All animal experiments were conducted in accordance with the rules issued by the State Veterinary Administration of the Slovak Republic, legislation No 377/2012 and with the regulations of the Animal Research and Care Committee of Centre of Experimental Medicine SAS – Project No. 2237/18-221/3, approved on 21 August 2018. This study was carried out in accordance with recommendations in the ARRIVE guidelines.
Male Wistar rats (Dobrá voda, Slovakia) aged 12-15 weeks and weighing 323 ± 28 g were used for our experiments. Animals were randomly divided into 4 experimental groups: (i) healthy control group (C), (ii) group affected by acute streptozotocin-induced diabetes (D), (iii) healthy control group with administration of metabolic regulator dichloroacetate (DCA), and (iiii) group with acute streptozotocin-induced diabetes modulated with DCA (D+DCA). Experimental animals were housed under natural mode conditions (22 ± 2 °C, humidity 55 ± 10 %, 12:12 dark/light cycle) with constant access to drinking water and standard pellet diet ad libitum.
An experimental model of acute diabetes was induced in male Wistar rats using a single intraperitoneal injection of streptozotocin (65 mg/kg of body weight, Sigma–Aldrich, USA) dissolved in 0.1 M citrate buffer (pH 4.0) 8 days prior to the experiment. Beginning from induction until the onset of the acute phase, glycosuria was monitored daily using GlukoPHAN strips (Pliva - Lachema, Brno, Czech Republic). Blood glucose, cholesterol and triacylglycerols were measured (MultiCare and appropriate test strips, Biochemical System International, Florence, Italy) on the eighth day after streptozotocin administration using blood collected from the tail vein as well as insulin in the serum (RIA kit, Linco Research, USA). Animals that revealed glycemia ≥ 20 mmol/L were considered diabetic.
DCA (Sigma–Aldrich, USA) was administered to animals intraperitoneally in two doses (150 mg/kg and 75 mg/kg) 60 minutes and 15 minutes before heart excision 74. DCA acts as an inhibitor of PDHK and ensures the activation of PDH, which is normally inhibited in the diabetic myocardium.
Administration of anesthesia
The experiment was performed on intraperitoneally anesthetized animals (thiopental, 50-60 mg/kg of body weight applied with heparin 500 IU). Prior to heart extraction, the animals were stabilized for 30 minutes.
Isolation of cardiac mitochondria
Mitochondria were isolated from rat hearts; immediately after extraction from the chest, hearts were immersed in cooled saline solution (0.9% NaCl, 4 °C) and washed to remove blood residues, aorta and fat. Differential centrifugation was used for mitochondrial isolation at 4 °C. After adding a small amount of cooled isolation solution (180 mM KCl, 4 mM ethylenediaminetetraacetic acid (EDTA) and 1% bovine serum albumin dissolved in dH2O, pH 7.4), the heart was milled with the GentleMACS Octo Dissociator (Miltenyi Biotec GmbH, Germany) (2 cycles per 1 min). Subsequently, cooled isolation solution was added to the sample up to 20 mL. This solution was homogenized using a manual homogenizer and centrifuged (1000 g, 10 min). The obtained mitochondria-containing supernatant was repeatedly centrifuged (6200 g, 10 min). The resulting sediment was suspended in a homogeneous state in 20 mL of cold isolation solution without albumin (180 mM KCl and 4 mM EDTA dissolved in dH2O, pH 7.4) and centrifuged again (6200 g, 10 minutes). The sediment (resulting mitochondrial fraction) was diluted with a small amount of isolation solution without albumin and homogenized 7. The concentration of proteins in the mitochondrial fraction was determined by a Synergy H1 multidetection reader (BioTek, USA) using Bradford’s method 75.
A schematic overview of the methodological procedures can be seen in Figure 8.
Measurement of mitochondrial membrane potential using the fluorescent dye JC-1
The freshly isolated mitochondrial suspension was diluted to a concentration of 1 mg protein/ml with cooled 20 mM MOPS buffer (3-(N-morpholino) propane sulfonic acid), pH 7.5, containing 110 mM KCl, 10 mM ATP, 10 mM MgCl2, 10 mM sodium succinate, and 1 mM EGTA. Samples were kept on ice until measurement. The mitochondrial membrane potential was analyzed using a FluoroLog FL3 spectrofluorometer (HORIBA Scientific, USA) and the fluorescent dye JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide, Enzo Life Sciences). The measurement was performed in the cuvette using a diluted suspension of 0.25 mg mitochondrial protein/ml 20 mM MOPS buffer with pH 7.5, while the resulting volume was 2 ml. JC-1 (2 µl, 0.3 mM) was added to the suspension, and the fluorescence intensity of the membrane potential was measured after 10 minutes of incubation at 25 °C. The fluorescence of each mitochondrial sample was monitored using an excitation wavelength of 490 nm and two emission wavelengths of 530 nm and 590 nm simultaneously. JC-1 represents a lipophilic cationic dye that is able to enter and redistribute in mitochondria depending on membrane potential; it can be in the form of monomers or J-aggregates. While JC-1 dominates in depolarized mitochondria in the form of monomers that emit green fluorescence (~530 nm), in energized and negatively charged mitochondria, it dominates in the form of J-aggregates that emit red fluorescence (~590 nm) 76. Next, 10 µl of 2,4-dinitrophenol (54.32 mM) was added, and the fluorescence was measured after 2 minutes of incubation at 25 °C. 2,4-dinitrophenol increases the proton permeability of the inner mitochondrial membrane, leading to the depolarization of membrane potential, which served as a positive control 77. The data are presented as a ratio of red J-aggregates and green monomers.
Measurement of the mitochondrial calcium retention capacity
For the monitoring of changes in extramitochondrial Ca2+ concentration and the ability of mitochondria to buffer exogenous Ca2+, a CRC assay modified by Gomez et al., Duicu et al. and Harisseh et al. was used 78–80. Fresh myocardial mitochondria were suspended in 2 ml of cooled incubation CRC buffer (50 µg protein/ml incubation CRC buffer composed of 150 mM sucrose, 50 mM KCl, 2 mM KH2PO4, 5 mM succinic acid in 20 mM Tris-HCl, pH 7.4 in 37 °C). The fluorescent probe Calcium GreenTM-5N (10 µl of 0.1 mM, Invitrogen, excitation-emission, 500-530 nm) was added to the diluted mitochondrial suspension, while the resulting volume in the cuvette was 2 ml. After consequent stabilization (200 s), 0.125 mM CaCl2 was added at regular intervals (60 s) in a constant amount of 4 µl at room temperature with stirring. The change in the extramitochondrial concentration of Ca2+ was continuously monitored using a FluoroLog FL3 spectrofluorometer (HORIBA Scientific, USA). The fluorescent probe Calcium GreenTM-5N has the characteristics of a low affinity impermeable dye that shows increased intensity of fluorescent emission after binding to Ca2+, while the probe does not fluoresce if Ca2+ is not present 81. Calcium GreenTM-5N also has a higher dissociation constant; thus, it serves as an appropriate indicator for monitoring the kinetics of the fast dynamics of Ca2+82. As a result of CaCl2 addition, an increased fluorescent signal associated with an increased Ca2+ concentration in the extramitochondrial space was detected. The signal decreased after uptake of Ca2+ to the mitochondrial matrix to almost baseline values. This effect was observable until mPTP opening due to Ca2+ overload in the mitochondrial matrix, shown as a sudden rise in extramitochondrial Ca2+ concentration accompanied by an increased fluorescent signal. Therefore, the amount of CaCl2 needed for the induction of mPTP opening serves as an indicator of the sensitivity of mPTP to Ca2+ overload in experimental models simulating pathological burden.
Proteomic analysis by nanoliquid chromatography and mass spectrometry (LC–MS/MS)
Mitochondrial samples containing 250 μg of protein were digested by trypsin in solution (Sigma–Aldrich, USA) at a ratio of 1:25 (a concentration of trypsin 0.2 µg/µL) overnight at 37 °C, and sample preparation was identical to that reported previously 49. Before proteomic analysis using a Nano System liquid chromatograph (Ultimate 3000 RSLC, Thermo Fisher Scientific, Germering, Germany) followed by mass spectrometry with electrospray ionization (ESI) and a 3D ion trap mass analyzer (Amazon SL, Bruker, Bremen, Germany), samples were desalted by using C18-U SPE columns (Strata, Phenomenex). All LC–MS parameters and database searching and protein identification methods were described previously 49.
Statistical analyses and interpretation
Descriptive and univariate analyses were performed on all selected animals' characteristics. Mean ± SEM (standard error of mean) is given for the normally distributed variables or a median and interquartile range if data showed substantial deviations from normality. Differences between the groups were tested with one-way analysis of variance (ANOVA), followed by the Dunnett test for multiple comparisons with the control or Tukey–Kramer test for all pairwise comparisons. In the case of nonnormality and/or unequal variances between groups being compared, a nonparametric alternative (the Kruskal–Wallis test and the post hoc pairwise comparisons with the Connover-Inman test) was performed. We used a two-way ANOVA to analyze data from factorial experiments: DCA treatment as the first main factor and experimentally induced diabetic condition as the second main factor.
Analysis of proteomic data was performed according to the model presented previously in Andelova et al. 49. In our analysis, we focused on mitochondrial proteins, which are considered structural and regulatory components of the mPTP complex, and 3 proteins related to oxidative damage or antioxidant potential, which occurred at the intersection of all 4 groups. Proteins with two or more identified peptides were further evaluated by the label-free method of the exponentially modified Protein Abundance Index (emPAI). The retrieved emPAI values were used to create a so-called “fold change” (FC) measure. The FC was defined as the ratio of abundances under 2 different experimental conditions averaged across replicates under these conditions. The log-transformed value of the 2 experimental group ratios - individually for each protein - was tested against the null hypothesis of no change. The absolute FC was calculated as 2log2value. Two-way repeated-measures ANOVA was used to verify the treatment effect. To evaluate differences in abundance levels of quantifiable proteins between two different experimental conditions, a two-sample t test was performed on log-transformed technical replicates. To estimate mutual relationships among the identified proteins within the groups, coexpression analysis was performed. Exploratory data analysis and using Pearson's correlation coefficient served for visual inspection of mutual correlation with significance. The resulting correlations presented in the form of heatmaps were obtained with Morpheus online software (https://software.broadinstitute.org/Morpheus/).
All p values were considered statistically significant at a two-tailed p value of <0.05. For more detailed information, see Andelova et al. 49.
Statistical analyses were performed using StatsDirect 3.0.191 software (Stats Direct Ltd., Cheshire, UK), Statistica 13 software (Dell-StatSoft, Inc. Tulsa, OK, TIBCO Software Inc. USA) and GraphPad Prism 8.0.1 (GraphPad Software, Inc., USA).