Luteolin improves heart preservation through inhibiting hypoxia-dependent L-type calcium channels in cardiomyocytes

Abstract

The current study aimed to evaluate whether luteolin could improve long-term heart preservation; this was achieved by evaluating the heart following long-term storage in University of Wisconsin solution (the control group) and in solutions containing three luteolin concentrations. The effects of different preservation methods were evaluated with respect to cardiac function while hearts were in custom-made ex vivo Langendorff perfusion systems. Different preservation methods were evaluated with respect to the histology, ultrastructure and apoptosis rate of the hearts, and the function of cardiomyocytes. In the presence of luteolin, the rate pressure product of the left ventricle was increased within 60 min of reperfusion following a 12-h preservation, coronary flow was higher within 30 min of reperfusion, cardiac contractile function was higher throughout reperfusion following 12- and 18-h preservations, and the left ventricle peak systolic pressure was significantly higher compared with the control group (all P<0.05). The expression levels of apoptosis regulator Bax and apoptosis regulator Bcl-2 in the luteolin groups were significantly decreased and increased, respectively. Lactate dehydrogenase, creatine kinase and malondialdehyde enzymatic activity was increased following long-term storage, while the activity of superoxide dismutase was significantly decreased. Furthermore, luteolin inhibited L-type calcium currents in ventricular myocytes under hypoxia conditions. Thus, luteolin demonstrated protective effects during long-term heart preservation in what appeared to be a dose-dependent manner, which may be accomplished through inhibiting hypoxia-dependent L-type calcium channels.

Keywords: L-type calcium channel; Langendorff isolated heart perfusion model; apoptosis; heart preservation; luteolin; patch-clamp.

Figure 1.

General workflow. UW, University of Wisconsin solution; KH, Krebs-Henseleit solution.

Figure 2.

Lu improves heart function 12 h after preservation. (A) LVPSP, (B) +dP/dt_max, (C) -dP/dt_max, (D) HR, (E) RPP and (F) CF were measured 15, 30, 45 and 60 min after a 12-h preservation in a low, medium or high Lu solution. (G) Water content was measured after a 12-h preservation in a low, medium or high Lu solution. *P<0.05 vs. Control. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l; LVPSP, left ventricle peak systolic pressure; dP/dt_max, maximal rate of rise of LVPSP; HR, heart rate; RPP, rate pressure product; CF, coronary flow.

Figure 3.

Lu improves heart function 18 h after preservation. (A) LVPSP, (B) +dP/dt_max, (C) -dP/dt_max, (D) HR, (E) RPP and (F) CF were measured 15, 30, 45 and 60 min after an 18-h preservation in a low, medium or high Lu solution. (G) Water content was measured after an 18-h preservation in a low, medium or high Lu solution. *P<0.05 vs. control. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l; LVPSP, left ventricle peak systolic pressure; dP/dt_max, maximal rate of rise of LVPSP; HR, heart rate; RPP, rate pressure product; CF, coronary flow.

Figure 4.

Lu protects rat cardiac muscle morphology from long-term storage-induced damage. The morphology of rat cardiac muscle (A) prior to preservation, in the (B) control, (C) Lu low, (D) Lu medium and (E) Lu high groups 12 h after preservation, and in the (F) control, (G) Lu low, (H) Lu medium, (I) Lu high groups 18 h after preservation. Magnification, ×400. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l.

Figure 5.

Lu protects rat cardiac muscle ultrastructure from long-term storage-induced damage. (A) The morphology of rat cardiac muscle prior to preservation (magnification, ×10,000). The morphology of rat cardiac muscle in the (B) control (magnification, ×10,000), (C) Lu low (magnification, ×10,000), (D) Lu medium (magnification, ×10,000) and (E) Lu high (magnification, ×10,000) groups 12 h after preservation. The morphology of rat cardiac muscle in the (F) control (magnification, ×10,000), (G) Lu low (magnification, ×10,000), (H) Lu medium (magnification, ×10,000), (I) Lu high (magnification, ×10,000) groups 18 h after preservation. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l.

Figure 6.

Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining reveals that Lu protects rat cardiac muscle from apoptosis. Rat cardiac muscle (A) prior to preservation, in the (B) control, (C) Lu low, (D) Lu medium and (E) Lu high groups 12 h after preservation, and in the (F) control, (G) Lu low, (H) Lu medium, (I) Lu high groups 18 h after preservation (magnification, ×400). Quantification of the number of apoptotic cells (J) 12 h and (K) 18 h after preservation. *P<0.05 vs. control. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l.

Figure 7.

Lu ameliorates long-term storage-induced increases in apoptosis regulator Bcl-2 expression. Rat cardiac muscle in the (A) control, (B) Lu low, (C) Lu medium and (D) Lu high groups 12 h after preservation, and in the (E) control, (F) Lu low, (G) Lu medium, (H) Lu high groups 18 h after preservation. Magnification, ×400. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l.

Figure 8.

Lu enhances long-term storage-induced decreases in apoptosis regulator BAX expression. Rat cardiac muscle in the (A) control, (B) Lu low, (C) Lu medium (D) Lu high groups 12 h after preservation, and in the (E) control, (F) Lu low, (G) Lu medium, (H) Lu high groups 18 h after preservation. Magnification, ×400. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l.

Figure 9.

Luteolin inhibits the enzymatic activity of LDH, CK and MDA, and promotes the enzymatic activity of SOD. The enzymatic activities of (A) LDH and (B) CK were measured 15, 30, 45 and 60 min after a 12-h preservation in a low, medium or high Lu solution. The enzymatic activities of (C) LDH and (D) CK were measured 15, 30, 45 and 60 min after an 18-h preservation in a low, medium or high Lu solution. The enzymatic activities of (E) SOD and (F) MDA were measured 15, 30, 45 and 60 min after a 12 and 18-h preservation in a low, medium or high Lu solution. *P<0.05 vs. control. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l; LDH, lactate dehydrogenase; CK, creatine kinase; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 10.

Luteolin inhibits L-type calcium channels during hypoxia. (A) Representative traces of the ramp currents recorded. (B) A summary of the amplitude-voltage association in rat cardiac cells. (C) Traces of calcium currents following the incubation of cells in (a) Lu high, (b) Lu medium and (c) Lu low solutions, (d) following washout and (e) in the control group. (D) Percentage of calcium current through L-type calcium channels. *P<0.05 vs. control. Lu, luteolin; low, 7.5 µmol/l; medium, 15 µmol/l; high, 30 µmol/l.