Novel Potentials of the DPP-4 Inhibitor Sitagliptin against Ischemia-Reperfusion (I/R) Injury in Rat Ex-Vivo Heart Model

Abstract

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a class of oral anti-diabetic drugs, implicated in pleiotropic secondary cardioprotective effects. The aim of the study was to unveil the unknown and possible cardioprotective targets that can be exerted by sitagliptin (Sitg) against ischemia-reperfusion (I/R) injury. Male wistar rats received 2 weeks' Sitg oral treatment of different doses (25, 50, 100, and 150 mg/kg/day), or saline as a Control. Hearts were then isolated and subjected to two different I/R injury protocols: 10 min perfusion, 45 min regional ischemia, and 120 min reperfusion for infarct size (IS) measurement, or: 10 min perfusion, 45 min regional ischemia and 10 min reperfusion for biochemical analysis: nitric oxide synthases (NOSs) and DPP-4 activity, glucagon-like peptide-1 (GLP-1), Calcium, transient receptor potential vanilloid (TRPV)-1 and calcitonin gene-related peptide (CGRP) levels, transient receptor potential canonical (TRPC)-1 and e-NOS protein expression. NOS inhibitor (L-NAME) and TRPV-1 inhibitor (Capsazepine) were utilized to confirm the implication of both signaling mechanisms in DPP-4 inhibition-induced at the level of IS. Findings show that Sitg (50 mg) resulted in significant decrease in IS and DPP-4 activity, and significant increase in GLP-1, NOS activity, e-NOS expression, TRPV-1 level and TRPC-1 expression, compared to controls. Results of CGRP are in line with TRPV-1, as a downstream regulatory effect. NOS system and transient receptor potential (TRP) channels can contribute to DPP-4 inhibition-mediated cardioprotection against I/R injury using Sitagliptin.

Keywords: Calcitonin gene related peptide; DPP-4 inhibitors; NOS activity; dipeptidyl-peptidase-4; endothelial nitric oxide synthase; infarct size; ischemia-reperfusion injury; transient receptor potential channels.

Figure 1

Schematic diagram showing the activation of the non-selective cation channels, transient receptor potential canonical (TRPC-1), increasing calcium (Ca²⁺) influx into the vascular smooth muscle cells (VSMC) and endothelial cells. Displayed data suggests that calcium influx induces the upregulation of endothelial nitric oxide synthase (e-NOS) and nitric oxide (NO) release, promoting endothelial protective effects.

Figure 2

Effect of different doses of Sitagliptin (Sitg) on infarct size (expressed in %). Results are shown as (Mean ± Standard error of mean (SEM)); (n = 8–16 animals/group). Statistical significance: *** p < 0.001 relative to the Control group. Sitg (50 mg) exhibited a cardioprotective effect against ischemia-reperfusion (I/R) injury, while no significance was reported in other doses (25, 100, and 150 mg/kg/day).

Figure 3

Changes in Dipeptidyl peptidase-4 (DPP-4) enzyme activity (expressed in microunits/mL) and Glucagon-like peptide 1 (GLP-1; expressed in ng/mL) in the heart tissues of Sitagliptin (Sitg 50 mg) treated animal groups. Data are represented as (Mean ± SEM); (n = 4–10 animals/group). Statistical significance: * p < 0.05 compared to the Control group.

Figure 4

Effect of Sitagliptin treatment on TRPV-1 (expressed in ng/mL) and CGRP (ng/mg protein) ischemic cardiac tissue levels, compared to the Control animal group. A clear significant increase is observed in both proteins levels observed comparing the treated group to the Control (** p < 0.01). Data are illustrated as (Mean ± SEM); (n = 5–10 animals/group).

Figure 5

Changes in calcium content of cardiac tissues excised from the Sitagliptin (Sitg (50 mg); n = 7) treated animals and the Control ones (n = 4). The bar chart displays an increase in calcium concentration in Sitg (50 mg) group, and values presented are in terms of (Mean ± SEM).

Figure 6

Upregulation of TRPC-1 protein expression level (expressed in Intensity × mm²) in the heart tissues of Sitagliptin (Sitg (50 mg); n = 5) treated group vs. Control (n = 6). Data are in terms of (Mean ± SEM). Statistical significance: * p < 0.05.

Figure 7

Increase in endothelial nitric oxide synthase (e-NOS (n = 5–6)) protein expression in ischemic cardiac tissues from Sitagliptin (Sitg (50 mg)) treated group compared to Control (** p < 0.01). Values are expressed in (Intensity × mm²). Presented data are (Mean ± SEM).

Figure 8

Increase in nitric oxide synthase activity (cNOS (n = 8)) in ischemic cardiac tissues from Sitagliptin (Sitg (50 mg)) treated group compared to Control (* p < 0.05). Values are expressed in (pmol/min/mg protein). Presented data are (Mean ± SEM).

Figure 9

Loss of cardioprotective effect mediated by NOS and increase in infarct size with intraperitoneal injection of NOS-inhibitor (l-NAME), infarct size expressed in (%). Comparing Control (Saline, n = 12) and Sitagliptin (Sitg (50 mg), n = 10) groups shows a significant decrease in infarct size (*** p < 0.001), while this protective effect was abolished comparing the Sitg (50 mg) + l-NAME (Sitagliptin 50 mg + Nω-Nitro-l-arginine methyl ester hydrochloride (i.p), n = 11) group with the Sitg (50 mg, n = 10) treated group (# p < 0.05), which means that cardioprotective effect of Sitagliptin against infarction is mediated through NOS. A significant decrease († p < 0.05) in infarct size was also observed in Control (Saline) + l-NAME (Saline + Nω-Nitro-l-arginine methyl ester hydrochloride (i.p), n = 11) group, compared to Control (Saline). Data plotted as (Mean ± SEM).

Figure 10

Loss of cardioprotective effect mediated by TRPV-1 and increase in infarct size with intraperitoneal injection of TRPV-1-inhibitor (Capsazepine), Infarct size expressed in (%). Comparing the 2 groups, Control (Saline) + DMSO (Control (Saline) + Dimethyl sulfoxide (i.p), n = 7) and Sitg (50 mg) + DMSO (Sitagliptin 50 mg + Dimethyl sulfoxide (i.p), n = 6), shows a significant decrease in infarct size (* p < 0.05), while this protective effect was abolished comparing the Sitg (50 mg) + CAP (Sitagliptin 50 mg + Capsazepine (i.p), n = 8) group with the Sitg (50 mg) + DMSO treated group (# p < 0.05), which means that cardioprotective effect of Sitagliptin against infarction is mediated by TRPV-1. A significant difference (†† p < 0.01) was observed in Sitg (50 mg) + DMSO (Sitagliptin (50 mg) + Dimethyl sulfoxide (i.p), n = 6) group, compared to Saline + CAP (Saline + Capsazepine (i.p), n = 8). Data plotted as (Mean ± SEM).

Figure 11

Schematic diagram showing the signaling pathways activated by the DPP-4 inhibitor sitagliptin, directly or upon binding to GLP-1 receptors. The diagram represents the traditional signaling mechanisms involved in cardioprotection, including cAMP/PKA, PI3K, Akt/P-Akt, ErK1/2, and cGMP, mediated NOS upregulation and e-NOS production. It also clarifies the novelty of this study (Upregulation of TRP channels and CGRP mediated by sitagliptin and GLP-1). Activation of TRP channels is either through the direct effect of DPP-4 inhibitor (1), or through GLP-1R and GLP-1 activation (2). The arrow sign () indicates mechanism’s activation, and the T bar sign () indicates the inhibitory effect.

Figure 12

Diagram illustrating 4 different I/R experimental protocols. (a) Heart tissues subjected to 45 min ischemia and 120 min of reperfusion, after 2 weeks of oral animal treatment with Saline and different doses of Sitagliptin, for infarct size measurement. (b) Hearts subjected to 45 min ischemia and 10 min brief reperfusion, after which the animals received a 2 weeks’ oral administration of Saline and Sitg (50 mg), for further biochemical measurements. Infarct size measurement from heart tissues exposed to prolonged ischemia-reperfusion injury, after which the animals received a 2 weeks’ co-treatment of Saline, Sitg (50 mg) and intraperitoneal injection of NOS-inhibitor (l-NAME) (c). In the 4th experimental protocol (d), the inhibitory effect of TRPV-1 against ischemia-reperfusion (IR) injury was assessed by heart infarct size measurement at the end of a prolonged reperfusion-injury, and after co-treating the animals intraperitoneally with Capsazepine (TRPV-1 inhibitor).

Figure 13

Schematic diagram clarifying the ischemia-reperfusion (I/R) protocol used with brief reperfusion time (10 min) for the purpose of biochemical measurements in experiment 2.

Figure 14

Representative photographs of transversely sectioned Evans-blue perfused, triphenyltetrazolium chloride (TTC)—stained heart tissues, outlining the area at risk (AAR; sum of white and red area); blue, healthy viable tissue; pale white, infarcted tissue. Myocardial infarct area (IS; white) was measured post-myocardial ischemia-reperfusion and TTC staining, in different treated groups and Controls.

Figure 15

Expression of e-NOS and TRPC-1 proteins in ischemic heart tissues treated with different doses of sitagliptin (Sitg (25, 50, 100, & 150 mg)), compared to the control (Saline) group. The blots show that both proteins are significantly expressed in sitagliptin (50 mg) treated groups.