced through the right ventricle at 180 beats/min. Coronary flow was measured by collecting coronary effluent from the pulmonary artery. Myocardial function assessment A latex balloon (Harvard Apparatus, MA, USA) sewn onto a fluid-filled catheter was inserted through the left atrial appendage and the mitral valve into the left ventricle. The volume of the water-filled balloon was set at a constant end-diastolic physiological pressure of 6–8 mmHg using a calibrated micro-syringe during equilibrium, and this balloon volume was maintained for the duration of the experiment. The perfusion pressure, temperature, heart rate, left ventricular developed pressure (LVdevP, mmHg), left ventricular end-diastolic pressure (LVEDP, mmHg) and maximum derivative of left ventricular pressure (dP/dt max, mmHg/s) were recorded continuously using a data acquisition hardware (PowerLab/8 SP by AD Instruments, Oxford, UK) and readings were visualized using the LabChart software (v7.3.8, AD Instruments, Oxford, UK). Experimental protocol The experimental protocol is shown in Fig. 1. Only hearts harvested from ZDF rats were used for ex vivo Langendorff perfusion. Hearts were perfused for 15 min (equilibration period for baseline data acquisition). Following 15 min of equilibrium, ZDF hearts were subjected to temporary warm global ischaemia by cross-clamping the aorta and submerging the heart in 37°C Krebs-Ringer solution. Figure 1: Open in new tabDownload slide Experimental protocol. Hearts were harvested from ZDF rats (n = 18) and perfused in the Langendorff mode. After 15 min of equilibration, warm temporary global ischaemia was induced by cross-clamping the aorta. Following 30 min of warm global ischaemia, the cross-clamp was released and the hearts were reperfused. Immediately at the onset of reperfusion, 1 ml of vehicle alone or 1 ml of vehicle containing mitochondria isolated from either ZDF (MTZDF) or ZL rats (MTZL) was delivered through the aortic cannula. Mitochondria were transplanted at 2 × 105 ± 0.3 × 105 mitochondria/g wet weight. Hearts were reperfused for 120 min and then used for biochemical and histological analysis. Myocardial function assessment time points are annotated (red triangles). ZDF: Zucker diabetic fatty; ZL: Zucker lean. Figure 1: Open in new tabDownload slide Experimental protocol. Hearts were harvested from ZDF rats (n = 18) and perfused in the Langendorff mode. After 15 min of equilibration, warm temporary global ischaemia was induced by cross-clamping the aorta. Following 30 min of warm global ischaemia, the cross-clamp was released and the hearts were reperfused. Immediately at the onset of reperfusion, 1 ml of vehicle alone or 1 ml of vehicle containing mitochondria isolated from either ZDF (MTZDF) or ZL rats (MTZL) was delivered through the aortic cannula. Mitochondria were transplanted at 2 × 105 ± 0.3 × 105 mitochondria/g wet weight. Hearts were reperfused for 120 min and then used for biochemical and histological analysis. Myocardial function assessment time points are annotated (red triangles). ZDF: Zucker diabetic fatty; ZL: Zucker lean. Experimental groups Following 15 min of equilibrium and 30 min of warm global ischaemia, the cross-clamp was released and the hearts received either vehicle alone (1 ml, VEH, n = 6) or vehicle containing mitochondria and the hearts were reperfused for 120 min [8]. Mitochondria were isolated from either ZDF rats (MTZDF, n = 6) or ZL rats (MTZL, n = 6). ZDF mitochondria were isolated from the same rats used for harvesting the hearts. Mitochondria delivery For delivery of the mitochondria, 1 ml of vehicle solution containing 2 × 105 ± 0.3 × 105 mitochondria/g wet weight was resuspended in 2 ml of 37°C Krebs-Ringer solution and administered antegradely to the coronary arteries through the aortic cannula over 10 s. Mitochondrial uptake 18F-Rhodamine 6G-labelled mitochondria In a separate set of experiments, ZDF rats (n = 4) were used for visualization of mitochondrial uptake in the heart. A donor rat (n = 1) was used to isolate syngeneic mitochondria from the pectoralis major muscle tissue as previously described [13, 14]. The isolated mitochondria were labelled with 18F-rhodamine 6G. Rats (n = 3) were anaesthetized and maintained on 2–3% inhaled isoflurane. A sternotomy was performed, and the ascending aorta was exposed. The 18F-rhodamine 6G-labelled mitochondria (1 × 109 in 1 ml respiration buffer) were delivered antegrade to the coronary arteries via injection to the coronary ostium using a tuberculin syringe with a 30-G needle. Ten minutes after the injection, the animals were euthanized in a CO2 chamber and examined using positron emission tomography (PET) and microcomputed tomography (μCT) [14]. Human mitochondria In a separate set of ZDF rats (n = 3), 1 × 109 mitochondria were isolated from human cardiac fibroblasts and were delivered retrogradely to the coronary arteries through the aortic cannula. After 10 min, tissue was collected. The use of human mitochondria in rodent heart tissue allowed differentiation between endogenous rat mitochondria and transplanted human mitochondria based on immune reactivity to a monoclonal anti-human mitochondrial antibody (biotin, MTCO2; Abcam, Cambridge, MA, USA) and visualized using Vectastain (Vector Laboratories, Burlingame, CA, USA) as previously described [13, 14]. Histopathological analysis and transmission electron microscopy Following 120 min of reperfusion, hearts were removed from the cannula, sectioned at 1-cm thickness and stored for further analysis. Myocardial tissue samples were fixed in 10% formalin and paraffin-embedded for histopathological analysis. Serial slides were used for haematoxylin and eosin staining and Masson’s trichrome staining. Haematoxylin and eosin-stained slides were evaluated for the area of necrosis and inflammatory cell infiltration. Masson’s trichrome stained slides were evaluated for cardiac fibrosis as previously described [3, 4, 13]. Transmission electron microscopy was used to analyse structural damage in the heart grafts, as described in previous studies [3, 4, 6]. Myocardial tissue samples were collected and fixed in 1.25% formaldehyde, 2.5% grade I glutaraldehyde and 0.03% picric acid. All analyses were performed by a blinded observer. Measurement of infarct size The infarct size was determined using 1% triphenyl tetrazolium chloride (Sigma-Aldrich, St. Louis, MO, USA) and the percentage of the area of infarcted tissue per total area of cardiac tissue was calculated digitally with ImageJ (https://imagej.nih.gov/ij/) as previously described [13, 15]. The determination of wet weight-to-dry weight ratios was also performed as previously described [4, 13]. ATP Following 120 min of reperfusion, myocardial tissue samples were collected, snap-frozen in liquid nitrogen and stored in −80°C for ATP and protein content analysis. ATP was determined using the Colorimetric ATP Assay Kit (Abcam) according to the manufacturer’s instructions [21]. Myocardial protein per mg wet weight was determined using the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. All samples were run in triplicate. ATP content was expressed as mmol/mg protein/mg tissue (wet weight). Statistical analysis The normality of all continuous variables was tested using the Shapiro–Wilk test and graphically assessed by histograms and Q–Q plots. Box-and-whisker plots and the non-parametric Mann–Whitney U-test followed by Bonferroni adjustment were used for comparing glucose, body weight, heart weight, heart-to-body weight ratio, mitochondria ATP, ATP content of the myocardial tissue, dry-to-wet weight and infarct size. All other variables conformed to a normal Gaussian-shaped distribution and are presented as mean ± standard error of the mean. For functional outcome variables (LVdevP, LVEDP, dP/dt max and CBF) application of parametric statistical tests was opted for to provide a more powerful and robust analysis for within- and between-group analysis throughout the experimental period. Longitudinal analysis for between-groups comparisons was performed using 2-way repeated-measures analysis of variance within the framework of fitting mixed-effects linear regression models. To reduce the probability of false-positive results (type I error) due to multiple comparisons, Benjamini and Hochberg's false discovery rate was applied to control the family-wise error to α <0.05. All tests reported are 2-tailed. Statistical analyses were performed with GraphPad Prism version 7.0 for Mac OS X (GraphPad Software, La Jolla, CA, USA) [22]. RESULTS Type 2 diabetes model ZDF 14-week-old male rats had significantly increased body weight (P < 0.001), heart-to-body weight ratio (P < 0.001) and blood glucose (P < 0.001) when compared to ZL male rats of the same age (Fig. 2A–C). ATP content was significantly lower (P < 0.001) in the mitochondria isolated from ZDF rats as compared to ZL rats (Fig. 2D). There was no difference, in body weight, heart weight, blood glucose and heart -to-body weight ratio between the 3 experimental groups of ZDF rats before ex vivo perfusion (P > 0.05) (Table 1). Figure 2: Open in new tabDownload slide Body and heart weight, blood glucose and mitochondrial ATP content. Box plots show data for (A) body weight (g), (B) heart-to-body weight ratio and (C) blood glucose (mg/dl) for 14-week-old ZDF rats and ZL rats. (D) ATP content of 1 × 109 mitochondria isolated from either ZDF or ZL rats is shown (n = 6 per group) with P-values obtained from Mann–Whitney U-tests. *P < 0.05: ZDF versus ZL. ZDF: Zucker diabetic fatty; ZL: Zucker lean. Figure 2: Open in new tabDownload slide Body and heart weight, blood glucose and mitochondrial ATP content. Box plots show data for (A) body weight (g), (B) heart-to-body weight ratio and (C) blood glucose (mg/dl) for 14-week-old ZDF rats and ZL rats. (D) ATP content of 1 × 109 mitochondria isolated from either ZDF or ZL rats is shown (n = 6 per group) with P-values obtained from Mann–Whitney U-tests. *P < 0.05: ZDF versus ZL. ZDF: Zucker diabetic fatty; ZL: Zucker lean. Table 1: Blood glucose, heart and body weight and mitochondria ATP content in 14-week-old ZDF and ZL rats ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 Blood glucose (mg/dl), body weight (g), heart weight (g), heart-to-body weight ratio, heart wet-to-dry weight ratio and mitochondrial ATP content in 14-week-old ZDF and ZL rats. All data are shown as mean ± standard error of the mean. The number of animals in each group is shown as n, in brackets. P-values are shown for each value. Significant differences between groups at P-value <0.05 are shown in bold. MTZDF: vehicle and ZDF mitochondria; MTZL: vehicle and ZL mitochondria; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. Open in new tab Table 1: Blood glucose, heart and body weight and mitochondria ATP content in 14-week-old ZDF and ZL rats ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 Blood glucose (mg/dl), body weight (g), heart weight (g), heart-to-body weight ratio, heart wet-to-dry weight ratio and mitochondrial ATP content in 14-week-old ZDF and ZL rats. All data are shown as mean ± standard error of the mean. The number of animals in each group is shown as n, in brackets. P-values are shown for each value. Significant differences between groups at P-value <0.05 are shown in bold. MTZDF: vehicle and ZDF mitochondria; MTZL: vehicle and ZL mitochondria; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. Open in new tab Mitochondrial uptake PET/μCT imaging of 18F-rhodamine 6G-labelled mitochondria injected directly into the heart demonstrated specific distribution throughout the heart (Fig. 3A). Radiolabelled mitochondria were not detectable in any other organ or region of the body. Figure 3: Open in new tabDownload slide Uptake and distribution of transplanted mitochondria. (A) 18F-Rhodamine 6G-labelled mitochondria were distributed throughout the heart and were not detectable in any other region of the body. Images are shown for 3-dimensional reconstructed views. (B) Representative immunohistochemical images (magnification ×20) demonstrating mitochondrial uptake in the isolated perfused hearts. (C) Expanded box area of (B). The majority of transplanted mitochondria are indicated with white arrows. Scale bars = 100 μm. Figure 3: Open in new tabDownload slide Uptake and distribution of transplanted mitochondria. (A) 18F-Rhodamine 6G-labelled mitochondria were distributed throughout the heart and were not detectable in any other region of the body. Images are shown for 3-dimensional reconstructed views. (B) Representative immunohistochemical images (magnification ×20) demonstrating mitochondrial uptake in the isolated perfused hearts. (C) Expanded box area of (B). The majority of transplanted mitochondria are indicated with white arrows. Scale bars = 100 μm. Analysis of mitochondrial uptake revealed that injected human mitochondria were distributed primarily within myocardial fibres (Fig. 3B and C) [14]. Myocardial function LVdevP, dP/dt max and LVEDP were not significantly different between groups at baseline. Following 30-min global ischaemia and 120-min reperfusion, MTZDF and MTZL showed significantly increased myocardial function (Fig. 4A–C). LVdevP was 44.3 ± 5.9 mmHg in VEH and was significantly increased to 92.8 ± 5.2 mmHg in MTZDF (P < 0.001 vs VEH) and to 110.7 ± 2.4 mmHg in MTZL (P < 0.001 vs VEH). dP/dt max following 120 min reperfusion was significantly increased to 5414.8 ± 529.2 mmHg/s in MTZDF and to 6332.7 ± 568.5 mmHg/s in MTZL as compared to 2584.8 ± 401.9 mmHg/s in VEH (P < 0.01 each vs VEH). Figure 4: Open in new tabDownload slide Myocardial function during baseline, global ischaemia and reperfusion. (A) Left ventricular developed pressure (mmHg), (B) left ventricular end-diastolic pressure (mmHg), (C) dP/dt max (mmHg) and (D) coronary blood flow (ml/min) at baseline, warm global ischaemia and reperfusion in ZDF rat hearts receiving VEH or vehicle containing mitochondria from either ZDF rats (MTZDF) or ZL rats (MTZL). Injection is denoted by dashed vertical line. All results are shown as mean ± standard error of the mean for n = 6 per group. The P-values were obtained from 2-way repeated-measures analysis of variance using mixed-effects linear regression models and corrected by Benjamini and Hochberg’s false discovery rate. *P < 0.05 MTZDF versus VEH; #P < 0.05 MTZL versus VEH. BL: baseline; dP/dt max: maximum derivative of left ventricular pressure; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. Figure 4: Open in new tabDownload slide Myocardial function during baseline, global ischaemia and reperfusion. (A) Left ventricular developed pressure (mmHg), (B) left ventricular end-diastolic pressure (mmHg), (C) dP/dt max (mmHg) and (D) coronary blood flow (ml/min) at baseline, warm global ischaemia and reperfusion in ZDF rat hearts receiving VEH or vehicle containing mitochondria from either ZDF rats (MTZDF) or ZL rats (MTZL). Injection is denoted by dashed vertical line. All results are shown as mean ± standard error of the mean for n = 6 per group. The P-values were obtained from 2-way repeated-measures analysis of variance using mixed-effects linear regression models and corrected by Benjamini and Hochberg’s false discovery rate. *P < 0.05 MTZDF versus VEH; #P < 0.05 MTZL versus VEH. BL: baseline; dP/dt max: maximum derivative of left ventricular pressure; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. LVEDP was significantly increased to 18.6 ± 1.5 mmHg in VEH after 120 min of reperfusion as compared to 12.1 ± 1.3 mmHg in MTZDF (P = 0.016 vs VEH) and to 8.6 ± 0.8 mmHg in MTZL (P < 0.01 vs VEH). No difference was observed between MTZDF and MTZL groups in any of the myocardial function indices except for LVdevP after 90 min (P = 0.028) and 120 min (P = 0.021) of reperfusion. No difference in CBF was observed between groups throughout the experiment (P > 0.05) (Fig. 4D). ATP Total tissue ATP content of the cardiac tissue was significantly increased in both MT groups compared to the VEH group (MTZDF: 18.9 ± 1.5 mmol/mg protein/mg tissue vs MTZL: 28.1 ± 2.3 mmol/mg protein/mg tissue vs VEH: 13.1 ± 0.5 mmol/mg protein/mg tissue; P = 0.018 for MTZDF vs VEH and P < 0.01 for MTZL vs VEH) (Fig. 5A). Total tissue ATP content of cardiac tissue was higher in the MTZL group than in the MTZDF group (P < 0.01). Figure 5: Open in new tabDownload slide ATP content, dry-to-wet weight and infarct size. (A) ATP content mmol/mg protein/mg tissue wet weight, (B) dry/wet weight ratio of the cardiac tissue and (C) infarct size as % of total cardiac mass in ZDF rat hearts following 30 min of warm global ischaemia and 120 min of reperfusion. (D) Representative TTC stained ZDF rat hearts receiving VEH, or MTZDF rats or from MTZL rats following 30 min of warm global ischaemia and 120 min of reperfusion. Unstained regions (white) represent the infarcted myocardium. Scale bar = 1 cm. All results are shown for n = 6 per group and P-values were obtained from Mann–Whitney U-test with Bonferroni correction. *P < 0.05: MTZDF versus VEH; #P < 0.05: MTZL versus VEH; **P < 0.05: MTZDF versus MTZL; ns: no significant difference at P < 0.05 detected. MTZDF; mitochondria isolated from ZDF; MTZL; mitochondria isolated from Zucker lean; TTC: tetrazolium chloride; VEH: vehicle alone; ZDF: Zucker diabetic fatty. Figure 5: Open in new tabDownload slide ATP content, dry-to-wet weight and infarct size. (A) ATP content mmol/mg protein/mg tissue wet weight, (B) dry/wet weight ratio of the cardiac tissue and (C) infarct size as % of total cardiac mass in ZDF rat hearts following 30 min of warm global ischaemia and 120 min of reperfusion. (D) Representative TTC stained ZDF rat hearts receiving VEH, or MTZDF rats or from MTZL rats following 30 min of warm global ischaemia and 120 min of reperfusion. Unstained regions (white) represent the infarcted myocardium. Scale bar = 1 cm. All results are shown for n = 6 per group and P-values were obtained from Mann–Whitney U-test with Bonferroni correction. *P < 0.05: MTZDF versus VEH; #P < 0.05: MTZL versus VEH; **P < 0.05: MTZDF versus MTZL; ns: no significant difference at P < 0.05 detected. MTZDF; mitochondria isolated from ZDF; MTZL; mitochondria isolated from Zucker lean; TTC: tetrazolium chloride; VEH: vehicle alone; ZDF: Zucker diabetic fatty. Infarct size No significant difference was observed in the wet weight-to-dry weight ratio between groups (P > 0.05) (Fig. 5B). A significant reduction in infarct size was observed between the MTZDF and MTZL groups as compared to the VEH group (MTZDF: 11.8 ± 0.7% vs MTZL: 9.9 ± 0.5% vs VEH: 52.0 ± 1.4%; P < 0.01 each vs VEH) (Fig. 5C and D). There was no significant difference in infarct size between MTZDF and MTZL. Histology Haematoxylin and eosin and Masson’s trichrome showed increased longitudinal and transverse interfibrillar separation in VEH hearts (Fig. 6A) compared with MT hearts (Fig. 6B and C), suggesting increased diffuse myocardial tissue injury and oedema in the hearts that did not receive mitochondria. No difference in collagen content was observed between groups (Fig. 6D–F). Figure 6: Open in new tabDownload slide Haematoxylin and eosin and Masson’s trichrome. Representative haematoxylin and eosin and Masson’s trichrome stained micrographs of Zucker diabetic fatty (ZDF) rat hearts. (A) ZDF hearts receiving vehicle alone show increased longitudinal and transverse interfibrillar separation as compared to ZDF hearts receiving (B) mitochondria isolated from ZDF rats or (C) mitochondria isolated from Zucker lean rats. No difference in collagen content was observed in (D) vehicle alone, (E) mitochondria isolated from ZDF or (F) mitochondria isolated from Zucker lean hearts. Scale bars = 100 μm. Figure 6: Open in new tabDownload slide Haematoxylin and eosin and Masson’s trichrome. Representative haematoxylin and eosin and Masson’s trichrome stained micrographs of Zucker diabetic fatty (ZDF) rat hearts. (A) ZDF hearts receiving vehicle alone show increased longitudinal and transverse interfibrillar separation as compared to ZDF hearts receiving (B) mitochondria isolated from ZDF rats or (C) mitochondria isolated from Zucker lean rats. No difference in collagen content was observed in (D) vehicle alone, (E) mitochondria isolated from ZDF or (F) mitochondria isolated from Zucker lean hearts. Scale bars = 100 μm. Electron microscopy Electron microscopy confirmed mitochondrial damage in VEH that was reduced or not present in MT hearts (Fig. 6). In VEH hearts, mitochondria were swollen and electron translucent, with enlarged intermembrane space and disrupted matrix and calcium accumulation (Fig. 7A). MTZDF hearts showed only traces of calcium accumulation and slight disorganization of the cristae (Fig. 7B), while MTZL hearts exhibited a preserved mitochondrial structure (Fig. 7C). Figure 7: Open in new tabDownload slide Transmission electron microscopy. Representative transmission electron microscopy images of Zucker diabetic fatty (ZDF) rat hearts receiving vehicle alone or mitochondria isolated from ZDF rats or mitochondria isolated from Zucker lean rats. (A) In ZDF hearts receiving vehicle alone the mitochondria are swollen and electron-translucent, with an enlarged intermembrane space, a disrupted matrix and calcium accumulation. (B) ZDF hearts receiving ZDF mitochondria show only traces of calcium accumulation in mitochondria and minimal disorganization of cristae. (C) ZDF hearts receiving Zucker lean mitochondria show preserved mitochondrial structure. Scale bars = 500 nm. Figure 7: Open in new tabDownload slide Transmission electron microscopy. Representative transmission electron microscopy images of Zucker diabetic fatty (ZDF) rat hearts receiving vehicle alone or mitochondria isolated from ZDF rats or mitochondria isolated from Zucker lean rats. (A) In ZDF hearts receiving vehicle alone the mitochondria are swollen and electron-translucent, with an enlarged intermembrane space, a disrupted matrix and calcium accumulation. (B) ZDF hearts receiving ZDF mitochondria show only traces of calcium accumulation in mitochondria and minimal disorganization of cristae. (C) ZDF hearts receiving Zucker lean mitochondria show preserved mitochondrial structure. Scale bars = 500 nm. Open in new tabDownload slide Open in new tabDownload slide DISCUSSION T2D increases myocardial susceptibility to IRI and significantly contributes to the poor prognosis of T2D patients after acute coronary events or cardiac interventions [23]. Despite advances in coronary intervention strategies, there are no viable methodologies to ameliorate IRI in T2D patients requiring cardiac surgery. In this study, we demonstrate the efficacy of mitochondrial transplantation in enhancing myocardial function, myocellular survival and increasing ATP content following warm global ischaemia and reperfusion in the isolated perfused diabetic rat heart. To perform these studies, we have used ZDF rats (and their controls ZL) as this is an established model of T2D [20]. ZDF male rats carry a mutation of the leptin receptor gene and share many common features found in patients with T2D, including the progressive development of hyperglycaemia, insulin resistance, loss of pancreatic β-cell mass and hyperglycaemia-induced and obesity-associated complications [20]. We have used 30 min of warm global ischaemia, consistent with previous studies conducted by us and others [3, 5, 7]. This length of warm global ischaemia provides for sufficient injury to the myocardium to allow for comparative analysis between treatment and non-treatment groups. The reperfusion period was chosen to allow for infarct size determination and to demonstrate the efficacy of mitochondrial transplantation to enhance post-ischaemic myocardial function and viability compared to untreated hearts [4]. Our results show that in vehicle hearts, 30 min of warm global ischaemia significantly decreased myocardial function. Our results agree with those observed in other studies using ZDF rat hearts for isolated Langendorff perfusion [2, 24]. In our studies, we have investigated the efficacy of mitochondrial transplantation using mitochondria isolated from the diabetic (ZDF) rat and mitochondria isolated from a non-diabetic (ZL) donor. The role of the mitochondrion in T2D has been previously shown by Raza et al. [25] who demonstrated that mitochondria in ZDF rats are dysfunctional, having significantly reduced respiratory capacity and ATP production as compared with those in ZL controls. This agrees with Minet and Gaster [26] who showed that in isolated mitochondria from lean subjects and T2D patients, the mitochondrial ATP production rate was significantly lower in diabetic mitochondria as compared to lean mitochondria. Our data agree with these studies and show that the ZDF mitochondria produced significantly less ATP than the ZL mitochondria. Based on these observations, we hypothesized that the efficacy of mitochondrial transplantation would be significantly decreased in ZDF hearts receiving ZDF mitochondria as compared to ZDF hearts receiving ZL (non-diabetic) mitochondria. However, our results demonstrate that the transplantation of mitochondria isolated from either ZDF or ZL rats provide similar cardioprotection from 30 min of warm global ischaemia. Both ZDF mitochondria and ZL mitochondria transplanted into ZDF hearts significantly increased LVdevP and dP/dt max following 30 min of warm global ischaemia and 120 min of reperfusion as compared to hearts that received VEH. In addition, we show that both ZDF mitochondria and ZL mitochondria transplanted into ZDF hearts significantly decreased myocardial infarct size as compared to the vehicle. There was no significant difference in infarct size reduction observed in ZDF hearts receiving either ZDF mitochondria or ZL mitochondria. These results demonstrate that despite the decrease in ATP synthesis in ZDF mitochondria as compared to ZL mitochondria, there were no significant differences in post-ischaemic myocardial function recovery or infarct size limitation observed between these groups. No evidence of inflammation due to mitochondrial transplantation was evident, recapitulating our previous studies in large animals and in humans [4, 6, 10, 11, 17, 18]. Our results show uptake and distribution of injected mitochondria throughout the myocardium in agreement with our previous studies demonstrating that exogenous mitochondria are rapidly taken up by cardiac cells through actin-dependent endocytosis and remain present and viable in the myocardium for 28 days [3–6, 9, 13, 22]. It is important to note that while both ZDF mitochondria and ZL mitochondria significantly increased total tissue ATP content as compared to vehicle hearts our data show that ZDF hearts receiving ZL mitochondria had significantly higher ATP levels as compared to those receiving ZDF mitochondria. We expect that these results reflect the elevated ATP content and the previously demonstrated increased oxygen consumption [25, 26] of the ZL mitochondria as compared to ZDF mitochondria as shown in Fig. 2 and in previous studies by others [26]. We suspect that although no significant differences in post-ischaemic myocardial contractile function or infarct size were seen between hearts receiving either ZDF mitochondria or ZL mitochondria that the differences in total tissue ATP content may suggest that the effects of ZL mitochondria may be more prolonged. In our studies, we have used only 120 min of reperfusion and have not investigated prolonged recovery. Additional studies may demonstrate that transplantation of ZL mitochondria would provide superior long-term cardioprotection in terms of myocardial function and myocellular salvation as compared to ZDF mitochondria. In vivo research protocols involving regional myocardial IRI and long-term follow-up should be implemented. Our results demonstrating no significant difference in the dry-to-wet weight ratio between groups are in agreement with previous research and recapitulate our previous findings that mitochondrial transplantation is not associated with macroscopic oedema of the myocardial tissue [3–5]. Limitations In our investigation, we have used only male rats since the male gender is associated with an increased risk of cardiovascular disease and represents the majority of the T2D population requiring cardiac intervention [27]. Further studies in premenopausal and postmenopausal females are also required. Although transplantation of mitochondria isolated from either ZDF or ZL rats showed equivalent efficacy in enhancing myocardial function and survival, these were ex vivo perfused rodent hearts perfused for 120 min and therefore long-term efficacy and superiority of these 2 modalities have yet to be investigated. CONCLUSION Our study provides evidence that mitochondrial transplantation in T2D rat hearts is cardioprotective. Although hearts that received ZL mitochondria had increased ATP content compared to hearts that received ZDF mitochondria, this did not translate into differences in post-ischaemic myocardial function or myocellular viability. This novel technique is safe and has considerable potential to reduce morbidity and mortality in T2D patients subjected to myocardial IRI. Funding This work was supported by grants from the Boston Children’s Hospital Anesthesia Foundation, the Ryan Family Endowment, the Cardiac Conduction Fund, a Richard A. and Susan F. Smith President’s Innovation Award, Michael B. Klein and Family, the Sidman Family Foundation, the Michael B. Rukin Charitable Foundation, the Kenneth C. Griffin Charitable Research Fund and the Boston Investment Council. Conflict of interest: Pedro J. del Nido and James D. McCully have patents pending for the isolation and use of mitochondria. All other authors declared no conflict of interest. Author contributions Ilias P. Doulamis: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Writing—original draft; Writing—review & Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation editing. Alvise Guariento: Data curation; Investigation; Methodology; Validation; Writing—review & editing. Thomas Duignan: Investigation; Methodology; Writing—review & editing. Arzoo Orfany: Investigation; Methodology; Validation; Writing—review & editing. Takashi Kido: Investigation; Methodology; Writing—review & editing. David Zurakowski: Data curation; Formal analysis; Software; Supervision; Validation; Writing—review & editing. Pedro J. del Nido: Funding acquisition; Supervision; Writing—review & editing. James D. McCully: Conceptualization; Funding acquisition; Investigation; Methodology; Project administration; Resources; Supervision; Validation; Writing—review & editing. Presented at the 33rd Annual Meeting of the European Association for Cardio-Thoracic Surgery, Lisbon, Portugal, 3–5 October 2019. REFERENCES 1 Chatterjee S Khunti K Davies MJ. Type 2 diabetes . Lancet 2017 ; 389 : 2239 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Pælestik KB Jespersen NR Jensen RV Johnsen J Bøtker HE Kristiansen SB. Effects of hypoglycemia on myocardial susceptibility to ischemia-reperfusion injury and preconditioning in hearts from rats with and without type 2 diabetes . Cardiovasc Diabetol 2017 ; 16 : 1 – 10 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Masuzawa A Black KM Pacak CA Ericsson M Barnett RJ Drumm C et al. Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury . Am J Physiol Circ Physiol 2013 ; 304 : H966 – 82 . Google Scholar Crossref Search ADS WorldCat 4 McCully JD Cowan DB Pacak CA Toumpoulis IK Dayalan H Levitsky S. Injection of isolated mitochondria during early reperfusion for cardioprotection . Am J Physiol Circ Physiol 2009 ; 296 : H94 – 105 . Google Scholar Crossref Search ADS WorldCat 5 Kaza AK Wamala I Friehs I Kuebler JD Rathod RH Berra I et al. Myocardial rescue with autologous mitochondrial transplantation in a porcine model of ischemia/reperfusion . J Thorac Cardiovasc Surg 2017 ; 153 : 934 – 43 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Cowan DB Yao R Akurathi V Snay ER Thedsanamoorthy JK Zurakowski D et al. Intracoronary delivery of mitochondria to the ischemic heart for cardioprotection . PLoS One 2016 ; 11 : e0160889 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Lim VG Bell RM Arjun S Kolatsi-Joannou M Long DA Yellon DM. SGLT2 inhibitor, canagliflozin, attenuates myocardial infarction in the diabetic and nondiabetic heart . JACC Basic Transl Sci 2019 ; 4 : 15 – 26 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Preble JM Pacak CA Kondo H MacKay AA Cowan DB McCully JD. Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration . J Vis Exp 2014 ; 8 : 2 – 8 . WorldCat 9 Pacak CA Preble JM Kondo H Seibel P Levitsky S Del Nido PJ et al. Actin-dependent mitochondrial internalization in cardiomyocytes: evidence for rescue of mitochondrial function . Biol Open 2015 ; 4 : 622 – 6 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Emani SM Piekarski BL Harrild D Del Nido PJ McCully JD. Autologous mitochondrial transplantation for dysfunction after ischemia-reperfusion injury . J Thorac Cardiovasc Surg 2017 ; 154 : 286 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Emani SM McCully JD. Mitochondrial transplantation: applications for pediatric patients with congenital heart disease . Transl Pediatr 2018 ; 7 : 169 – 75 . Google Scholar Crossref Search ADS PubMed WorldCat 12 McCully JD Cowan DB Emani SM del Nido PJ. Mitochondrial transplantation: from animal models to clinical use in humans . Mitochondrion 2017 ; 34 : 127 – 34 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Orfany A Arriola CG Doulamis IP Guariento A Ramirez-Barbieri G Moskowitzova K et al. Mitochondrial transplantation ameliorates acute limb ischemia . J Vasc Surg 2019 ; doi:10.1016/j.jvs.2019.03.079. [Epub ahead of print]. WorldCat 14 Moskowitzova K Shin B Liu K Ramirez-Barbieri G Guariento A Blitzer D et al. Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation . J Heart Lung Transplant 2019 ; 38 : 92 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Shin B Cowan DB Emani SM Del Nido PJ McCully JD. Mitochondrial transplantation in myocardial ischemia and reperfusion injury . Adv Exp Med Biol 2017 ; 982 : 595 – 619 . Google Scholar Crossref Search ADS PubMed WorldCat 16 McCully JD Levitsky S del Nido PJ Cowan DB. Mitochondrial transplantation for therapeutic use . Clin Transl Med 2016 ; 5 : 16 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Masuzawa A Black KM Pacak CA Ericsson M Barnett RJ Drumm C et al. Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury . Am J Physiol Heart Circ Physiol 2013 ; 304 : H966 – 82 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Ramirez-Barbieri G Moskowitzova K Shin B Blitzer D Orfany A Guariento A et al. Alloreactivity and allorecognition of syngeneic and allogeneic mitochondria . Mitochondrion 2019 ; 46 : 103 – 15 . Google Scholar Crossref Search ADS PubMed WorldCat 19 https://www.nap.edu/catalog/12910/guide-for-the-care-and-use-of-laboratory-animals-eighth (14 November 2019, date last accessed). 20 Shiota M Printz RL. Diabetes in Zucker diabetic fatty rat . Methods Mol Biol 2012 ; 933 : 103 – 23 . Google Scholar PubMed WorldCat 21 Smith PK Krohn RI Hermanson GT Mallia AK Gartner FH Provenzano MD et al. Measurement of protein using bicinchoninic acid . Anal Biochem 1985 ; 150 : 76 – 85 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Guariento A Blitzer D Doulamis I Shin B Moskowitzova K Orfany A et al. Preischemic autologous mitochondrial transplantation by intracoronary injection for myocardial protection . J Thorac Cardiovasc Surg 2019 . [Epub ahead of print]. WorldCat 23 Lejay A Fang F John R Van JA Barr M Thaveau F et al. Ischemia reperfusion injury, ischemic conditioning and diabetes mellitus . J Mol Cell Cardiol 2016 ; 91 : 11 – 22 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Kristiansen SB Løfgren B Støttrup NB Khatir D Nielsen-Kudsk JE Nielsen TT , et al. Ischaemic preconditioning does not protect the heart in obese and lean animal models of type 2 diabetes . Diabetologia 2004 ; 47 : 1716 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Raza H John A Howarth FC. Increased oxidative stress and mitochondrial dysfunction in Zucker diabetic rat liver and brain . Cell Physiol Biochem 2015 ; 35 : 1241 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Minet AD Gaster M. ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects . Biochem Biophys Res Commun 2010 ; 402 : 70 – 4 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Murphy E Steenbergen C. Gender-based differences in mechanisms of protection in myocardial ischemia-reperfusion injury . Cardiovasc Res 2007 ; 75 : 478 – 86 . Google Scholar Crossref Search ADS PubMed WorldCat ABBREVIATIONS ABBREVIATIONS BL Baseline CBF Coronary blood flow dP/dt max Maximum derivative of left ventricular pressure IRI Ischaemia-reperfusion injury LVdevP Left ventricular developed pressure LVEDP Left ventricular end-diastolic pressure PET Positron emission tomography T2D Type 2 diabetes TTC Tetrazolium chloride VEH Vehicle alone ZDF Zucker diabetic fatty ZL Zucker lean μCT Microcomputed tomography © The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Journal of Cardio-Thoracic Surgery Oxford University Press

Mitochondrial transplantation for myocardial protection in diabetic hearts

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Oxford University Press
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© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.
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1010-7940
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1873-734X
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10.1093/ejcts/ezz326
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Abstract

Abstract OBJECTIVES Type 2 diabetes causes mitochondrial dysfunction, which increases myocardial susceptibility to ischaemia–reperfusion injury. We investigated the efficacy of transplantation of mitochondria isolated from diabetic or non-diabetic donors in providing cardioprotection from warm global ischaemia and reperfusion in the diabetic rat heart. METHODS Ex vivo perfused hearts from Zucker diabetic fatty (ZDF fa/fa) rats (n = 6 per group) were subjected to 30 min of warm global ischaemia and 120 min reperfusion. Immediately prior to reperfusion, vehicle alone (VEH) or vehicle containing mitochondria isolated from either ZDF (MTZDF) or non-diabetic Zucker lean (ZL +/?) (MTZL) skeletal muscle were delivered to the coronary arteries via the aortic cannula. RESULTS Following 30-min global ischaemia and 120-min reperfusion, left ventricular developed pressure was significantly increased in MTZDF and MTZL groups compared to VEH group (MTZDF: 92.8 ± 5.2 mmHg vs MTZL: 110.7 ± 2.4 mmHg vs VEH: 44.3 ± 5.9 mmHg; P < 0.01 each); and left ventricular end-diastolic pressure was significantly decreased (MTZDF 12.1 ± 1.3 mmHg vs MTZL 8.6 ± 0.8 mmHg vs VEH: 18.6 ± 1.5 mmHg; P = 0.016 for MTZDF vs VEH and P < 0.01 for MTZL vs VEH). Total tissue ATP content was significantly increased in both MT groups compared to VEH group (MTZDF: 18.9 ± 1.5 mmol/mg protein/mg tissue vs MTZL: 28.1 ± 2.3 mmol/mg protein/mg tissue vs VEH: 13.1 ± 0.5 mmol/mg protein/mg tissue; P = 0.018 for MTZDF vs VEH and P < 0.01 for MTZL vs VEH). Infarct size was significantly decreased in the MT groups (MTZDF: 11.8 ± 0.7% vs MTZL: 9.9 ± 0.5% vs VEH: 52.0 ± 1.4%; P < 0.01 each). CONCLUSIONS Mitochondrial transplantation significantly enhances post-ischaemic myocardial functional recovery and significantly decreases myocellular injury in the diabetic heart. Mitochondrial transplantation , Myocardial ischaemia–reperfusion injury , Diabetes INTRODUCTION Type 2 diabetes (T2D) affects 30 million people in the USA and is increasing by ∼1.5 million per year [1]. There are sufficient data to show that T2D increases the susceptibility of the myocardium to ischaemia–reperfusion injury (IRI) [2]. We and others have demonstrated that mitochondria play a key role in the modulation of IRI in the heart [3–6]. Our studies have shown that IRI detrimentally affects mitochondrial function and cellular energetics. These events occur during ischaemia and persist despite the restoration of coronary blood flow and tissue reperfusion, ultimately negatively impacting post-ischaemic myocardial contractile function and cellular viability [3–6]. These effects are exacerbated by T2D and significantly increase morbidity and mortality in T2D patients requiring cardiac interventions [7]. Recently, we have pioneered a novel therapy, mitochondrial transplantation, that replaces native mitochondria damaged by IRI with viable, structurally intact, respiration competent mitochondria isolated from non-ischaemic tissue obtained from the patient’s own body [3–6, 8–16]. Mitochondrial transplantation has been clinically proven to enhance post-ischaemic cardiac viability and function. We have validated the efficacy of mitochondrial transplantation in vitro and in perfused large animal heart models, and finally in human paediatric cardiac patients [4, 8–11, 14, 15, 17]. Our studies have demonstrated that there is no direct or indirect, acute or chronic alloreactivity, allorecognition or damage-associated molecular pattern reaction to single or serial injections of either syngeneic or allogeneic mitochondria. These studies demonstrated that heterologous mitochondrial transplantation is safe and that there is no immunoreaction between the donor mitochondria and the host [18]. We hypothesized that mitochondrial transplantation will significantly enhance myocardial function, viability and energy production following IRI in the ex vivo isolated perfused diabetic heart. In this study, we investigate the efficacy of mitochondrial transplantation isolated from diabetic or non-diabetic donors in providing cardioprotection from IRI in a model of T2D. MATERIALS AND METHODS Animal care and biosafety This investigation was conducted under the National Institutes of Health’s guidelines on animal care and use and was approved by the Boston Children’s Hospital’s Animal Care and Use Committee (Protocol no. 15-01-2722) [19]. Animal model Zucker diabetic fatty rats (ZDFLeprfa/Crl; ZDF) and non-diabetic Zucker lean rats (Lean fa/+; ZL) were used [20]. All rats were males, aged to 14 weeks old (n = 24), obtained from Charles River Laboratory (Wilmington, MA, USA) and had an average weight of 344 ± 16 g. ZDF rats develop T2D at the age of 12 weeks, which is fully manifested at 14 weeks and lasts up to 24 weeks [20]. ZL rats do not spontaneously develop T2D and maintain normal serum glucose and normal body weight [7, 20]. Experimental model All animals were anaesthetized in an isoflurane chamber (2–4%). Following confirmation of anaesthesia, the chest was opened with an inverted T-shaped incision. Heparin (500 U.I.) was injected into the abdominal aorta and allowed to circulate for 10 s. The heart was removed enbloc with the lungs. Upon harvesting of the heart, the pectoralis major muscle was dissected, and two #6 biopsy punch samples were removed for the isolation of mitochondria [8]. Mitochondrial isolation Tissue samples were obtained from the pectoralis major using a 6-mm biopsy punch. Tissue weight was 0.18 ± 0.04 g (wet weight). Mitochondria were isolated with the use of a commercial tissue dissociator and differential filtration in <30 min as previously described [8]. The isolated mitochondria were suspended in a vehicle [250 mmol/l sucrose, 20 mmol/l K+-HEPES buffer (pH 7.2), 0.5 mmol/l K+-EGTA (pH 8.0)] and were used for mitochondrial transplantation [3, 6]. Langendorff perfusion The heart was excised and placed in a 4°C bath of Krebs-Ringer containing 100 mM NaCl, 4.7 mM KCl, 1.1 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 1.7 mM CaCl2, 11.5 mM glucose, 4.9 mM pyruvic acid and 5.4 mM fumaric acid bubbled with 5% CO2 and 95% O2 (pH 7.4 at 37°C), where spontaneous beating ceased within seconds. The heart was then immediately secured onto a metallic cannula and perfused in the Langendorff mode as previously described [4]. Hearts were perfused at a constant pressure of 60 mmHg. The temperature of the perfusate was maintained at 37°C. Hearts were paced through the right ventricle at 180 beats/min. Coronary flow was measured by collecting coronary effluent from the pulmonary artery. Myocardial function assessment A latex balloon (Harvard Apparatus, MA, USA) sewn onto a fluid-filled catheter was inserted through the left atrial appendage and the mitral valve into the left ventricle. The volume of the water-filled balloon was set at a constant end-diastolic physiological pressure of 6–8 mmHg using a calibrated micro-syringe during equilibrium, and this balloon volume was maintained for the duration of the experiment. The perfusion pressure, temperature, heart rate, left ventricular developed pressure (LVdevP, mmHg), left ventricular end-diastolic pressure (LVEDP, mmHg) and maximum derivative of left ventricular pressure (dP/dt max, mmHg/s) were recorded continuously using a data acquisition hardware (PowerLab/8 SP by AD Instruments, Oxford, UK) and readings were visualized using the LabChart software (v7.3.8, AD Instruments, Oxford, UK). Experimental protocol The experimental protocol is shown in Fig. 1. Only hearts harvested from ZDF rats were used for ex vivo Langendorff perfusion. Hearts were perfused for 15 min (equilibration period for baseline data acquisition). Following 15 min of equilibrium, ZDF hearts were subjected to temporary warm global ischaemia by cross-clamping the aorta and submerging the heart in 37°C Krebs-Ringer solution. Figure 1: Open in new tabDownload slide Experimental protocol. Hearts were harvested from ZDF rats (n = 18) and perfused in the Langendorff mode. After 15 min of equilibration, warm temporary global ischaemia was induced by cross-clamping the aorta. Following 30 min of warm global ischaemia, the cross-clamp was released and the hearts were reperfused. Immediately at the onset of reperfusion, 1 ml of vehicle alone or 1 ml of vehicle containing mitochondria isolated from either ZDF (MTZDF) or ZL rats (MTZL) was delivered through the aortic cannula. Mitochondria were transplanted at 2 × 105 ± 0.3 × 105 mitochondria/g wet weight. Hearts were reperfused for 120 min and then used for biochemical and histological analysis. Myocardial function assessment time points are annotated (red triangles). ZDF: Zucker diabetic fatty; ZL: Zucker lean. Figure 1: Open in new tabDownload slide Experimental protocol. Hearts were harvested from ZDF rats (n = 18) and perfused in the Langendorff mode. After 15 min of equilibration, warm temporary global ischaemia was induced by cross-clamping the aorta. Following 30 min of warm global ischaemia, the cross-clamp was released and the hearts were reperfused. Immediately at the onset of reperfusion, 1 ml of vehicle alone or 1 ml of vehicle containing mitochondria isolated from either ZDF (MTZDF) or ZL rats (MTZL) was delivered through the aortic cannula. Mitochondria were transplanted at 2 × 105 ± 0.3 × 105 mitochondria/g wet weight. Hearts were reperfused for 120 min and then used for biochemical and histological analysis. Myocardial function assessment time points are annotated (red triangles). ZDF: Zucker diabetic fatty; ZL: Zucker lean. Experimental groups Following 15 min of equilibrium and 30 min of warm global ischaemia, the cross-clamp was released and the hearts received either vehicle alone (1 ml, VEH, n = 6) or vehicle containing mitochondria and the hearts were reperfused for 120 min [8]. Mitochondria were isolated from either ZDF rats (MTZDF, n = 6) or ZL rats (MTZL, n = 6). ZDF mitochondria were isolated from the same rats used for harvesting the hearts. Mitochondria delivery For delivery of the mitochondria, 1 ml of vehicle solution containing 2 × 105 ± 0.3 × 105 mitochondria/g wet weight was resuspended in 2 ml of 37°C Krebs-Ringer solution and administered antegradely to the coronary arteries through the aortic cannula over 10 s. Mitochondrial uptake 18F-Rhodamine 6G-labelled mitochondria In a separate set of experiments, ZDF rats (n = 4) were used for visualization of mitochondrial uptake in the heart. A donor rat (n = 1) was used to isolate syngeneic mitochondria from the pectoralis major muscle tissue as previously described [13, 14]. The isolated mitochondria were labelled with 18F-rhodamine 6G. Rats (n = 3) were anaesthetized and maintained on 2–3% inhaled isoflurane. A sternotomy was performed, and the ascending aorta was exposed. The 18F-rhodamine 6G-labelled mitochondria (1 × 109 in 1 ml respiration buffer) were delivered antegrade to the coronary arteries via injection to the coronary ostium using a tuberculin syringe with a 30-G needle. Ten minutes after the injection, the animals were euthanized in a CO2 chamber and examined using positron emission tomography (PET) and microcomputed tomography (μCT) [14]. Human mitochondria In a separate set of ZDF rats (n = 3), 1 × 109 mitochondria were isolated from human cardiac fibroblasts and were delivered retrogradely to the coronary arteries through the aortic cannula. After 10 min, tissue was collected. The use of human mitochondria in rodent heart tissue allowed differentiation between endogenous rat mitochondria and transplanted human mitochondria based on immune reactivity to a monoclonal anti-human mitochondrial antibody (biotin, MTCO2; Abcam, Cambridge, MA, USA) and visualized using Vectastain (Vector Laboratories, Burlingame, CA, USA) as previously described [13, 14]. Histopathological analysis and transmission electron microscopy Following 120 min of reperfusion, hearts were removed from the cannula, sectioned at 1-cm thickness and stored for further analysis. Myocardial tissue samples were fixed in 10% formalin and paraffin-embedded for histopathological analysis. Serial slides were used for haematoxylin and eosin staining and Masson’s trichrome staining. Haematoxylin and eosin-stained slides were evaluated for the area of necrosis and inflammatory cell infiltration. Masson’s trichrome stained slides were evaluated for cardiac fibrosis as previously described [3, 4, 13]. Transmission electron microscopy was used to analyse structural damage in the heart grafts, as described in previous studies [3, 4, 6]. Myocardial tissue samples were collected and fixed in 1.25% formaldehyde, 2.5% grade I glutaraldehyde and 0.03% picric acid. All analyses were performed by a blinded observer. Measurement of infarct size The infarct size was determined using 1% triphenyl tetrazolium chloride (Sigma-Aldrich, St. Louis, MO, USA) and the percentage of the area of infarcted tissue per total area of cardiac tissue was calculated digitally with ImageJ (https://imagej.nih.gov/ij/) as previously described [13, 15]. The determination of wet weight-to-dry weight ratios was also performed as previously described [4, 13]. ATP Following 120 min of reperfusion, myocardial tissue samples were collected, snap-frozen in liquid nitrogen and stored in −80°C for ATP and protein content analysis. ATP was determined using the Colorimetric ATP Assay Kit (Abcam) according to the manufacturer’s instructions [21]. Myocardial protein per mg wet weight was determined using the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. All samples were run in triplicate. ATP content was expressed as mmol/mg protein/mg tissue (wet weight). Statistical analysis The normality of all continuous variables was tested using the Shapiro–Wilk test and graphically assessed by histograms and Q–Q plots. Box-and-whisker plots and the non-parametric Mann–Whitney U-test followed by Bonferroni adjustment were used for comparing glucose, body weight, heart weight, heart-to-body weight ratio, mitochondria ATP, ATP content of the myocardial tissue, dry-to-wet weight and infarct size. All other variables conformed to a normal Gaussian-shaped distribution and are presented as mean ± standard error of the mean. For functional outcome variables (LVdevP, LVEDP, dP/dt max and CBF) application of parametric statistical tests was opted for to provide a more powerful and robust analysis for within- and between-group analysis throughout the experimental period. Longitudinal analysis for between-groups comparisons was performed using 2-way repeated-measures analysis of variance within the framework of fitting mixed-effects linear regression models. To reduce the probability of false-positive results (type I error) due to multiple comparisons, Benjamini and Hochberg's false discovery rate was applied to control the family-wise error to α <0.05. All tests reported are 2-tailed. Statistical analyses were performed with GraphPad Prism version 7.0 for Mac OS X (GraphPad Software, La Jolla, CA, USA) [22]. RESULTS Type 2 diabetes model ZDF 14-week-old male rats had significantly increased body weight (P < 0.001), heart-to-body weight ratio (P < 0.001) and blood glucose (P < 0.001) when compared to ZL male rats of the same age (Fig. 2A–C). ATP content was significantly lower (P < 0.001) in the mitochondria isolated from ZDF rats as compared to ZL rats (Fig. 2D). There was no difference, in body weight, heart weight, blood glucose and heart -to-body weight ratio between the 3 experimental groups of ZDF rats before ex vivo perfusion (P > 0.05) (Table 1). Figure 2: Open in new tabDownload slide Body and heart weight, blood glucose and mitochondrial ATP content. Box plots show data for (A) body weight (g), (B) heart-to-body weight ratio and (C) blood glucose (mg/dl) for 14-week-old ZDF rats and ZL rats. (D) ATP content of 1 × 109 mitochondria isolated from either ZDF or ZL rats is shown (n = 6 per group) with P-values obtained from Mann–Whitney U-tests. *P < 0.05: ZDF versus ZL. ZDF: Zucker diabetic fatty; ZL: Zucker lean. Figure 2: Open in new tabDownload slide Body and heart weight, blood glucose and mitochondrial ATP content. Box plots show data for (A) body weight (g), (B) heart-to-body weight ratio and (C) blood glucose (mg/dl) for 14-week-old ZDF rats and ZL rats. (D) ATP content of 1 × 109 mitochondria isolated from either ZDF or ZL rats is shown (n = 6 per group) with P-values obtained from Mann–Whitney U-tests. *P < 0.05: ZDF versus ZL. ZDF: Zucker diabetic fatty; ZL: Zucker lean. Table 1: Blood glucose, heart and body weight and mitochondria ATP content in 14-week-old ZDF and ZL rats ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 Blood glucose (mg/dl), body weight (g), heart weight (g), heart-to-body weight ratio, heart wet-to-dry weight ratio and mitochondrial ATP content in 14-week-old ZDF and ZL rats. All data are shown as mean ± standard error of the mean. The number of animals in each group is shown as n, in brackets. P-values are shown for each value. Significant differences between groups at P-value <0.05 are shown in bold. MTZDF: vehicle and ZDF mitochondria; MTZL: vehicle and ZL mitochondria; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. Open in new tab Table 1: Blood glucose, heart and body weight and mitochondria ATP content in 14-week-old ZDF and ZL rats ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 ZDF (n = 18) ZL (n = 6) P-value VEH (n = 6) MTZDF (n = 6) MTZL (n = 6) P-value Blood glucose (mg/dl) 168.4 ± 9.5 505.9 ± 14.1 <0.001 483.4 ± 24.1 544.0 ± 23.1 492.8 ± 22.1 0.188 Body weight (g) 340.7 ± 4.2 306.3 ± 12.1 <0.001 343.2 ± 6.5 337.0 ± 5.5 351.5 ± 8.7 0.554 Heart weight (g) 1.6 ± 0.1 1.0 ± 0.1 <0.001 1.7 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.11 Heart-to-body weight ratio 0.5 ± 0.1 0.3 ± 0.1 <0.001 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.069 Heart wet-to-dry weight ratio 8.1 ± 1.0 7.7 ± 0.9 0.078 7.7 ± 0.4 8.9 ± 1.7 7.5 ± 0.9 0.234 Mitochondrial ATP content (μmol/109 mitochondria) 229.7 ± 11.8 327 ± 13.3 <0.001 Blood glucose (mg/dl), body weight (g), heart weight (g), heart-to-body weight ratio, heart wet-to-dry weight ratio and mitochondrial ATP content in 14-week-old ZDF and ZL rats. All data are shown as mean ± standard error of the mean. The number of animals in each group is shown as n, in brackets. P-values are shown for each value. Significant differences between groups at P-value <0.05 are shown in bold. MTZDF: vehicle and ZDF mitochondria; MTZL: vehicle and ZL mitochondria; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. Open in new tab Mitochondrial uptake PET/μCT imaging of 18F-rhodamine 6G-labelled mitochondria injected directly into the heart demonstrated specific distribution throughout the heart (Fig. 3A). Radiolabelled mitochondria were not detectable in any other organ or region of the body. Figure 3: Open in new tabDownload slide Uptake and distribution of transplanted mitochondria. (A) 18F-Rhodamine 6G-labelled mitochondria were distributed throughout the heart and were not detectable in any other region of the body. Images are shown for 3-dimensional reconstructed views. (B) Representative immunohistochemical images (magnification ×20) demonstrating mitochondrial uptake in the isolated perfused hearts. (C) Expanded box area of (B). The majority of transplanted mitochondria are indicated with white arrows. Scale bars = 100 μm. Figure 3: Open in new tabDownload slide Uptake and distribution of transplanted mitochondria. (A) 18F-Rhodamine 6G-labelled mitochondria were distributed throughout the heart and were not detectable in any other region of the body. Images are shown for 3-dimensional reconstructed views. (B) Representative immunohistochemical images (magnification ×20) demonstrating mitochondrial uptake in the isolated perfused hearts. (C) Expanded box area of (B). The majority of transplanted mitochondria are indicated with white arrows. Scale bars = 100 μm. Analysis of mitochondrial uptake revealed that injected human mitochondria were distributed primarily within myocardial fibres (Fig. 3B and C) [14]. Myocardial function LVdevP, dP/dt max and LVEDP were not significantly different between groups at baseline. Following 30-min global ischaemia and 120-min reperfusion, MTZDF and MTZL showed significantly increased myocardial function (Fig. 4A–C). LVdevP was 44.3 ± 5.9 mmHg in VEH and was significantly increased to 92.8 ± 5.2 mmHg in MTZDF (P < 0.001 vs VEH) and to 110.7 ± 2.4 mmHg in MTZL (P < 0.001 vs VEH). dP/dt max following 120 min reperfusion was significantly increased to 5414.8 ± 529.2 mmHg/s in MTZDF and to 6332.7 ± 568.5 mmHg/s in MTZL as compared to 2584.8 ± 401.9 mmHg/s in VEH (P < 0.01 each vs VEH). Figure 4: Open in new tabDownload slide Myocardial function during baseline, global ischaemia and reperfusion. (A) Left ventricular developed pressure (mmHg), (B) left ventricular end-diastolic pressure (mmHg), (C) dP/dt max (mmHg) and (D) coronary blood flow (ml/min) at baseline, warm global ischaemia and reperfusion in ZDF rat hearts receiving VEH or vehicle containing mitochondria from either ZDF rats (MTZDF) or ZL rats (MTZL). Injection is denoted by dashed vertical line. All results are shown as mean ± standard error of the mean for n = 6 per group. The P-values were obtained from 2-way repeated-measures analysis of variance using mixed-effects linear regression models and corrected by Benjamini and Hochberg’s false discovery rate. *P < 0.05 MTZDF versus VEH; #P < 0.05 MTZL versus VEH. BL: baseline; dP/dt max: maximum derivative of left ventricular pressure; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. Figure 4: Open in new tabDownload slide Myocardial function during baseline, global ischaemia and reperfusion. (A) Left ventricular developed pressure (mmHg), (B) left ventricular end-diastolic pressure (mmHg), (C) dP/dt max (mmHg) and (D) coronary blood flow (ml/min) at baseline, warm global ischaemia and reperfusion in ZDF rat hearts receiving VEH or vehicle containing mitochondria from either ZDF rats (MTZDF) or ZL rats (MTZL). Injection is denoted by dashed vertical line. All results are shown as mean ± standard error of the mean for n = 6 per group. The P-values were obtained from 2-way repeated-measures analysis of variance using mixed-effects linear regression models and corrected by Benjamini and Hochberg’s false discovery rate. *P < 0.05 MTZDF versus VEH; #P < 0.05 MTZL versus VEH. BL: baseline; dP/dt max: maximum derivative of left ventricular pressure; VEH: vehicle alone; ZDF: Zucker diabetic fatty; ZL: Zucker lean. LVEDP was significantly increased to 18.6 ± 1.5 mmHg in VEH after 120 min of reperfusion as compared to 12.1 ± 1.3 mmHg in MTZDF (P = 0.016 vs VEH) and to 8.6 ± 0.8 mmHg in MTZL (P < 0.01 vs VEH). No difference was observed between MTZDF and MTZL groups in any of the myocardial function indices except for LVdevP after 90 min (P = 0.028) and 120 min (P = 0.021) of reperfusion. No difference in CBF was observed between groups throughout the experiment (P > 0.05) (Fig. 4D). ATP Total tissue ATP content of the cardiac tissue was significantly increased in both MT groups compared to the VEH group (MTZDF: 18.9 ± 1.5 mmol/mg protein/mg tissue vs MTZL: 28.1 ± 2.3 mmol/mg protein/mg tissue vs VEH: 13.1 ± 0.5 mmol/mg protein/mg tissue; P = 0.018 for MTZDF vs VEH and P < 0.01 for MTZL vs VEH) (Fig. 5A). Total tissue ATP content of cardiac tissue was higher in the MTZL group than in the MTZDF group (P < 0.01). Figure 5: Open in new tabDownload slide ATP content, dry-to-wet weight and infarct size. (A) ATP content mmol/mg protein/mg tissue wet weight, (B) dry/wet weight ratio of the cardiac tissue and (C) infarct size as % of total cardiac mass in ZDF rat hearts following 30 min of warm global ischaemia and 120 min of reperfusion. (D) Representative TTC stained ZDF rat hearts receiving VEH, or MTZDF rats or from MTZL rats following 30 min of warm global ischaemia and 120 min of reperfusion. Unstained regions (white) represent the infarcted myocardium. Scale bar = 1 cm. All results are shown for n = 6 per group and P-values were obtained from Mann–Whitney U-test with Bonferroni correction. *P < 0.05: MTZDF versus VEH; #P < 0.05: MTZL versus VEH; **P < 0.05: MTZDF versus MTZL; ns: no significant difference at P < 0.05 detected. MTZDF; mitochondria isolated from ZDF; MTZL; mitochondria isolated from Zucker lean; TTC: tetrazolium chloride; VEH: vehicle alone; ZDF: Zucker diabetic fatty. Figure 5: Open in new tabDownload slide ATP content, dry-to-wet weight and infarct size. (A) ATP content mmol/mg protein/mg tissue wet weight, (B) dry/wet weight ratio of the cardiac tissue and (C) infarct size as % of total cardiac mass in ZDF rat hearts following 30 min of warm global ischaemia and 120 min of reperfusion. (D) Representative TTC stained ZDF rat hearts receiving VEH, or MTZDF rats or from MTZL rats following 30 min of warm global ischaemia and 120 min of reperfusion. Unstained regions (white) represent the infarcted myocardium. Scale bar = 1 cm. All results are shown for n = 6 per group and P-values were obtained from Mann–Whitney U-test with Bonferroni correction. *P < 0.05: MTZDF versus VEH; #P < 0.05: MTZL versus VEH; **P < 0.05: MTZDF versus MTZL; ns: no significant difference at P < 0.05 detected. MTZDF; mitochondria isolated from ZDF; MTZL; mitochondria isolated from Zucker lean; TTC: tetrazolium chloride; VEH: vehicle alone; ZDF: Zucker diabetic fatty. Infarct size No significant difference was observed in the wet weight-to-dry weight ratio between groups (P > 0.05) (Fig. 5B). A significant reduction in infarct size was observed between the MTZDF and MTZL groups as compared to the VEH group (MTZDF: 11.8 ± 0.7% vs MTZL: 9.9 ± 0.5% vs VEH: 52.0 ± 1.4%; P < 0.01 each vs VEH) (Fig. 5C and D). There was no significant difference in infarct size between MTZDF and MTZL. Histology Haematoxylin and eosin and Masson’s trichrome showed increased longitudinal and transverse interfibrillar separation in VEH hearts (Fig. 6A) compared with MT hearts (Fig. 6B and C), suggesting increased diffuse myocardial tissue injury and oedema in the hearts that did not receive mitochondria. No difference in collagen content was observed between groups (Fig. 6D–F). Figure 6: Open in new tabDownload slide Haematoxylin and eosin and Masson’s trichrome. Representative haematoxylin and eosin and Masson’s trichrome stained micrographs of Zucker diabetic fatty (ZDF) rat hearts. (A) ZDF hearts receiving vehicle alone show increased longitudinal and transverse interfibrillar separation as compared to ZDF hearts receiving (B) mitochondria isolated from ZDF rats or (C) mitochondria isolated from Zucker lean rats. No difference in collagen content was observed in (D) vehicle alone, (E) mitochondria isolated from ZDF or (F) mitochondria isolated from Zucker lean hearts. Scale bars = 100 μm. Figure 6: Open in new tabDownload slide Haematoxylin and eosin and Masson’s trichrome. Representative haematoxylin and eosin and Masson’s trichrome stained micrographs of Zucker diabetic fatty (ZDF) rat hearts. (A) ZDF hearts receiving vehicle alone show increased longitudinal and transverse interfibrillar separation as compared to ZDF hearts receiving (B) mitochondria isolated from ZDF rats or (C) mitochondria isolated from Zucker lean rats. No difference in collagen content was observed in (D) vehicle alone, (E) mitochondria isolated from ZDF or (F) mitochondria isolated from Zucker lean hearts. Scale bars = 100 μm. Electron microscopy Electron microscopy confirmed mitochondrial damage in VEH that was reduced or not present in MT hearts (Fig. 6). In VEH hearts, mitochondria were swollen and electron translucent, with enlarged intermembrane space and disrupted matrix and calcium accumulation (Fig. 7A). MTZDF hearts showed only traces of calcium accumulation and slight disorganization of the cristae (Fig. 7B), while MTZL hearts exhibited a preserved mitochondrial structure (Fig. 7C). Figure 7: Open in new tabDownload slide Transmission electron microscopy. Representative transmission electron microscopy images of Zucker diabetic fatty (ZDF) rat hearts receiving vehicle alone or mitochondria isolated from ZDF rats or mitochondria isolated from Zucker lean rats. (A) In ZDF hearts receiving vehicle alone the mitochondria are swollen and electron-translucent, with an enlarged intermembrane space, a disrupted matrix and calcium accumulation. (B) ZDF hearts receiving ZDF mitochondria show only traces of calcium accumulation in mitochondria and minimal disorganization of cristae. (C) ZDF hearts receiving Zucker lean mitochondria show preserved mitochondrial structure. Scale bars = 500 nm. Figure 7: Open in new tabDownload slide Transmission electron microscopy. Representative transmission electron microscopy images of Zucker diabetic fatty (ZDF) rat hearts receiving vehicle alone or mitochondria isolated from ZDF rats or mitochondria isolated from Zucker lean rats. (A) In ZDF hearts receiving vehicle alone the mitochondria are swollen and electron-translucent, with an enlarged intermembrane space, a disrupted matrix and calcium accumulation. (B) ZDF hearts receiving ZDF mitochondria show only traces of calcium accumulation in mitochondria and minimal disorganization of cristae. (C) ZDF hearts receiving Zucker lean mitochondria show preserved mitochondrial structure. Scale bars = 500 nm. Open in new tabDownload slide Open in new tabDownload slide DISCUSSION T2D increases myocardial susceptibility to IRI and significantly contributes to the poor prognosis of T2D patients after acute coronary events or cardiac interventions [23]. Despite advances in coronary intervention strategies, there are no viable methodologies to ameliorate IRI in T2D patients requiring cardiac surgery. In this study, we demonstrate the efficacy of mitochondrial transplantation in enhancing myocardial function, myocellular survival and increasing ATP content following warm global ischaemia and reperfusion in the isolated perfused diabetic rat heart. To perform these studies, we have used ZDF rats (and their controls ZL) as this is an established model of T2D [20]. ZDF male rats carry a mutation of the leptin receptor gene and share many common features found in patients with T2D, including the progressive development of hyperglycaemia, insulin resistance, loss of pancreatic β-cell mass and hyperglycaemia-induced and obesity-associated complications [20]. We have used 30 min of warm global ischaemia, consistent with previous studies conducted by us and others [3, 5, 7]. This length of warm global ischaemia provides for sufficient injury to the myocardium to allow for comparative analysis between treatment and non-treatment groups. The reperfusion period was chosen to allow for infarct size determination and to demonstrate the efficacy of mitochondrial transplantation to enhance post-ischaemic myocardial function and viability compared to untreated hearts [4]. Our results show that in vehicle hearts, 30 min of warm global ischaemia significantly decreased myocardial function. Our results agree with those observed in other studies using ZDF rat hearts for isolated Langendorff perfusion [2, 24]. In our studies, we have investigated the efficacy of mitochondrial transplantation using mitochondria isolated from the diabetic (ZDF) rat and mitochondria isolated from a non-diabetic (ZL) donor. The role of the mitochondrion in T2D has been previously shown by Raza et al. [25] who demonstrated that mitochondria in ZDF rats are dysfunctional, having significantly reduced respiratory capacity and ATP production as compared with those in ZL controls. This agrees with Minet and Gaster [26] who showed that in isolated mitochondria from lean subjects and T2D patients, the mitochondrial ATP production rate was significantly lower in diabetic mitochondria as compared to lean mitochondria. Our data agree with these studies and show that the ZDF mitochondria produced significantly less ATP than the ZL mitochondria. Based on these observations, we hypothesized that the efficacy of mitochondrial transplantation would be significantly decreased in ZDF hearts receiving ZDF mitochondria as compared to ZDF hearts receiving ZL (non-diabetic) mitochondria. However, our results demonstrate that the transplantation of mitochondria isolated from either ZDF or ZL rats provide similar cardioprotection from 30 min of warm global ischaemia. Both ZDF mitochondria and ZL mitochondria transplanted into ZDF hearts significantly increased LVdevP and dP/dt max following 30 min of warm global ischaemia and 120 min of reperfusion as compared to hearts that received VEH. In addition, we show that both ZDF mitochondria and ZL mitochondria transplanted into ZDF hearts significantly decreased myocardial infarct size as compared to the vehicle. There was no significant difference in infarct size reduction observed in ZDF hearts receiving either ZDF mitochondria or ZL mitochondria. These results demonstrate that despite the decrease in ATP synthesis in ZDF mitochondria as compared to ZL mitochondria, there were no significant differences in post-ischaemic myocardial function recovery or infarct size limitation observed between these groups. No evidence of inflammation due to mitochondrial transplantation was evident, recapitulating our previous studies in large animals and in humans [4, 6, 10, 11, 17, 18]. Our results show uptake and distribution of injected mitochondria throughout the myocardium in agreement with our previous studies demonstrating that exogenous mitochondria are rapidly taken up by cardiac cells through actin-dependent endocytosis and remain present and viable in the myocardium for 28 days [3–6, 9, 13, 22]. It is important to note that while both ZDF mitochondria and ZL mitochondria significantly increased total tissue ATP content as compared to vehicle hearts our data show that ZDF hearts receiving ZL mitochondria had significantly higher ATP levels as compared to those receiving ZDF mitochondria. We expect that these results reflect the elevated ATP content and the previously demonstrated increased oxygen consumption [25, 26] of the ZL mitochondria as compared to ZDF mitochondria as shown in Fig. 2 and in previous studies by others [26]. We suspect that although no significant differences in post-ischaemic myocardial contractile function or infarct size were seen between hearts receiving either ZDF mitochondria or ZL mitochondria that the differences in total tissue ATP content may suggest that the effects of ZL mitochondria may be more prolonged. In our studies, we have used only 120 min of reperfusion and have not investigated prolonged recovery. Additional studies may demonstrate that transplantation of ZL mitochondria would provide superior long-term cardioprotection in terms of myocardial function and myocellular salvation as compared to ZDF mitochondria. In vivo research protocols involving regional myocardial IRI and long-term follow-up should be implemented. Our results demonstrating no significant difference in the dry-to-wet weight ratio between groups are in agreement with previous research and recapitulate our previous findings that mitochondrial transplantation is not associated with macroscopic oedema of the myocardial tissue [3–5]. Limitations In our investigation, we have used only male rats since the male gender is associated with an increased risk of cardiovascular disease and represents the majority of the T2D population requiring cardiac intervention [27]. Further studies in premenopausal and postmenopausal females are also required. Although transplantation of mitochondria isolated from either ZDF or ZL rats showed equivalent efficacy in enhancing myocardial function and survival, these were ex vivo perfused rodent hearts perfused for 120 min and therefore long-term efficacy and superiority of these 2 modalities have yet to be investigated. CONCLUSION Our study provides evidence that mitochondrial transplantation in T2D rat hearts is cardioprotective. Although hearts that received ZL mitochondria had increased ATP content compared to hearts that received ZDF mitochondria, this did not translate into differences in post-ischaemic myocardial function or myocellular viability. This novel technique is safe and has considerable potential to reduce morbidity and mortality in T2D patients subjected to myocardial IRI. Funding This work was supported by grants from the Boston Children’s Hospital Anesthesia Foundation, the Ryan Family Endowment, the Cardiac Conduction Fund, a Richard A. and Susan F. Smith President’s Innovation Award, Michael B. Klein and Family, the Sidman Family Foundation, the Michael B. Rukin Charitable Foundation, the Kenneth C. Griffin Charitable Research Fund and the Boston Investment Council. Conflict of interest: Pedro J. del Nido and James D. McCully have patents pending for the isolation and use of mitochondria. All other authors declared no conflict of interest. Author contributions Ilias P. Doulamis: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Writing—original draft; Writing—review & Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation editing. Alvise Guariento: Data curation; Investigation; Methodology; Validation; Writing—review & editing. Thomas Duignan: Investigation; Methodology; Writing—review & editing. Arzoo Orfany: Investigation; Methodology; Validation; Writing—review & editing. Takashi Kido: Investigation; Methodology; Writing—review & editing. David Zurakowski: Data curation; Formal analysis; Software; Supervision; Validation; Writing—review & editing. Pedro J. del Nido: Funding acquisition; Supervision; Writing—review & editing. James D. McCully: Conceptualization; Funding acquisition; Investigation; Methodology; Project administration; Resources; Supervision; Validation; Writing—review & editing. Presented at the 33rd Annual Meeting of the European Association for Cardio-Thoracic Surgery, Lisbon, Portugal, 3–5 October 2019. REFERENCES 1 Chatterjee S Khunti K Davies MJ. Type 2 diabetes . Lancet 2017 ; 389 : 2239 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Pælestik KB Jespersen NR Jensen RV Johnsen J Bøtker HE Kristiansen SB. Effects of hypoglycemia on myocardial susceptibility to ischemia-reperfusion injury and preconditioning in hearts from rats with and without type 2 diabetes . Cardiovasc Diabetol 2017 ; 16 : 1 – 10 . 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Google Scholar Crossref Search ADS PubMed WorldCat ABBREVIATIONS ABBREVIATIONS BL Baseline CBF Coronary blood flow dP/dt max Maximum derivative of left ventricular pressure IRI Ischaemia-reperfusion injury LVdevP Left ventricular developed pressure LVEDP Left ventricular end-diastolic pressure PET Positron emission tomography T2D Type 2 diabetes TTC Tetrazolium chloride VEH Vehicle alone ZDF Zucker diabetic fatty ZL Zucker lean μCT Microcomputed tomography © The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

European Journal of Cardio-Thoracic Surgery – Oxford University Press

Published: May 1, 2020

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