The cardioprotective efficacy of TVP1022 in a rat model of ischaemia/reperfusion

Abstract

Background and purpose: Because myocardial infarction is a major cause of morbidity and mortality worldwide, protecting the heart from the ischaemia and reperfusion (I/R) damage is the focus of intense research. Based on our in vitro findings showing that TVP1022 (the S-enantiomer of rasagiline, an anti-Parkinsonian drug) possesses cardioprotective effects, in the present study we investigated the hypothesis that TVP1022 can attenuate myocardial damage in an I/R model in rats.

Experimental approach: The model consisted of 30-min occlusion of the left anterior descending artery followed by 4 or 24 h reperfusion. In addition, we investigated the possible mechanisms of cardioprotection in H9c2 cells and neonatal rat ventricular myocytes (NRVM) exposed to oxidative stress induced by H(2) O(2) .

Key results: TVP1022 (20 and 40 mg·kg(-1) ) administered 5 min before reperfusion followed by an additional dose 4 h after reperfusion reduced the infarct size and attenuated the decline in ventricular function. TVP1022 also attenuated I/R-induced deterioration in cardiac mitochondrial integrity evaluated by mitochondrial swelling capacity. In vitro, using H9c2 cells and NRVM, TVP1022 attenuated both serum free- and H(2) O(2) -induced damage, preserved mitochondrial membrane potential and Bcl-2 levels, inhibited mitochondrial cytochrome c release and the increase in cleaved caspase 9 and 3 levels, and enhanced the phosphorylation of protein kinase C and glycogen synthase kinase-3β.

Conclusions and implications: TVP1022 provided cardioprotection in a model of myocardial infarction, and therefore should be considered as a novel adjunctive therapy for attenuating myocardial damage resulting from I/R injuries.

Figure 1

TVP1022 reduced the infarct size in ischaemia and reperfusion (I/R) rats. (A) Representative pictures of ventricular slices from rats treated with saline or TVP1022 (40 mg·kg⁻¹) as described in Methods. The area at risk (AAR) and infarct regions were determined at 24 h after reperfusion. Dark stain – viable area; pale stain – area at risk (AAR). (B) The AAR normalized to LV area (%). (C) The infarct size normalized to the AAR (%). Each bar represents the mean ± SEM of the I/R + saline (n = 13), I/R + TVP1022, 20 mg·kg⁻¹ (n = 12) and MI + TVP1022, 40 mg·kg⁻¹ (n = 6) groups. *P < 0.05 versus control.

Figure 2

TVP1022 attenuated the decline in ventricular function in ischaemia and reperfusion (I/R rats). (A) Representative echocardiographic 2D-mode records obtained at the papillary muscle level at baseline and 24 h post-I/R, in rats treated with saline (upper panel) or TVP1022 40 mg·kg⁻¹ (lower panel). (B) Ejection fraction and (C) fractional shortening measured at baseline and 24 h post-I/R in the different groups: I/R + Saline (n = 10), I/R + TVP1022, 20 mg·kg⁻¹ (n = 9) and I/R + TVP1022, 40 mg·kg⁻¹ (n = 10). *P < 0.05. versus baseline measurement.

Figure 3

TVP1022 preserved global circumferential strain 24 h post-ischaemia and reperfusion (I/R). (A) Two sets of representative circumferential strain maps (coloured circles) implanted on the echocardiographic records. Each set includes baseline and 24 h post-I/R records of three B-mode short-axis records. Upper panel – apex (AP); middle panel – papillary muscle (PM); lower panel – mitral valve (MV). Negative circumferential strain (blue) indicates myocardial contraction. (B) AP and (C) PM circumferential strain measured at baseline and 24 h post-I/R. Results were expressed as mean ± SEM obtained from the I/R + saline (n = 16), TVP1022 20 mg·kg⁻¹ (n = 14) and TVP1022 40 mg·kg⁻¹ (n = 9) groups. *P < 0.05 versus baseline measurement.

Figure 4

TVP1022 attenuated ischaemia and reperfusion (I/R)-induced mitochondrial damage. Mitochondria were isolated from intact ventricular tissue (n = 5), and from the area at risk from I/R + saline (n = 5) and I/R + TVP1022 40 mg·kg⁻¹ (n = 6) groups. The absorbance at 520 nm of isolated mitochondria was recorded every 10 s for 5 min after the addition of 200 µM Ca²⁺. The relative absorbance is presented as mean ± SEM. #P < 0.001 versus intact; *P < 0.001 versus I/R + saline.

Figure 5

TVP1022 protected neonatal rat ventricular myocytes (NRVM) against serum starvation-induced apoptosis. NRVM were incubated with or without TVP1022 in serum free medium. (A) Apoptotic nuclei were identified by TUNEL analysis. Absolute values of 5–10 separate fields were averaged, and apoptotic cells were expressed as percentage of total cells in three independent experiments. #P < 0.05 versus full serum; *P < 0.05 versus serum-free medium. (B) Representative Western blots of cleaved caspase 9, cleaved caspase 3 and Bcl-2. Equal loading was checked with an antibody against β-actin (n = 3). DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling.

Figure 6

TVP1022 protected H9c2 cells from H₂O₂-induced cardiotoxicity. H9c2 cells were treated with H₂O₂ (300 µM) in the absence or presence of TVP1022 (0.1, 1 and 10 µM) for 1 h. (A) Cell viability was measured using the MTT assay. (B) Representative images of H9c2 cells stained with JC-1. (C) Representative immunofluorescence images of cytochrome c and a quantitative summary of four independent experiments. Cytochrome c (green); mitochondrial staining with MitoTracker (red); nuclear staining with Topro (blue) and merged images of cytochrome c and mitochondrial staining showing co-localization in yellow. Co-localization is presented as percentage of stained area per total cell area (n = 20–30 cells). #P < 0.05 versus control; *P < 0.05 versus H₂O₂. MTT, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium.

Figure 7

The involvement of the PKC/GSK-3β and PI3K/AKT pathways in the cardioprotective efficacy of TVP1022 in H9c2 cells. (A) H9c2 cells were pre-incubated without or with chelerythrine (5 µM) or LY294002 (10 µM) for 1 h, and then further incubated with H₂O₂ (300 µM) in the absence or presence of TVP1022 (10 µM) for 1 h. Cell viability was measured by the MTT assay. Results are presented as percent of control untreated cells. n = 3 experiments. *P < 0.05 versus H₂O₂ alone; ^P < 0.05 versus H₂O₂+ TVP1022. (B) H9c2 cells were incubated without or with TVP1022 (1 µM and 10 µM) or PMA (100 nM) for 30 min. (C) H9c2 cells were pre-incubated with a vehicle alone, or with chelerythrine (5 µM) for 1 h, and then incubated without or with TVP1022 (10 µM) for 30 min. Phosphorylated PKC levels were analysed in cell lysates and were normalized to β-tubulin levels. (D) H9c2 cells were incubated without or with TVP1022 (1 µM and 10 µM) for 30 min. (E) H9c2 cells were pre-incubated with a vehicle alone, or with LY294002 (10 µM) or GF109203X (2.5 µM), and then incubated without or with TVP1022 (10 µM) for 30 min. Phosphorylated GSK-3β (Ser9) levels were analysed in cell lysates and normalized to GSK-3β levels. In panels (B)–(E), upper lanes show representative Western blots of the respective proteins in the different experimental groups. Equal loading was checked with an antibody against β-tubulin or GSK-3β. All blots are representative of three independent experiments, summarized in bar graphs below. #P < 0.05 versus control untreated cells; *P < 0.05 versus TVP1022. MTT, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium; PMA, 12-myristate 13-acetate.

Figure 8

The effect of TVP1022 on phosphorylation of PKC and GSK-3β in neonatal rat ventricular myocytes (NRVM). NRVM were incubated without or with TVP1022 (1 or 10 µM) for 30 min. Phosphorylation levels of (A) PKC and (C) GSK-3β (Ser9) were analysed in NRVM lysates by Western blotting. NRVM were pre-incubated with vehicle alone, or with (B) chelerythrine (5 µM) or (D) LY294002 (10 µM) for 1 h, and then incubated without or with TVP1022 (10 µM) for 30 min. Phosphorylated PKC and GSK-3β levels were analysed in cell lysates and normalized to β-tubulin or to GSK-3β levels respectively. In panels (A)–(D) upper lanes show representative Western blots of the respective proteins in the different experimental groups. Equal loading was checked with an antibody against β-tubulin or GSK-3β. All blots are representative of three independent experiments, summarized in bar graphs below. #P < 0.05 versus control untreated cells; *P < 0.05 versus TVP1022. (E) A schematic model for the cardioprotective effect of TVP1022. TVP1022 inactivates GSK-3β via PKC and PI3K signalling pathways, resulting in cardioprotection against MPTP opening, and mitochondrial cytochrome c release into the cytosol. In turn, TVP1022 increases the levels of anti-apoptotic protein Bcl-2 and inhibits the activation of caspases 9 and 3. MPTP, mitochondrial permeability transition pore.