Cardioprotective effects of lixisenatide in rat myocardial ischemia-reperfusion injury studies

Abstract

Background: Lixisenatide is a glucagon-like peptide-1 analog which stimulates insulin secretion and inhibits glucagon secretion and gastric emptying. We investigated cardioprotective effects of lixisenatide in rodent models reflecting the clinical situation.

Methods: The acute cardiac effects of lixisenatide were investigated in isolated rat hearts subjected to brief ischemia and reperfusion. Effects of chronic treatment with lixisenatide on cardiac function were assessed in a modified rat heart failure model after only transient coronary occlusion followed by long-term reperfusion. Freshly isolated cardiomyocytes were used to investigate cell-type specific mechanisms of lixisenatide action.

Results: In the acute setting of ischemia-reperfusion, lixisenatide reduced the infarct-size/area at risk by 36% ratio without changes on coronary flow, left-ventricular pressure and heart rate. Treatment with lixisenatide for 10 weeks, starting after cardiac ischemia and reperfusion, improved left ventricular end-diastolic pressure and relaxation time and prevented lung congestion in comparison to placebo. No anti-fibrotic effect was observed. Gene expression analysis revealed a change in remodeling genes comparable to the ACE inhibitor ramipril. In isolated cardiomyocytes lixisenatide reduced apoptosis and increased fractional shortening. Glucagon-like peptide-1 receptor (GLP1R) mRNA expression could not be detected in rat heart samples or isolated cardiomyocytes. Surprisingly, cardiomyocytes isolated from GLP-1 receptor knockout mice still responded to lixisenatide.

Conclusions: In rodent models, lixisenatide reduced in an acute setting infarct-size and improved cardiac function when administered long-term after ischemia-reperfusion injury. GLP-1 receptor independent mechanisms contribute to the described cardioprotective effect of lixisenatide. Based in part on these preclinical findings patients with cardiac dysfunction are currently being recruited for a randomized, double-blind, placebo-controlled, multicenter study with lixisenatide.

Trial registration: (ELIXA, ClinicalTrials.gov Identifier: NCT01147250).

Figures

Figure 1

Effect of lixisenatide on infarct size and cardiac function in isolated rat hearts subjected to ischemia (45 min) and reperfusion (120 min). (A) infarct to risk area ratio, (B) risk area, and (C, D) selected coronary flow and rate pressure product immediately after onset of reperfusion (5 min) and at the end of reperfusion (120 min). *) denotes p < 0.05 versus placebo.

Figure 2

Effect of long-term treatment with lixisenatide or ramipril after transient ischemia-reperfusion injury: Heart weight. (A), lung weight (B), left-ventricular end diastolic pressure (LVedP, C) and relaxation time (tau Weiss, D). *) denotes p < 0.05 versus sham and #) p < 0.05 versus placebo.

Figure 3

Effect of long-term treatment with either lixisenatide or ramipril after transient ischemia-reperfusion injury on heart morphology. (A) Picrosirius red staining at the papillary muscle level of representative animals, Bar = 5 mm; (B) quantification of left ventricular fibrotic to total area; (C) thickness of the left ventricular, septal and right ventricular wall; and (D) gene expression analysis by realtime PCR of selected fibrosis genes. *) denotes p < 0.05 versus sham and #) p < 0.05 versus placebo.

Figure 4

Gene expression patterns 10 weeks after ischemia reperfusion injury performed on all samples and comparing non-infarct area versus infarct area. (A) Determination of 92 representative genes followed by 2 dimensional hierarchical clustering and visualization as heatmap. Each sample forms a column and each gene forms a row with the heatmap intensities standardized within each gene. Overall 5 independent samples were measured for each group and two different tissue areas (infarct and non-infarct ventricular tissue) resulting in a final number of 40 samples. (B) Principle component analysis (PCA) of the same data reflecting the total expression changes into two uncorrelated principal components.

Figure 5

Effect of lixisenatide treatment on apoptosis and contractility of isolated cardiomyocytes. (A) Caspase-3/7 activity is reduced in cardiomyocytes incubated with either insulin or lixisenatide (each at 100 nM). (B, C) Lixisenatide (Lixi) but not GLP-1 (6–36)-amide increases fractional shortening. Isoprenaline (Iso, 1nM) served as positive control. (D). Cardiomyocytes isolated from GLP-1 receptor knockout mice respond to lixisenatide. Mean values and SEM are given. In the fractional shortening experiments, n > 18 single cardiomyocytes were measured for each group.

Figure 6

Expression of 5 genes involved in GLP-1 incretin biology in different rat tissues assessed by quantitative Taqman PCR. Two biologically different samples were analyzed and averaged (shown are mean values and SD). The geometric mean of three housekeeping genes (GAPDH, Actb and B2M) was used to normalize expression of specific genes using the formula 2^-Δc(t). For detection of GLP1R two different specific Taqman primer assays were used spanning different part of the receptor mRNA. GLP1R (P1) denotes Rn00562406_m1 and detect exon 3–4. GLP1R (P2) is the label for Rn01640381_m1 which detects a sequence spanning exon 8–9. The term n.a. indicates that no specific amplification could be achieved at the maximum cycle number (n=40).