Chloroquine differentially modulates coronary vasodilation in control and diabetic mice

Abstract

Background and purpose: Chloroquine is a traditional medicine to treat malaria. There is increasing evidence that chloroquine not only induces phagocytosis but regulates vascular tone. Few reports investigating the effect of chloroquine on vascular responsiveness of coronary arteries have been made. In this study, we examined how chloroquine affected endothelium-dependent relaxation in coronary arteries under normal and diabetic conditions.

Experimental approach: We isolated coronary arteries from mice and examined endothelium-dependent relaxation (EDR). Human coronary endothelial cells and mouse coronary endothelial cells isolated from control and diabetic mouse (TALLYHO/Jng [TH] mice, a spontaneous type 2 diabetic mouse model) were used for the molecular biological or cytosolic NO and Ca²⁺ measurements.

Key results: Chloroquine inhibited endothelium-derived NO-dependent relaxation but had negligible effect on endothelium-derived hyperpolarization (EDH)-dependent relaxation in coronary arteries of control mice. Chloroquine significantly decreased NO production in control human coronary endothelial cells partly by phosphorylating eNOS^Thr495 (an inhibitory phosphorylation site of eNOS) and attenuating the rise of cytosolic Ca²⁺ concentration after stimulation. EDR was significantly inhibited in diabetic mice in comparison to control mice. Interestingly, chloroquine enhanced EDR in diabetic coronary arteries by, specifically, increasing EDH-dependent relaxation due partly to its augmenting effect on gap junction activity in diabetic mouse coronary endothelial cells.

Conclusions and implications: These data indicate that chloroquine affects vascular relaxation differently under normal and diabetic conditions. Therefore, the patients' health condition such as coronary macrovascular or microvascular disease, with or without diabetes, must be taken account into the consideration when selecting chloroquine for the treatment of malaria.

Figure 1

Endothelium‐dependent relaxation in coronary artery is attenuated by chloroquine. (a) Endothelium‐dependent relaxation (EDR) induced by ACh in the absence (Cont, open circle) or presence (CQ, closed circle) of chloroquine (CQ, 10 μM). n _mice = 6 in each group. Data are mean ± SE. *P < .05 versus Cont. (b) Smooth muscle (SM)‐dependent relaxation induced by sodium nitroprusside (SNP, an NO donor) in the absence (Cont) or presence (CQ) of CQ. n _mice = 7 in each group. (c) SM‐dependent relaxation induced by papaverine (Pap, a PDE inhibitor) in the absence (Cont) or presence (CQ) of CQ. n _mice = 6 in each group. (d)Endothelium‐derived hyperpolarization (EDH)‐dependent relaxation in the absence (Cont) or presence (CQ) of CQ. EDH‐dependent relaxation was conducted by ACh administration in the presence of indomethacin (Indo, a COX inhibitor, 10 μM) and L‐NAME (an eNOS inhibitor, 100 μM). n _mice = 8 in each group. (e) Endothelium‐derived NO (EDNO)‐dependent relaxation in the absence (Cont) or presence (CQ) of CQ. EDNO‐dependent relaxation was accessed by ACh administration in the presence of Indo, TRAM34 (an inhibitor of intermediate‐conductance Ca²⁺‐activated K⁺ channel, 100 nM), and apamin (an inhibitor of small conductance Ca²⁺ activated K⁺ channel, 100 nM). n _mice = 13 in each group. (f) Pretreatment with Indo (to inhibit PGI₂), L‐NAME (to inhibit NO), and TRAM34 and apamin (to inhibit EDH) abolishes ACh‐induced relaxation in coronary artery (CA). n _mice = 8 in each group. *P < .05 compared to control (i.e., w/o inhibitors). Statistical comparison between dose–response curves was made by two‐way ANOVA with Bonferroni post hoc test

Figure 2

CQ decreases cytosolic NO level, increases phosphorylated eNOS⁴⁹⁵, and attenuates the rise in cytosolic Ca²⁺ concentration after stimulation in human coronary endothelial cells. (a) Representative images showing NO signals, determined by DAF, in human coronary endothelial cells (HCECs) with or without (w/o) CQ treatment for 30 min. Bar = 50 μm. (b) Summarized data showing DAF intensity in control cells (Cont, n _experiment = 15, n _cells = 127) and CQ‐treated cells (CQ, n _experiment = 13, n _cells = 118). Data are mean ± SE. *P < .05 compared to Cont. (c) WB analysis on total eNOS, phosphorylated eNOS at Ser1177 (p‐eNOS^ser1177), and Thr495 (p‐eNOS^Thr495) in control HCECs (Cont) and CQ‐treated HCECs (CQ). Actin was used as a loading control. The scattered plots show the level of p‐eNOS normalized to total eNOS (left panels) and the level of total eNOS normalized to actin (right panel). n _experiment = 7. Data are mean ± SE. *P < .05 compared to Cont. (d) Representative records showing cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) in HCECs before, during, and after stimulation with cyclopiazonic acid (CPA, an inhibitor of sarcoplasmic reticulum Ca²⁺‐ATPase to increase [Ca²⁺]_cyto, 10 μM) in the absence (vehicle, blue tracing) or presence (CQ, red tracing) of CQ. (e,f) Summarized data showing the amplitude (∆Peak, e) and the AUC (f) of CPA‐induced increase in [Ca²⁺]_cyto in control cells (Cont, n _experiment = 12, n _cells = 120) and CQ‐treated cells (CQ, n _experiment = 11, n _cells = 110). Data are mean ± SE. *P < .05 compared to Cont. (g) The level of p‐eNOS^Thr495 after the treatment of CPA with or without CQ. n _experiment = 6. Data are mean ± SE. *P < .05 compared to CPA. Unpaired Student's t‐test was used for comparisons of two experimental groups in Figure 2b,c,g. Mann–Whitney test was used for Figures 2e,f

Figure 3

CQ enhances EDH‐dependent coronary vasodilation in diabetic mice. (a): Oral glucose tolerance test in Wt mice (n _mice = 7) and TH mice (n _mice = 7). *P < .05 compared to Wt mice. (b) EDR in CAs from Wt and TH mice without CQ treatment (w/o CQ). Wt w/o CQ, n _mice = 13; TH w/o CQ, n _mice = 12. Data are mean ± SE. *P < .05 compared to Wt w/o CQ. (c) Effect of CQ on EDR in CAs from Wt mice. Wt w/o CQ, n _mice = 13; Wt with CQ, n _mice = 9. Data are mean ± SE. *P < .05 compared to Wt w/o CQ. (d) Effect of CQ on EDR in CAs from TH mice. TH w/o CQ, n _mice = 12; TH with CQ, n _mice = 8. Data are mean ± SE. *P < .05 compared to TH w/o CQ. (e) SM‐dependent relaxation induced by SNP in CAs from Wt (n _mice = 5) and TH mice (n _mice = 5) in the absence of CQ treatment (w/o CQ). (f) EDH‐dependent relaxation in CAs isolated from Wt mice in the absence or presence of CQ. n _mice = 7 in each group. (g) EDH‐dependent relaxation in CAs from TH mice in the absence or presence of CQ. n _mice = 7 in each group. *P < .05 compared to w/o CQ. (h) EDNO‐dependent relaxation in CAs from Wt mice in the absence or presence of CQ. n _mice = 6 in each group. *P < .05 versus w/o CQ. (i) EDNO‐dependent relaxation in CAs from TH mice in the absence or presence of CQ. n _mice = 5 in each group. Indo and L‐NAME were used to inhibit PGI₂ and NO in experiments shown in (f) and (g), while Indo, TRAM34, and apamin were used to inhibit PGI₂ and EDH in experiments shown in (h) and (i). (j) EDH‐dependent relaxation in CAs isolated from Wt mice in the presence of CQ with vehicle (0.25% ethanol) or 18GA (a non‐specific Gap junction inhibitor, 50 μM). n _mice = 8 in each group. (k) EDH‐dependent relaxation in CAs from TH mice in the presence of CQ with vehicle or 18GA. n _mice = 8 in each group. *P < .05 versus w/o 18GA. Statistical comparison between dose–response curves were made by two‐way ANOVA with Bonferroni post hoc test

Figure 4

CQ increases gap junction activity but does not change the CPA‐induced [Ca²⁺]_cyto increase, in MCECs isolated from TH mice. (a,b) Lucifer yellow dye transfer experiment in MCECs was conducted to examine the gap junction activity. (a) Representative photographs showing the images of LY dye transfer in ECs. The red line indicates the edge of the scrape. The yellow line indicates the farthest site of cells showing visual uptake of dye. The distance between yellow and red lines was calculated as a dye transfer. (b) The scattered dots show the normalized data of dye transfer by the distance in MCECs isolated from Wt without CQ (Wt‐MCECs w/o CQ). Bar = 100 μ. n _mice = 6 in each group. (c,d) Summarized data showing ∆Peak (c) and the AUC (d) of CPA‐induced increase in [Ca²⁺]_cyto in Wt MCECs w/o CQ (Wt, n _mice = 5, n _cells = 43), Wt MCECs with CQ (Wt + CQ, n _mice = 5, n _cells = 43), TH MCECs w/o CQ (TH, n _mice = 5, n _cells = 44), and TH MCECs with CQ (TH + CQ, n _mice = 5, n _cells = 44). *P < .05 between groups as indicated. Statistical comparison between the groups was made by one‐way ANOVA with Bonferroni post hoc test