Effects of sildenafil and/or muscle derived stem cells on myocardial infarction

Abstract

Background: Previous studies have shown that long-term oral daily PDE 5 inhibitors (PDE5i) counteract fibrosis, cell loss, and the resulting dysfunction in tissues of various rat organs and that implantation of skeletal muscle-derived stem cells (MDSC) exerts some of these effects. PDE5i and stem cells in combination were found to be more effective in non-MI cardiac repair than each treatment separately. We have now investigated whether sildenafil at lower doses and MDSC, alone or in combination are effective to attenuate LV remodeling after MI in rats.

Methods: MI was induced in rats by ligature of the left anterior descending coronary artery. Treatment groups were: "Series A": 1) untreated; 2) oral sildenafil 3 mg/kg/day from day 1; and "Series B": intracardiac injection at day 7 of: 3) saline; 4) rat MDSC (106 cells); 5) as #4, with sildenafil as in #2. Before surgery, and at 1 and 4 weeks, the left ventricle ejection fraction (LVEF) was measured. LV sections were stained for collagen, myofibroblasts, apoptosis, cardiomyocytes, and iNOS, followed by quantitative image analysis. Western blots estimated angiogenesis and myofibroblast accumulation, as well as potential sildenafil tachyphylaxis by PDE 5 expression. Zymography estimated MMPs 2 and 9 in serum.

Results: As compared to untreated MI rats, sildenafil improved LVEF, reduced collagen, myofibroblasts, and circulating MMPs, and increased cardiac troponin T. MDSC replicated most of these effects and stimulated cardiac angiogenesis. Concurrent MDSC/sildenafil counteracted cardiomyocyte and endothelial cells loss, but did not improve LVEF or angiogenesis, and upregulated PDE 5.

Conclusions: Long-term oral sildenafil, or MDSC given separately, reduce the MI fibrotic scar and improve left ventricular function in this rat model. The failure of the treatment combination may be due to inducing overexpression of PDE5.

Figure 1

Experimental protocol. Myocardial infarction (MI) was induced in rats by left anterior descending coronary artery (LAD) ligation. Arrows indicate the time points for treatments with sildenafil alone, MDSC, and MDSC + sildenafil, performance of surgical procedure, and measurement of various parameters (listed under each arrow), and final sacrifice.

Figure 2

Long-term oral sildenafil improved left ventricular function and reduced infarction size after LAD occlusion. Sildenafil was given continuously for 4 weeks in the drinking water (3 mg/kg/day) (n = 8/group).  Top:  The LVEF was measured before MI (basal), and at 1 and 4 weeks. UT: untreated control; S: sildenafil.  Middle:  representative micrographs (4 X) for the histochemical detection of collagen by Picrosirius red in paraffin-embedded sections.  Bottom:  quantitative image analysis of infarction area. Statistical differences are stated for untreated versus basal, and sildenafil versus untreated; *p < 0.05; ***p < 0.005.

Figure 3

Long-term oral sildenafil reduced myofibroblast accumulation in the infarction area, but did not significantly counteract the cardiomyocyte loss in this region. (n = 8/group). Paraffin-embedded sections were used.  Top:  quantitative image analysis of myofibroblasts by immunohistochemistry for ASMA.  Middle:  representative micrographs for the immunohistochemical detection of troponin T.  Bottom:  quantitative image analysis of troponin T. UT: untreated control; S: sildenafil; ***p < 0.005.

Figure 4

Intracardiac implantation of MDSC improved the LVEF and reduced infarction size after LAD, but concurrent chronic sildenafil abrogated these effects. Intracardiac injection of saline or MDSC was conducted at 1 week (n = 7/group). Sildenafil was then given continuously in the drinking water until sacrifice at 4 weeks.  Top:  Ejection fraction before MI (basal), and at 1 and 4 weeks. NaCl: control injected with saline; MDSC: injection with MDSC; MDSC + S: MDSC with sildenafil.  Bottom:  quantitative image analysis of infarction area by Picrosirius red histochemistry. Statistical differences are stated for untreated versus basal, and MDSC, and MDSC + sildenafil versus untreated; *p < 0.05.

Figure 5

MDSC implanted in the MI area survived after 4 weeks and reduced the apoptotic index, in a process stimulated by sildenafil that also partially counteracted the cardiomyocyte loss. (n = 7/group).  Top:  representative picture of frozen sections from MI regions that received DAPI-labeled MDSC, visualized with blue (DAPI) and red (Texas red) fluorescence filters for implanted nuclei and troponin-T stained cardiomyocytes, respectively. Arrows: cardiomyocyte/MDSC nuclei overlapping.  Middle and lower panels:  quantitative image analysis by Troponin T and TUNEL (apoptosis) immuno-histochemistry in paraffin-embedded sections, respectively. Abbreviations as in Figure 4. Statistical differences are stated for untreated versus basal, and MDSC, and MDSC + sildenafil versus untreated; *p < 0.05; **p < 0.01;***p < 0.005.

Figure 6

Implanted MDSC stimulated angiogenesis and reduced myofibroblasts, without affecting PDE 5 expression, while concurrent sildenafil protected the endothelium but increased myofibroblasts and upregulated PDE5. (n = 7/group). Protein extracts were obtained from region #3 adjacent to the infarction area and subjected to western blot analysis.  A:  representative immunoblots (n = 8), indicating band sizes.  B:  PDE 5 assayed in MDSC cultures in duplicate.  C:  Densitometric values corrected by GAPDH. Statistical differences are stated for untreated versus basal, and MDSC, and MDSC + sildenafil versus untreated; *p < 0.05; **p < 0.01;***p < 0.005.

Figure 7

Implanted MDSC reduced left ventricular remodelling, as indicated by the decrease of both pro MMP-2 and 9 in serum, and concurrent long-term oral sildenafil, did not modified these effects. MMPs were analyzed by zymography (n = 7/group). The Y axes indicate relative densitometric intensities. Abbreviations as in Figures 1 and 3; *p < 0.05.