Pretreatment with low-dose gadolinium chloride attenuates myocardial ischemia/reperfusion injury in rats

Abstract

Aim: We have shown that low-dose gadolinium chloride (GdCl3) abolishes arachidonic acid (AA)-induced increase of cytoplasmic Ca(2+), which is known to play a crucial role in myocardial ischemia/reperfusion (I/R) injury. The present study sought to determine whether low-dose GdCl3 pretreatment protected rat myocardium against I/R injury in vitro and in vivo.

Methods: Cultured neonatal rat ventricular myocytes (NRVMs) were treated with GdCl3 or nifedipine, followed by exposure to anoxia/reoxygenation (A/R). Cell apoptosis was detected; the levels of related signaling molecules were assessed. SD rats were intravenously injected with GdCl3 or nifedipine. Thirty min after the administration the rats were subjected to LAD coronary artery ligation followed by reperfusion. Infarction size, the release of serum myocardial injury markers and AA were measured; cell apoptosis and related molecules were assessed.

Results: In A/R-treated NRVMs, pretreatment with GdCl3 (2.5, 5, 10 μmol/L) dose-dependently inhibited caspase-3 activation, death receptor-related molecules DR5/Fas/FADD/caspase-8 expression, cytochrome c release, AA release and sustained cytoplasmic Ca(2+) increases induced by exogenous AA. In I/R-treated rats, pre-administration of GdCl3 (10 mg/kg) significantly reduced the infarct size, and the serum levels of CK-MB, cardiac troponin-I, LDH and AA. Pre-administration of GdCl3 also significantly decreased the number of apoptotic cells, caspase-3 activity, death receptor-related molecules (DR5/Fas/FADD) expression and cytochrome c release in heart tissues. The positive control drug nifedipine produced comparable cardioprotective effects in vitro and in vivo.

Conclusion: Pretreatment with low-dose GdCl3 significantly attenuates I/R-induced myocardial apoptosis in rats by suppressing activation of both death receptor and mitochondria-mediated pathways.

Figure 1

GdCl₃ attenuated A/R-induced cell apoptosis in NRVMs. (A) Vehicle or GdCl₃ (2.5, 5, and 10 μmol/L) was incubated with A/R-treated cardiomyocytes as indicated for 24 h, and cell apoptosis was assessed on the basis of caspase-3 activity; (B) Apoptosis was also determined on the basis of annexin V-FITC/PI staining; (Q1: Necrotic cells; Q2: Early apoptotic cells; Q3: Viable cells; Q4: Late apoptotic cells). (C) Statistical analysis of flow cytometric data. All values are presented as the mean±SD. n=3. ^cP<0.01 vs control. ^eP<0.05, ^fP<0.01 vs A/R.

Figure 2

GdCl₃ 5 μmol/L and nifedipine 1 μmol/L inhibited A/R-induced cardiomyocytes apoptosis via the inhibition of the death receptor-related signaling pathway. (A) Representative images of Western blots and quantitative analyses of cleaved caspase-8; (B) Representative images of Western blots and quantitative analyses of DR5, Fas, and FADD. Mean±SD. n=3. ^bP<0.05, ^cP<0.01 vs control. ^eP<0.05, ^fP<0.01 vs A/R.

Figure 3

GdCl₃ inhibited A/R-induced cardiomyocyte apoptosis via inhibition of the mitochondria-related signaling pathway. (A) Representative images of Western blots and quantitative analyses of cytochrome c in mitochondrial/cytosol fractions; (B) Typical traces represent the spontaneous Ca²⁺ transients in NRVMs before and after AA (10 μmol/L) treatment for 2 min in the presence or absence of GdCl₃ (5 μmol/L) or nifedipine (1 μmol/L) for 2 min as indicated. (C) GdCl₃ (5 μmol/L) inhibited AA-induced increase in [Ca²⁺]_i in NRVMs. (D) Representative traces illustrate global Ca²⁺ transients before and after 50 μmol/L PE treatment in normal cells (Con) and the disability of spontaneous pacing in NRVMs with A/R or GdCl₃+A/R treatment. (E) Basal [Ca²⁺]_i levels, the mean fluorescence from 4 images at the beginning of recording normalized by the control F₀, were significantly enhanced as a result of A/R, which was partially prevented by 5 μmol/L GdCl₃. All values are presented as the mean±SD. n=3 for each bar in A. n=4–6 for each bar in B–E. ^bP<0.05, ^cP<0.01 vs control (A, E). ^fP<0.01 vs AA (C). ⁱP<0.01 vs A/R.

Figure 4

GdCl₃ reduced I/R-induced myocardial injury in a rat model. (A) The panel shows representative photomicrographs of LV sections from different groups after 30-min occlusion of the left anterior descending artery followed by 2 h of reperfusion. Blue-stained portion: non-ischemic normal region; red-stained portion: ischemic/reperfused but not infarcted region; unstained portion (white area): ischemic/reperfused, infarcted region; (B) Analysis of the percentage of myocardial infarct size in each group. Area-at-risk (AAR), infarct size (IS), left ventricle (LV); (C) Representative photomicrographs of H&E staining in myocardial tissues from sham-operated rats or rats with different pretreatments before I/R, bar = 50 μm; (D) Levels of CK-MB in rat sera; (E) Levels of cTn-I in rat sera; (F) LDH levels in rat sera. All values are presented as the mean±SD. n=8. ^bP<0.05, ^cP<0.01 vs sham. ^eP<0.05, ^fP<0.01 vs I/R.

Figure 5

GdCl₃ attenuated I/R-induced myocardial apoptosis in vivo. (A) Representative photomicrographs of TUNEL staining in myocardial tissues from sham-operated rats or rats with different pretreatments before I/R as indicated. Scale bar=30 μm; arrows in each panel indicate cells positive for apoptosis; (B) Bar graph shows the percentages of TUNEL-positive nuclei in the sham, I/R and drug-treated groups; (C) Cardiomyocyte apoptosis was assessed on the basis of caspase-3 activity; (D) Cardiomyocyte apoptosis was assessed on the basis of caspase-8 activity; (E) Cardiomyocyte apoptosis was assessed on the basis of Fas levels; (F–H) Representative images of Western blots and quantitative analyses of apoptosis-related signaling molecules (DR5, FADD, and cytochrome c). All values are presented as the means±SD. n=6–8. ^bP<0.05, ^cP<0.01 vs sham. ^eP<0.05, ^fP<0.01 vs I/R.

Figure 6

GdCl₃ decreased AA levels in the A/R and I/R models. (A) AA levels in NRVM A/R model with different treatments. All values are presented as the mean±SD. n=8. ^cP<0.01 vs Control. ^fP<0.01 vs A/R. (B) Serum AA levels in the I/R rat model with different treatments. All values are presented as the mean±SD. n=8. ^cP<0.01 vs sham. ^eP<0.05 vs I/R.