Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice

Abstract

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Figure 1.

DPP-4 inhibition abrogates Ang II–induced cardiac diastolic dysfunction. Indices of cardiac diastolic function, specifically ratio of (A) early-to-late septal annulus motion in diastole (E’/A’) and (B) IVRT. Values are mean ± standard error; n = 6 to 7. *P < 0.05 vs Con/Con+Saxa; **P < 0.05 vs all other groups; ^‡P = 0.08 vs Ang II.

Figure 2.

Ang II–induced cardiac periarterial fibrosis and fibroblast activation, but not hypertrophy, is resolved by DPP-4 inhibition. Cardiac hypertrophy, assessed by (A) heart weight–to–tibia length (HW:TL) and (B) cardiomyocyte cross-sectional area (CSA), and fibrosis, assessed by (C) periarterial PR staining, (D) collagen I (Col I) mRNA expression, and (E) periostin expression by immunoblot as an index of fibroblast activation. Representative wheat germ agglutinin (WGA) and PR images for CSA and periarterial fibrosis, respectively, in the middle panel. Scale bar is 100 µm. Periarterial fibrosis quantified as the ratio of adventitial PR staining to lumen area (adventitia:lumen). Stained arterioles indicated by arrows. Representative immunoblot with matching GAPDH control in lower panel. Values are mean ± standard error; n = 4 to 10. *P < 0.05 vs Con/Con+Saxa; **P < 0.05 vs all other groups; ^§P = 0.06 vs Con+Saxa; ^‡P = 0.09 vs Ang II.

Figure 3.

Activation of cardiac inflammatory and fibrotic signaling by Ang II is ameliorated by DPP-4 inhibition. (A) Cardiac gene expression of TLR4 and (B) immunoblot assessment of cardiac AP-1 signaling by Ser⁶³ p-c-Jun to total c-Jun, (C) NF-κB signaling by Ser⁵³⁶ phospho-p65 (p-p65) to total p65, (D) expression of TRAF3IP2, (E) mature IL-18, and (F) IL-17A. Representative immunoblots with matching GAPDH controls in left panel; GAPDH immunoblot for IL-17A is identical to that in Fig. 2 for periostin, as these targets were examined on the same membrane. Values are mean ± standard error; n = 4 to 5. **P < 0.05 vs all other groups.

Figure 4.

Impact of Ang II and DPP-4 inhibition on cardiac DPP-4 substrates. Immunoblot analysis of cardiac CXCL12 and CXCL10 expression in cardiac lysates. Representative immunoblots with matching GAPDH controls in bottom panel; GAPDH gel for CXCL12 is identical to that in Fig. 3 for IL-18, as these targets were examined on the same membrane. Values are mean ± standard error; n = 4 to 5. *P < 0.05 vs Con/Con+Saxa; ^‡P = 0.065 vs Ang II.

Figure 5.

DPP-4 inhibition differentially modulates Ang II–induced shifts in cardiac myeloid populations. (A) Flow cytometric analysis of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and (B) gene expression of myeloid marker mRNAs assessed in cardiac tissue. Values are mean ± standard error; n = 4 to 5. *P < 0.05 vs Con/Con+Saxa; **P < 0.05 vs all other groups; ^†P < 0.05 vs Con; ^‡P = 0.06 vs Con; ^§P = 0.052 vs Con; ^¥P = 0.065 vs Con.

Figure 6.

DPP-4 inhibition differentially modulates Ang II–induced shifts in cardiac lymphocyte subpopulations. Flow cytometric analysis of (A) cardiac T cells, (B) activated T cells, and (C) gene expression of T cell marker mRNAs in cardiac tissue. Values are mean ± standard error; n = 4 to 6. *P < 0.05 vs Con/Con+Saxa; **P < 0.05 vs all other groups; ^†P < 0.05 vs Con.