PDE5 Inhibitor Tadalafil and Hydroxychloroquine Cotreatment Provides Synergistic Protection against Type 2 Diabetes and Myocardial Infarction in Mice

Abstract

Diabetes is associated with a high risk for ischemic heart disease. We have previously shown that phosphodiesterase 5 inhibitor tadalafil (TAD) induces cardioprotection against ischemia/ reperfusion (I/R) injury in diabetic mice. Hydroxychloroquine (HCQ) is a widely used antimalarial and anti-inflammatory drug that has been reported to reduce hyperglycemia in diabetic patients. Therefore, we hypothesized that a combination of TAD and HCQ may induce synergistic cardioprotection in diabetes. We also investigated the role of insulin-Akt-mammalian target of rapamycin (mTOR) signaling, which regulates protein synthesis and cell survival. Adult male db/db mice were randomized to receive vehicle, TAD (6 mg/kg), HCQ (50 mg/kg), or TAD + HCQ daily by gastric gavage for 7 days. Hearts were isolated and subjected to 30-minute global ischemia, followed by 1-hour reperfusion in Langendorff mode. Cardiac function and myocardial infarct size were determined. Plasma glucose, insulin and lipid levels, and relevant pancreatic and cardiac protein markers were measured. Treatment with TAD + HCQ reduced myocardial infarct size (17.4% ± 4.3% vs. 37.8% ± 4.9% in control group, P < 0.05) and enhanced the production of ATP. The TAD + HCQ combination treatment also reduced fasting blood glucose, plasma free fatty acids, and triglyceride levels. Furthermore, TAD + HCQ increased plasma insulin levels (513 ± 73 vs. 232 ± 30 mU/liter, P < 0.05) with improved insulin sensitivity, larger pancreatic β-cell area, and pancreas mass. Insulin-like growth factor-1 (IGF-1) levels were also elevated by TAD + HCQ (343 ± 14 vs. 262 ± 22 ng/ml, P < 0.05). The increased insulin/IGF-1 resulted in activation of downstream Akt/mTOR cellular survival pathway. These results suggest that combination treatment with TAD and HCQ could be a novel and readily translational pharmacotherapy for reducing cardiovascular risk factors and protecting against myocardial I/R injury in type 2 diabetes.

Fig. 1.

Experimental design and protocol. The schematic diagram shows the sequence and timing of various experimental procedures, including the drug treatment, OGTT, insulin tolerance test (ITT), harvest of tissue, global I/R in Langendorff mode, histology analysis, as well as plasma and cardiac tissue biochemical measurements.

Fig. 2.

Effect of TAD, HCQ, and combination treatment on postischemic infarct size in db/db mice. Top: Representative images of transverse sections of TTC-stained hearts collected after 1-week respective drug treatments and ex vivo global I/R. Bottom: Bar diagram showing myocardial infarct size presented as % of risk area (mean ± S.E.M., n = 6 for CTRL and n = 5 for TAD, HCQ, and TAD + HCQ). *P < 0.05 versus CTRL group.

Fig. 3.

Effect of TAD, HCQ, and combination treatment on cardiac function. Post-I/R ventricular developed force (A), rate-force product (B), heart rate (C), and coronary flow rate (D) are presented as % of preischemia baseline (mean ± S.E.M.; n = 6 for CTRL and n = 5 for TAD, HCQ, and TAD + HCQ). No statistical significance was observed among the four treatment groups in any of the cardiac function indices.

Fig. 4.

Changes in blood glucose, plasma insulin, IGF-1 levels, and lipid profile after treatment with TAD and HCQ. (A, B) Plasma insulin and IGF-1 levels measured with ELISAs. (C) Blood glucose levels measured before and after the 1-week drug treatment, respectively (mean ± S.E.M.; n = 13 for CTRL and HCQ, n = 15 for TAD, and n = 12 for TAD + HCQ). (D–H) Plasma and cardiac levels of free fatty acids, triglycerides, and total cholesterol measured using enzymatic assays (mean ± S.E.M., n = 8 for TAD, n = 9 for HCQ, and n = 12 for CTRL and TAD + HCQ). *P < 0.05; ***P < 0.001 versus CTRL group.

Fig. 5.

Insulin tolerance test (ITT) and OGTT after treatment with TAD, HCQ, and a combination of TAD + HCQ. Animals were fasted overnight before the OGTT. Glucose (2 mg/kg) was administered via oral gavage, and blood samples were taken from the tails. (A) Glucose levels; (B) area under the curve; (C) insulin levels; and (D) area under the curve. The ITT was performed after the animals were fasted for 6 hours. Insulin (0.9 IU/kg regular human) was given i.p.; (E) blood glucose levels; (F) area under the curve; (G) insulin response curve is presented as percentage to baseline glucose. Data are mean ± S.E.M. (n = 5/group,). *P < 0.05 versus CTRL group.

Fig. 6.

Effect of TAD, HCQ, or combination treatment on pancreatic islets. (A) Representative pictures of immunofluorescent-stained paraffin sections of pancreata. Goat anti-insulin antibody was used to detect insulin inside islets, and a secondary antibody conjugated with fluorescein isothiocyanate was used. (B) Representative pictures of H&E-stained pancreas, paraffin-fixed sections, with further magnified representative images of the pancreatic islets at lower-right corners. (C) Insulin-positive β-cell area versus pancreas area (n = 5/group). (D) Bar diagram showing pancreatic islet number per mm² pancreas area in all treatment groups (n = 8 for TAD; n = 9 for CTRL, HCQ, and TAD + HCQ). (E) Bar diagram showing the percentage of pancreas mass versus body weight (n = 5/group). Data are mean ± S.E.M. *P < 0.05; **P < 0.01 versus CTRL.

Fig. 7.

Effect of TAD, HCQ, or combination treatment on mTOR activation after I/R. (A) Representative Western blot images; (B) bar diagram showing quantitative analysis of cardiac p-Akt^Thr308/Akt; (C) p-mTOR/mTOR; (D) expression of Raptor; (E) p-S6/S6; (F) Rictor; and (G) p-Akt^Ser473/Akt after I/R. Data are presented as mean ± S.E.M. (n = 4/group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 versus CTRL group.

Fig. 8.

Effect of TAD, HCQ, and combination treatment on myocardial ATP production. ATP levels are normalized with respective protein concentration for each sample. Data are presented as mean ± S.E.M. (n = 4/group). **P < 0.01 versus CTRL group.