Prenatal nicotine increases matrix metalloproteinase 2 (MMP-2) expression in fetal guinea pig hearts

Abstract

This study tested the hypothesis that maternal nicotine ingestion increases matrix metalloproteinase (MMP) expression in fetal hearts, which is mediated by the generation of reactive oxygen species. Timed pregnant guinea pigs were administered either water alone, nicotine (200 μg/mL), N-acetylcysteine (NAC), or nicotine plus NAC in their drinking water for 10 days at 52-day gestation (term = 65 days). Near-term (62 days), anesthetized fetuses were extracted, hearts were excised, and left cardiac ventricles snap frozen for analysis of MMP-2/-9/-13 protein and activity levels. Interstitial collagens were identified by Picrosirius red stain to assess changes in the extracellular matrix. Prenatal nicotine increased active MMP-2 forms and interstitial collagen but had no effect on either pro- or active MMP-9 or MMP-13 forms. In the presence of nicotine, NAC decreased active MMP-2 protein levels and reversed the nicotine-induced increase in collagen staining. We conclude that prenatal nicotine alters MMP-2 expression in fetal hearts that may be mediated by reactive oxygen species generation.

Figure 1.

Western analysis (left) and gelatin zymography (right) of matrix metalloproteinase 2 (MMP-2) of control (untreated, N = 4) and nicotine-treated (N = 4) fetal heart ventricles. Matrix metalloproteinase 2 bands were identified in Western immunoblots at ∼72 kDa and normalized to bands corresponding to α-actin as a loading control. Gel zymography identified both pro (72 kDa) and active (62 kDa) bands for MMP-2. Band densities were quantified by densitometry and expressed as arbitrary values. Values are mean ± standard error of mean (SEM). *Indicates P < .05 versus control.

Figure 2.

Western analysis of matrix metalloproteinase 9 ([MMP-9], left graph) and MMP-13 (right graph) of control (untreated) and nicotine-treated (N = 4) fetal heart ventricles. Separate gels were run for both MMP-9 and MMP-13. Bands corresponding to both pro (92 kDa for MMP-9 and 57 kDa for MMP-13) and active (86 kDa for MMP-9 and 45 kDa for MMP-13) forms were identified. Density values of each band were analyzed relative to either α-actin (for MMP-9) or glyceraldehyde 3-phosphate dehydrogenase ([GAPDH]; for MMP-13). Values are mean ± standard error of mean (SEM).

Figure 3.

The effect of N-acetylcysteine (NAC) on fetal cardiac matrix metalloproteinase 2 (MMP-2) expression of nicotine-treated animals by Western analysis (top left) and gelatin zymography (right). Negative controls (no nicotine) of NAC alone on MMP-2 expression in fetal hearts is shown in the bottom left graph. Matrix metalloproteinase 2 bands were identified in Western immunoblots at ∼72 kDa and normalized to bands corresponding to α-actin as a loading control. Gel zymography identified both pro (72 kDa) and active (62 kDa) bands for MMP-2. Band densities for gel zymography were quantified by densitometry and expressed as arbitrary values. Values are mean ± standard error of mean (SEM). *Indicates P < .05 versus control.

Figure 4.

Immunostaining of interstitial collagen of fetal heart ventricles by Picrosirius red stain. Interstitial collagen of hearts of age-matched fetal guinea pigs (N = 3 for each group) was identified by Picrosirius stain in control (top left), nicotine (top right), and in the presence (+) and absence (−) of N-acetylcysteine (NAC). Interstitial collagen fibers are identified as red stain (black arrows) in cardiac muscle. Magnification = ×200. Bottom graph illustrates the mean density values per area (×1000) of Picrosirius red stain of fetal guinea pig hearts quantified by NIH Image J software. *Indicates a significance difference (P < .05) from C (control), NAC, and NIC (nicotine) +NAC; ⁺ indicates a significance difference from NAC alone and untreated control (C).