Folic acid mitigated cardiac dysfunction by normalizing the levels of tissue inhibitor of metalloproteinase and homocysteine-metabolizing enzymes postmyocardial infarction in mice

Abstract

Myocardial infarction (MI) results in significant metabolic derangement, causing accumulation of metabolic by product, such as homocysteine (Hcy). Hcy is a nonprotein amino acid generated during nucleic acid methylation and demethylation of methionine. Folic acid (FA) decreases Hcy levels by remethylating the Hcy to methionine, by 5-methylene tetrahydrofolate reductase (5-MTHFR). Although clinical trials were inconclusive regarding the role of Hcy in MI, in animal models, the levels of 5-MTHFR were decreased, and FA mitigated the MI injury. We hypothesized that FA mitigated MI-induced injury, in part, by mitigating cardiac remodeling during chronic heart failure. Thus, MI was induced in 12-wk-old male C57BL/J mice by ligating the left anterior descending artery, and FA (0.03 g/l in drinking water) was administered for 4 wk after the surgery. Cardiac function was assessed by echocardiography and by a Millar pressure-volume catheter. The levels of Hcy-metabolizing enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 5-MTHFR, were estimated by Western blot analyses. The results suggest that FA administered post-MI significantly improved cardiac ejection fraction and induced tissue inhibitor of metalloproteinase, CBS, CSE, and 5-MTHFR. We showed that FA supplementation resulted in significant improvement of myocardial function after MI. The study eluted the importance of homocysteine (Hcy) metabolism and FA supplementation in cardiovascular disease.

Fig. 1.

Changes in left ventricle (LV), diastolic and systolic diameters (LVDd and LVDs, respectively) in sham-operated (sham), myocardial infarction-induced (MI), sham-operated and treated with folic acid (sham + FA), and myocardial infarction-induced and treated with folic acid (MI + FA) mice. The red arrow represents diameter in diastole; the white arrow represents diameter in systole. A: examples of M-mode echocardiograms obtained with 2-dimensional echocardiography from a short-axis midventrical view of hearts of the experimental animals. B: bar graphs of changes of LV diameters during diastole (LVDd) and systole (LVDs). Notice increased left ventricular cavity dimensions (LVDd and LVDs) in mice with MI. P < 0.05 vs. sham (*) and vs. MI (#); n = 9 animals for all groups.

Fig. 2.

Percent changes in ejection fraction (EF) in sham, MI, sham + FA, and MI + FA animals. A: examples of pressure-volume loops in experimental animals recorded after 4 wk of surgery. B: bar graphs of changes in EF, calculated from ventricles' volume {[(EDV − ESV)/EDV] × 100%, where EDV is end-diastolic volume and ESV is end-systolic volume}. P < 0.05 vs. sham (*) and vs. MI (#); n = 3 for all groups.

Fig. 3.

Myocyte contractility in sham, MI, sham + FA, and MI + FA animals. A: examples of cell shortening traces. B: changes in percent peak shortening presented as changes in baseline percent peak (bl% peak) and in peak height (Peak H). C: rates of contraction (+dL/dt) and relaxation (−dL/dt) of cardiomyocytes. The values are the means of measurements of at least five myocytes from each animal in each experimental group. The mean value of contractility was calculated from at least five contractions of each cardiomyocyte analyzed. P < 0.05 vs. the sham group (*) and vs. MI (#); n = 5 for all groups.

Fig. 4.

Heart wall anatomical changes in sham, MI, sham + FA, and MI + FA animals. A: examples of cross-sectional view of the whole hearts at the ventricle level. Note: right and left ventricles are distinctly visible. Magnified areas are the left ventricular walls of sham and sham + FA hearts (left) and MI and MI + FA hearts (right). Note: No visible necroses were found in the left ventricular wall of hearts from sham and sham + FA. B: collagen-associated (blue) intensity changes in hearts from experimental animals. P < 0.05 vs. the sham group (*) and vs. MI (#); n = 4 for all groups.

Fig. 5.

Expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of MMP (TIMP)-2, and TIMP-4 in myocytes from sham, MI, sham + FA, and MI + FA mice. A: examples of images indicating expression of MMP-2 (green) and TIMP-4 (red). The last column indicates colocalization of MMP-2 and TIMP-4. B: examples of images indicating expression of MMP-9 (red) and TIMP-2 (green). The last column indicates colocalization of MMP-9 and TIMP-2. Cellular nuclei in all experiments were stained with 4′,6-diamidino-2-phenylindole (DAPI). The micrographs were taken under the identical set of conditions for all groups. C: bar graph of changes in integrated optical density (IOD) in expression of MMP-2, MMP-9, TIMP-2, and TIMP-4 in myocytes. The micrographs were taken under the identical set of conditions for all groups. *P < 0.05 vs. sham, sham + FA, and MI + FA. D: Western blots and bar graphs of IOD changes in the levels of MMP-2, MMP-9, TIMP-2, and TIMP-4. P < 0.05 vs. sham, sham + FA, and MI + FA (*) vs. MI (#). Although we observed variations in the expression of various enzymes, i.e., MMPs and TIMPs, n = 6 in each group. E: Western blots and bar graphs of IOD changes in the levels of TIMP-1 and TIMP-3. P < 0.05 vs. sham, sham + FA, and MI + FA (*) and vs. MI (#); n = 6 for all groups.

Fig. 6.

Changes in expression of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and methylene tetrahydrofolate reductase (MTHFR) protein contents, by Western blot analysis, in hearts from sham, MI, sham + FA, and MI + FA mice. A: examples of Western blot images of the proteins studied and contents of β-actin in the respective samples. B: results of the Western blot analysis. Relative protein expression is reported as a ratio of IOD of each band to the IOD of the respective β-actin band. *P < 0.05 vs. sham, sham + FA, and MI + FA; n = 5 for all groups.

Fig. 7.

Barium-contrast X-ray images of the hearts from sham, MI, sham + FA, and MI + FA. Note: there is an increase in collateral vessels in the MI heart after folic acid (FA) treatment compared with MI.

Fig. 8.

Schematic presentation of possible mechanism involved in FA-induced cardiac function protection during MI. MI caused decrease in CBS, CSE, MTHFR, TIMP-2, and TIMP-4, but it increased expression of MMP-2 and MMP-9. Treatment with FA, which induced expression of CBS, 5-MTHFR, and CSE, mitigated the abnormal extracellular matrix remodeling in MI hearts. The activation of CBS and CSE generated hydrogen sulfide (H₂S), a most potent anti-oxidant and vasorelaxing agent that may counterbalance deteriorating effects caused by development of MI.