Chemicals and reagents

5-fluorouracil (5-FU) was procured from Sigma–Aldrich, USA. DCFH-DA, and Rhodamine 123. Rabbit 8-OHdG (bs-1278R) polyclonal antibody, Goat anti-rabbit IgG secondary antibody (65–6120), IL-6 rabbit polyclonal antibody (bs-0782R), IL-10 rabbit polyclonal antibody (PA5-85660) and TNF alpha rabbit polyclonal antibody (bs-2081R) were procured from Thermo Fisher Scientific (Rockford, USA). TUNEL assay kit (TACS-XL In Situ Apoptosis Detection Kit) was procured from R & D systems, USA. Other chemicals utilized in the current research were of the highest quality and obtained from local Indian firms.

Animals and treatment protocol

Male BALB/c mice (25–30 g), 6–8 weeks old, were obtained from Institutional central animal facility. Animals were housed under controlled conditions (room temperature 25 ± 1 °C; humidity (65–80%); 12/12 h alternate light/dark cycle). All animals were fed normal pellet and water ad libitum. Prior to the start of the experiment, all animals were acclimatized for one week. All animal experiments were approved by Institutional animal ethics committee (IAEC/KMC/54/2021). Animal experimentation was conducted in compliance with CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Gov. of India) regulations, and institutional animal ethics committee guidelines.

Animals were randomly divided into four groups viz; Control, Mito-TEMPO, 5-FU, and 5-FU + Mito- TEMPO group. All animals were acclimatized for one week before starting the experimental protocol. 5-FU group received intraperitoneal injection (12 mg/kg b.w.) of 5-FU once a day for four consecutive days. Mito-TEMPO group received intraperitoneal dose of Mito-TEMPO (0.1 mg/kg b.w. dissolved in 0.9% NaCl (aq.)) daily till the termination of the study. 5-FU + Mito-TEMPO group animals received mito-TEMPO and 5-FU as described for 5-FU and Mito-TEMPO groups, respectively. Mito-TEMPO administration started one week prior to the 5-FU administration (Fig. 1). The animals were assessed for change in body weight, diet, and water intake daily. All mice were sacrificed after 24 h of the last injection of 5-FU and heart tissue samples were processed for further investigations.

Fig. 1

Schematic diagram for treatment protocol

Cardiac injury marker

Blood samples of mice were obtained from retro-orbital plexus and the levels of serum CK-MB and AST were assessed using standard commercial kit (Liqui check, Agappe diagnostics, India) to confirm the induction of myocardial injury by 5-FU and the probable beneficial effect of mito-TEMPO treatment.

Histopathological analysis

Histopathological changes in mice heart were determined by Hematoxylin and Eosin (H & E) staining. Briefly, tissue samples were fixed in formalin and processed for H & E staining as per the standard laboratory protocol [16]. The stained tissue sections were examined under light microscope (Lx 300, Labomed, USA).

TTC staining for detection non-viable myocardial tissue

Non-viable myocardial tissue was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining of heart tissue [16]. Briefly, excised heart tissues were washed with ice-cold 0.9% normal saline and thin transverse sections of the tissue were obtained. The tissue sections were incubated in TTC solution (10 mg TTC in 0.1 M sodium phosphate buffer) for 20 min. Further, tissue fixation was carried out by immersing the tissue in 10% formalin, overnight. The infarcted area was photographed and analyzed using Image J software (Version 1.53k, National Institute of Health, USA).

8-hydroxy-2-deoxyguanosine (8-OHdG) Immunohistochemistry

The immunohistochemistry for 8-OHdG was performed as described earlier [17]. Briefly, paraffin embedded cardiac tissue sections were deparaffinized and rehydrated in descending grades of alcohol. The epitope retrieval was carried out by immersing the slide in sodium citrate solution (10 Mm, pH 6.0). Slides were incubated in primary antibody 8-OHdG (diluted to 1:400) at room temperature. Further, tissue sections were incubated in HRP-labeled secondary antibody (diluted to 1:3000) for 1 h at room temperature and visualized under light microscope (Lx 300, Labomed, USA).

TUNEL staining

For cell death analysis, TUNEL staining of cardiac tissue section was performed using commercially available kit as per manufacturer’s protocol. Briefly, deparaffinised and rehydrated tissue sections were covered with proteinase K and allowed to incubate at 37 °C for 15 min. After washing and quenching, slides were immersed in 1X TdT labeling buffer and covered with B-dNTP labeling reaction mixture at 37 °C for 30 min in a humidifying chamber. Further, slides were covered with Anti-BrdU antibody solution and incubated for 30 min at 37 °C. Further, slides were washed in PBS-Tween 20 solution for 2 min and immersed in Strep-HRP solution. Finally, after washing tissue section in PBS and deionized water slides were processed for counterstaining in methyl green and then observed under light microscope (Lx 300, Labomed, USA).

Estimation of mitochondrial oxidative stress and activity of mitochondrial enzymes

Mitochondria from cardiomyocytes were isolated as described previously [18]. Mitochondrial oxidative stress was assessed in terms of mitochondrial lipid peroxidation (mtLPO) and mitochondrial reactive oxygen species (mtROS) as described earlier [17]. Briefly, levels of mtLPO were estimated by suspending mitochondrial fraction in Buege and Aust reagent, and centrifuged at 1000 × g (10 min). Optical density of the obtained supernatant was read at 532 nm and level of mtLPO was expressed as nmol MDA/mg protein (molar extinction coefficient 1.56 × 105 M−1 cm−1). The levels of mtROS were estimated by incubating the mitochondrial fraction in DCFH-DA (1.25 mM in methanol; 37 °C for 30 min). Optical density of the sample was read at excitation wavelength 500 nm and emission wavelength 520 nm in fluorimeter (FP8300, Jasco, USA). Further, activities of mitochondrial complex-I, complex-II, and complex-IV, malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in cardiac tissue were estimated according to the procedure described previously [19,20,21].

Estimation of mitochondrial antioxidant defence status

Mitochondrial antioxidant defence status of mice heart was estimated in terms of activities of mitochondrial antioxidant defence enzymes. The ativity of mitochondrial reduced glutathione (mtGSH) was estimated by mixing 5,5′-dithiobis-2-nitrobenzoic acid (Ellman’s reagent) in 0.1 M phosphate buffer (pH 8) containing mitochondrial fraction [22]. Similarly, activity of mtGR was estimated by suspending mitochondrial fraction in substrate buffer (0.1 mM NADPH, 1 mM GSSG in 0.2 M phosphate buffer, pH 7.6), O.D. was noted at 340 nm for 3 min and activity of mtGR was expressed as nmol NADPH consumed/min/mg protein [23]. Further, activity of mtGPx was estimated by suspending mitochondrial fraction in reaction mixture (1 mM GSH, 0.22 mM NADPH, 5 mM EDTA, 50 mM Tris–HCl; pH 7.6). 0.22 mM tert-butyl hydroperoxide was added to the mixture, O.D. was noted at 340 nm for three minutes, and the activity of mtGPx was expressed as nmol NADPH oxidized/ min/mg protein [21]. The activity of MnSOD was estimated by suspending mitochondrial suspension in reaction mixture (0.2 mM pyrogallol, 1 mM EDTA, 50 mM Tris–HCl; pH 7.6) and O.D. of the sample was noted at 420 nm for three minutes. One unit enzyme activity was defined as the amount which decreased the autooxidation of pyrogallol by 50%. Enzyme activity was expressed as IU/min/mg protein [24].

Estimation of mitochondrial membrane potential

Mitochondrial membrane potential in cardiac tissue was assessed according to the procedure described previously [19]. Briefly, mitochondrial fraction was suspended in the reaction mixture (pH 7.4) containing sucrose (150 mM), MgCl2 (4 mM), HEPES KOH (30 mM) and K2HPO4 (5 mM) and incubated at 37 °C for 5 min. Rhodamine 123 (5 μM) was added to initiate the reaction and the sample was read in fluorometer (FP8300, Jasco, USA) at wavelength of 507 nm (excitation) and 527 nm (emission).

Inflammatory markers

Levels of inflammatory cytokines (IL-6, IL-10 and TNF-α) were assessed by ELISA technique. Briefly, antigen from cardiac tissues of mice was extracted as described earlier [25]. 96-well polystyrene flat-bottom ELISA plates (Himedia, India) were incubated with 50 μl of antigen overnight at 4 °C. After washing the plates with wash buffer (PBSTw), 1% bovine serum albumin was used as blocking reagent and100 μl of primary antibodies (diluted to 1:1000) of IL-6, IL-10 and TNF-α were added to respective well. Further, plates were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (diluted to 1:3000) and O.D. was noted at 450 nm in ELISA plate reader.

Protein estimation

Estimation of protein concentration present in mitochondrial fractions was carried out as described by previously [26]. The absorbance was noted at 620 nm using spectrophotometer.