Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy

Abstract

Background: Relevant preclinical models are necessary for further mechanistic and translational studies of c-kit+ cardiac stem cells (CSCs). The present study was undertaken to determine whether intracoronary CSCs are beneficial in a porcine model of chronic ischemic cardiomyopathy.

Methods and results: Pigs underwent a 90-minute coronary occlusion followed by reperfusion. Three months later, autologous CSCs (n=11) or vehicle (n=10) were infused into the infarct-related artery. At this time, all indices of left ventricular (LV) function were similar in control and CSC-treated pigs, indicating that the damage inflicted by the infarct in the 2 groups was similar; 1 month later, however, CSC-treated pigs exhibited significantly greater LV ejection fraction (echocardiography) (51.7±2.0% versus 42.9±2.3%, P<0.01), systolic thickening fraction in the infarcted LV wall, and maximum LV dP/dt, as well as lower LV end-diastolic pressure. Confocal microscopy showed clusters of small α-sarcomeric actin-positive cells expressing Ki67 in the scar of treated pigs, consistent with cardiac regeneration. The origin of these cycling myocytes from the injected cells was confirmed in 4 pigs that received enhanced green fluorescent protein -labeled CSCs, which were positive for the cardiac markers troponin I, troponin T, myosin heavy chain, and connexin-43. Some engrafted CSCs also formed vascular structures and expressed α-smooth muscle actin.

Conclusions: Intracoronary infusion of autologous CSCs improves regional and global LV function and promotes cardiac and vascular regeneration in pigs with old myocardial infarction (scar). The results mimic those recently reported in humans (Stem Cell Infusion in Patients with Ischemic CardiOmyopathy [SCIPIO] trial) and establish this porcine model of ischemic cardiomyopathy as a useful and clinically relevant model for studying CSCs.

Keywords: angiogenesis inducers; heart failure; muscle development; myocardial infarction; stem cells.

Figure 1

Experimental protocol. Three groups of pigs were studied (groups I–III). Four days after a baseline echocardiogram, pigs underwent a 90-min coronary occlusion followed by reperfusion or sham surgery. At three to four months after MI (97 ± 12 d in vehicle-treated and 96 ± 6 d in CSC-treated groups), pigs received intracoronary infusion of vehicle (group II), or autologous CSCs into the infarct-related artery using a balloon catheter (group III). Group I served as noninfarcted controls. Echocardiographic and hemodynamic assessment of cardiac function was performed before treatment and at the time of sacrifice. At 31 days after vehicle/CSC therapy, pigs were euthanized for morphometric and histological studies.

Figure 2

Plasma troponin T (A) and CKMB (B) levels at baseline (−1), immediately after i.c. cell/vehicle delivery (0), and 6, 12, 24 and 48 h after delivery. Data are means ± SEM.

Figure 3

Assessment of LV function before and after vehicle or CSC therapy: hemodynamic variables [LV end-diastolic pressure (A) and LV dP/dt_max (B)], representative M-mode echocardiographic images at 30 d after treatment in pigs that were given vehicle (C) and CSCs (D), and quantitative echocardiographic analysis of LV function (IW thickening fraction and infarcted wall thickening fraction) (E and F). Compared with the vehicle-treated pig, the CSC-treated animal exhibited a smaller LV cavity, a thicker infarcted wall, and improved motion of the infarcted wall (C and D), Quantitative echocardiographic analysis shows improvement in LV functional parameters at 30 d after CSC treatment (E and F). Data are means ± SEM. *, P<0.05 versus noninfarcted controls and §, P<0.05 vs. vehicle-treated pigs (unpaired t test).

Figure 3

Assessment of LV function before and after vehicle or CSC therapy: hemodynamic variables [LV end-diastolic pressure (A) and LV dP/dt_max (B)], representative M-mode echocardiographic images at 30 d after treatment in pigs that were given vehicle (C) and CSCs (D), and quantitative echocardiographic analysis of LV function (IW thickening fraction and infarcted wall thickening fraction) (E and F). Compared with the vehicle-treated pig, the CSC-treated animal exhibited a smaller LV cavity, a thicker infarcted wall, and improved motion of the infarcted wall (C and D), Quantitative echocardiographic analysis shows improvement in LV functional parameters at 30 d after CSC treatment (E and F). Data are means ± SEM. *, P<0.05 versus noninfarcted controls and §, P<0.05 vs. vehicle-treated pigs (unpaired t test).

Figure 3

Assessment of LV function before and after vehicle or CSC therapy: hemodynamic variables [LV end-diastolic pressure (A) and LV dP/dt_max (B)], representative M-mode echocardiographic images at 30 d after treatment in pigs that were given vehicle (C) and CSCs (D), and quantitative echocardiographic analysis of LV function (IW thickening fraction and infarcted wall thickening fraction) (E and F). Compared with the vehicle-treated pig, the CSC-treated animal exhibited a smaller LV cavity, a thicker infarcted wall, and improved motion of the infarcted wall (C and D), Quantitative echocardiographic analysis shows improvement in LV functional parameters at 30 d after CSC treatment (E and F). Data are means ± SEM. *, P<0.05 versus noninfarcted controls and §, P<0.05 vs. vehicle-treated pigs (unpaired t test).

Figure 4

Impact of CSC therapy on LV anatomy. Representative transverse sections of hearts from a vehicle-treated (A) and a CSC-treated (B) pig after 30 d of follow-up. Scar tissue (whitish patch) is highlighted in both the sections. Note that the scar area is smaller and the infarct wall thicker in the CSC-treated heart.

Figure 5

Representative slides of transmural blocks from the core infarct zone from a vehicle-treated (A) and a CSC-treated (B) pig after 30 d of follow-up (hematoxylin and eosin stain). The lower panel (C) shows a higher magnification of the section from a CSC-treated pig. A, Dense transmural fibrosis in a vehicle-treated pig with a homogeneous pattern of scar with less viable tissue; B and C, Mid wall fibrosis surrounded by thick viable myocyte bundles in a pig treated with CSCs.

Figure 6

Representative confocal microscopic image from a CSC-treated pig showing small cycling Ki67-positive myocytes in the infarcted region at 30 d after CSC infusion. Positivity for α-sarcomeric actin (red) identifies cardiomyocytes.

Figure 7

Intracoronary administration of CSCs promotes myocardial regeneration. Regenerated EGFP-positive myocytes in the infarcted region in a CSC-treated heart are labeled with α-sarcomeric actin (red) (A) and EGFP (green) (B). Panel (C) shows the combination of EGFP and α-sarcomeric actin (yellow-green).

Figure 8

Expression of cardiac-specific TnI, TnT, MHC, Cnx43, and vascular smooth muscle protein (α-smooth muscle actin) in EGFP-positive cells. Representative confocal microscopic images showing colocalization of EGFP with TnI (A), TnT (B), MHC (C), Cnx43 (D), and α-smooth muscle actin (E) in the infarct zone of a CSC-treated pig. Positivity for α-sarcomeric actin (red) identifies cardiomyocytes. In Figure 8E, the structures illustrated are most likely represent arterioles.

Figure 8

Expression of cardiac-specific TnI, TnT, MHC, Cnx43, and vascular smooth muscle protein (α-smooth muscle actin) in EGFP-positive cells. Representative confocal microscopic images showing colocalization of EGFP with TnI (A), TnT (B), MHC (C), Cnx43 (D), and α-smooth muscle actin (E) in the infarct zone of a CSC-treated pig. Positivity for α-sarcomeric actin (red) identifies cardiomyocytes. In Figure 8E, the structures illustrated are most likely represent arterioles.

Figure 8

Expression of cardiac-specific TnI, TnT, MHC, Cnx43, and vascular smooth muscle protein (α-smooth muscle actin) in EGFP-positive cells. Representative confocal microscopic images showing colocalization of EGFP with TnI (A), TnT (B), MHC (C), Cnx43 (D), and α-smooth muscle actin (E) in the infarct zone of a CSC-treated pig. Positivity for α-sarcomeric actin (red) identifies cardiomyocytes. In Figure 8E, the structures illustrated are most likely represent arterioles.