Galanin Coordinates Macrophage-Associated Fibro-Inflammatory Response and Mitochondrial Integrity in Myocardial Infarction Reperfusion Injury

Abstract

Myocardial infarction activates an intense fibro-inflammatory reaction that is essential for cardiac remodeling and heart failure (HF). Bioactive peptide galanin plays a critical role in regulating cardiovascular homeostasis; however, its specific functional relevance in post-infarction fibro-inflammatory reprogramming remains obscure. Here, we show that galanin coordinates the fibro-inflammatory trajectory and mitochondrial integrity in post-infarction reperfusion injury. Aberrant deposition of collagen was associated with a marked increase in CD68-positive macrophage infiltration in cardiac tissue in mice subjected to myocardial ischemia/reperfusion (I/R) for 14 days compared to sham controls. Furthermore, we found that the myocardial expression level of a specific marker of M2 macrophages, CD206, was significantly down-regulated in I/R-challenged mice. In contrast, galanin treatment started during the reperfusion phase blunted the fibro-inflammatory responses and promoted the expression of CD206 in I/R-remodeled hearts. In addition, we found that the anti-apoptotic and anti-hypertrophic effects of galanin were associated with the preservation of mitochondrial integrity and promotion of mitochondrial biogenesis. These findings depict galanin as a key arbitrator of fibro-inflammatory responses to cardiac I/R injury and offer a promising therapeutic trajectory for the treatment of post-infarct cardiovascular complications.

Keywords: apoptosis; cardiac remodeling; fibrosis; inflammation; mitochondria.

Figure 1

Galanin blunts cardiac fibrosis in myocardial infarction reperfusion injury. (A) Representative images of cardiac sections stained with Sirius Red (top panel; collagen fibers: red color) and visualized by polarized light microscopy (bottom panel; collagen fibers: white color) from mice subjected to sham operation or 30 min of cardiac ischemia and 14 days of reperfusion (I/R). (B) Quantification of myocardial fibrosis in (A). (C,D) Myocardial mRNA levels of collagen 1 (Col 1) and collagen 3 (Col 3) in vehicle- or galanin-treated (G) mice after 14 days of I/R injury, n = 5. Data presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. sham, # p < 0.05 vs. I/R according to one-way ANOVA followed by Bonferroni’s post hoc test.

Figure 2

Galanin inhibits macrophage-associated inflammatory reactions in myocardial infarction reperfusion injury. (A) CD68 immunostaining (green fluorescence) was performed to detect inflammatory cell populations in cardiac sections from vehicle- or galanin-treated mice subjected to 30 min of cardiac ischemia and 14 days of reperfusion. Nuclei were counterstained with DAPI (blue). (B) Quantification of CD68-positive cells from (A), n = 5. (C) Myocardial CD68 mRNA levels in vehicle- or galanin-treated mice subjected to I/R or sham, n = 5. Data presented as the mean ± SEM. * p < 0.05, ** p < 0.01 according to one-way ANOVA followed by Bonferroni’s post hoc test.

Figure 3

Galanin promotes the anti-inflammatory trajectory in I/R-remodeled hearts. Mice were exposed to 30 min of cardiac ischemia and 14 days of reperfusion. (A,B) Representative images and quantification of CD206 (red) in vehicle- or galanin-treated mice subjected to I/R damage, n = 4. Nuclei were counterstained with DAPI (blue). (C,D) Myocardial mRNA levels of CD206 and TNFα in vehicle- or galanin-treated control sham and I/R-challenged mice, n = 5. Data presented as the mean ± SEM; ns: not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, according to one-way ANOVA followed by Bonferroni’s post hoc test.

Figure 4

Galanin reduces cardiac hypertrophy in myocardial infarction reperfusion injury. (A) Representative photomicrographs of cardiac tissue sections with H&E staining and (B) quantitative analysis of the cross-sectional area. (C) Myocardial mRNA levels of β-MHC in vehicle- or galanin-treated control sham and I/R-challenged mice, n = 5. Data presented as the mean ± SEM; ns: not significant. ** p < 0.01, **** p < 0.001 according to one-way ANOVA followed by Bonferroni’s post hoc test.

Figure 5

Galanin prevents myocardial apoptosis in I/R-challenged hearts. (A) Representative photomicrographs of TUNEL staining of cardiac sections from vehicle- or galanin-treated mice exposed to 30 min of cardiac ischemia and 14 days of reperfusion. The green cells indicate the TUNEL-positive apoptotic cells. (B) Quantification of TUNEL-positive cells from (A), n = 5. (C) Myocardial mRNA levels (C) of caspase 3 in vehicle- or galanin-treated mice exposed to I/R injury. Data presented as the mean ± SEM; ns: not significant. * p < 0.05, **** p < 0.0001 according to one-way ANOVA followed by Bonferroni’s post hoc test.

Figure 6

Galanin preserves mitochondrial integrity in myocardial infarction reperfusion injury. (A) Electron micrographs of hearts from vehicle- or galanin (G)-treated mice subjected to cardiac I/R injury. Cardiac tissues were harvested for examination of the myocardial ultrastructure by transmission electron microscopy. Magnification: 14,000×. (M)—mitochondria; (C)—cristae; (S)—sarcomere; (*)—damaged mitochondria; (∧)—disrupted myofibrils. (B) Myocardial TFAM mRNA levels in vehicle- or galanin-treated mice subjected to I/R or sham. n = 5. Data are expressed as the mean ± SEM. * p < 0.05 according to one-way ANOVA followed by Bonferroni’s post hoc test.

Figure 7

Galanin promotes mitochondrial fission in I/R-remodeled hearts. (A) Representative Western blot of DRP1 protein expression and (B) quantification of (A). Myocardial mRNA levels of (C) DRP1, (D) MFN1, and (E) MFN2 in vehicle- or galanin-treated control sham and I/R-challenged mice, n = 5. Mice were exposed to 30 min of cardiac ischemia and 14 days of reperfusion. Data presented as the mean ± SEM; ns: not significant. ** p < 0.01 according to one-way ANOVA followed by Bonferroni’s post hoc test.