Extracorporeal shock wave therapy reverses ischemia-related left ventricular dysfunction and remodeling: molecular-cellular and functional assessment

Abstract

An optimal treatment for patients with diffuse obstructive arterial disease unsuitable for catheter-based or surgical intervention is still pending. This study tested the hypothesis that extracorporeal shock wave (ECSW) therapy may be a therapeutic alternative under such clinical situation. Myocardial ischemia was induced in male mini-pigs through applying an ameroid constrictor over mid-left anterior descending artery (LAD). Twelve mini-pigs were equally randomized into group 1 (Constrictor over LAD only) and group 2 (Constrictor over LAD plus ECSW [800 impulses at 0.09 mJ/mm(2)] once 3 months after the procedure). Results showed that the parameters measured by echocardiography did not differ between two groups on days 0 and 90. However, echocardiography and left ventricular (LV) angiography showed higher LV ejection fraction and lower LV end-systolic dimension and volume in group 2 on day 180 (p<0.035). Besides, mRNA and protein expressions of CXCR4 and SDF-1α were increased in group 2 (p<0.04). Immunofluorescence staining also showed higher number of vWF-, CD31-, SDF-1α-, and CXCR4-positive cells in group 2 (all p<0.04). Moreover, immunohistochemical staining showed notably higher vessel density but lower mean fibrosis area, number of CD40-positive cells and apoptotic nuclei in group 2 (all p<0.045). Mitochondrial protein expression of oxidative stress was lower, whereas cytochrome-C was higher in group 2 (all p<0.03). Furthermore, mRNA expressions of MMP-9, Bax and caspase-3 were lower, whereas Bcl-2, eNOS, VEGF and PGC-1α were higher in group 2 (all p<0.01). In conclusion, ECSW therapy effectively reversed ischemia-elicited LV dysfunction and remodeling through enhancing angiogenesis and attenuating inflammation and oxidative stress.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Detailed protocol and procedure.

Schematic illustration of the detailed protocol on preparative procedure of extracorporeal shock wave (ECSW) therapy. LAD = left anterior descending artery.

Figure 2. Expression of oxidative stress- and angiogenesis-related proteins in LV myocardium.

A) Protein expression of oxidative stress. †p<0.001 between the indicated groups. B) Protein expression of cytochrome C in mitochondrial level. †p<0.04 between the indicated groups. C) Protein expression of cytochrome C in cytosolic level. †p<0.04 between the indicated groups. D) Protein expression of CXCR4. †p<0.03 between the indicated groups. E) Protein expression of stromal cell-derived factor (SDF)-1α. †p<0.05 between the indicated groups. F) Protein expression of VEGF. †p<0.03 between the indicated groups. G) Protein expression of connexin43 (Cx43). †p<0.005 between the indicated groups. Symbols (*, †, ‡) indicate significance (at 0.05 level) (by one-way ANOVA and Bonferroni multiple comparison post hoc test, n = 6 in each group).

Figure 3. Immunohistochemical staining of fibrosis and apoptotic nuclei in LV myocardium.

Upper Panel: TUNEL assay (400x) showing notably higher number of apoptotic nuclei (red arrows) after ischemic insult (B) compared with normal controls (A) and ischemia following shock wave treatment (C). †p<0.001 between the indicated groups (D). Scale bars in right lower corner represent 20 µm. n = 6 in each group. Lower Panel: Masson's Trichrome staining (100×) showing remarkably higher fibrotic area (blue) following ischemia (F) than in normal controls (E) and ischemia with shock wave treatment (G). †p<0.0001 between the indicated groups (H). Scale bars in right lower corner represent 100 µm. n = 6 in each group.

Figure 4. Immunofluorescent staining of vWF and CD31 in LV myocardium.

Upper Panel: Immunofluorescence staining (400x) showing significantly lower number of vWF-positive cells (yellow arrows) after ischemia (B) than in normal controls (A) and ischemia with shock wave treatment (C). †p<0.001 between the indicated groups (D). Scale bars in right lower corner represent 20 µm. Lower Panel: Immunofluorescence staining (400x) showing notably lower number of CD31-positive cells (yellow arrows) after ischemia (F) than in normal controls (E) and ischemia with shock wave treatment (G). †p<0.01 between the indicated groups (H). Scale bars in right lower corner represent 20 µm.

Figure 5. Immunofluorescent staining of CXCR4 and SDF-1α in LV myocardium.

Upper Panel: Immunofluorescence staining (400x) showing significantly lower number of CXCR4-positive cells (yellow arrows) in normal controls (A) than in ischemic animals without (B) and with (C) shock wave treatment. *p<0.01 between the indicated groups (D). Scale bars in right lower corner represent 20 µm. Lower Panel: Immunofluorescence staining (400x) showing significantly lower number of SDF-1α-positive cells (yellow arrows) in normal controls (E) compared with animals after ischemia without (F) and with (G) shock wave treatment. *p<0.01 between the indicated groups (H). Scale bars in right lower corner represent 20 µm.

Figure 6. Immunohistochemical staining of α-SMA and CD40 nuclei in LV myocardium.

Upper panel: α-SMA immunohistochemical staining (200x) showing notably lower number of small vessel (red arrows) after ischemia (B) than in normal controls (A) and ischemia with shock wave treatment (C). †p<0.002 between the indicated groups (D). Scale bars in right lower corner represent 50 µm. Lower Panel: IHC staining (400x) showing the CD40-positively stained cells were remarkably higher after ischemia (F) than in normal controls (E) and ischemia with shock wave treatment (G). †p<0.001 between the indicated group (H). Scale bars in right lower corner represent 20 µm.

Figure 7. Profiles of mRNA expression in LV myocardium.

1) Notably higher mRNA expressions of vWF (A), SDF-1α (B), CXCR4 (C) and interleukin (IL)-8/Gro (D) in ischemic animals with shock wave treatment compared with other groups. ‡p<0.01 between indicated groups. 2) Remarkably lower mRNA expressions of eNOS (E) and IL-10 (G) after ischemia compared with other groups. †p<0.03 between indicated groups. 3) Significantly higher mRNA expressions of matrix metalloproteinase (MMP)-9 (F), Bax (H), and caspase 3 (I) after ischemia than in other groups. †p<0.04 between indicated groups. Notably higher mRNA expressions of Bcl-2 (J) and PGC-1α (K) in animals after ischemic insult with shock wave treatment compared to those without. †p<0.02 between indicated groups.

Figure 8. The proposed mechanisms.

Proposed mechanisms of extra-corporeal shock wave therapy on improving left ventricular (LV) function and attenuating LV remodeling in a porcine model of ischemic heart disease.