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Comparative Study
. 2011 May 13;108(10):1226-37.
doi: 10.1161/CIRCRESAHA.110.239046. Epub 2011 Mar 31.

Repair of the injured adult heart involves new myocytes potentially derived from resident cardiac stem cells

David Angert  1 , Remus M Berretta, Hajime Kubo, Hongyu Zhang, Xiongwen Chen, Wei Wang, Barbara Ogorek, Mary Barbe, Steven R Houser
Affiliations
Comparative Study

Repair of the injured adult heart involves new myocytes potentially derived from resident cardiac stem cells

David Angert et al. Circ Res. .

Abstract

Rationale: The ability of the adult heart to generate new myocytes after injury is not established.

Objective: Our purpose was to determine whether the adult heart has the capacity to generate new myocytes after injury, and to gain insight into their source.

Methods and results: Cardiac injury was induced in the adult feline heart by infusing isoproterenol (ISO) for 10 days via minipumps, and then animals were allowed to recover for 7 or 28 days. Cardiac function was measured with echocardiography, and proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU; 7-day minipump infusion). BrdU was infused for 7 days before euthanasia at days 10, 17, and 38 or during injury and animals euthanized at day 38. ISO caused reduction in cardiac function with evidence of myocyte loss from necrosis. During this injury phase there was a significant increase in the number of proliferative cells in the atria and ventricle, but there was no increase in BrdU+ myocytes. cKit+ cardiac progenitor cells were BrdU labeled during injury. During the first 7 days of recovery there was a significant reduction in cellular proliferation (BrdU incorporation) but a significant increase in BrdU+ myocytes. There was modest improvement in cardiac structure and function during recovery. At day 38, overall cell proliferation was not different than control, but increased numbers of BrdU+ myocytes were found when BrdU was infused during injury.

Conclusions: These studies suggest that ISO injury activates cardiac progenitor cells that can differentiate into new myocytes during cardiac repair.

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Figures

Figure 1
Figure 1. Cardiac function in the ISO injured heart
Echocardiography (ECHO) was performed at Baseline, Day 7 (during injury), Day 10 (end of injury phase), Day 17 (early recovery) and Day 38 (late recovery). Representative ECHO data is shown in (A). B–D: Cardiac systolic and diastolic function was increasingly depressed from Baseline through Day 10 (N=19) and showed modest improvement after removal (at Day 10) of ISO minipumps by Day 17 (N=12) and at Day 38 (N=6). The transmitral E to A ratio was significantly depressed, indicating diastolic stiffening of the heart at Days 7 (N=15), 10 (N=17) and 17 (N=9), with modest recovery at Day 38 (N=6). *P<0.05; **P<0.01; ***P<0.001 versus Baseline. Data are presented as mean ± SEM.
Figure 2
Figure 2. Left ventricular (A/B) and left atrial (C/D) end-diastolic and end-systolic volumes increase after ISO injury
ISO caused significant dilatation of the left atrium and ventricle. Left ventricular and atrial end-systolic and diastolic volumes increased significantly from Baseline through Day 10 (N=19) with moderate recovery at Day 17 (N=12; LAESV, LAEDV N=11) and Day 38 (N=6). *P<0.05; **P<0.01; ***P<0.001 versus Baseline. Data are presented as mean ± SEM.
Figure 3
Figure 3. ISO injury caused cardiac and myocyte hypertrophy
A/B: Cardiac hypertrophy was present by Day 17. D: An example of a section of normal heart is shown. This section was stained for cardiac actin (Red), DAPI (Nucleus; Blue) and laminin (Cell Perimeters; Green). Myocyte cross-sectional area was measured with ImageJ Software (NIH). Myocytes cut in cross section, with centrally located nuclei, were chosen for study. C: Approximately 300 cells were analyzed from each heart. Control-Day 10 (N=7); Day 17 (N=6); Day 38 (N=6). HW/TL and HW/BW: Control (N=7); Day 10 (N=10); Day 17-Day 38 (N=6). *P<0.05; **P<0.01; ***P<0.001 versus Control. Data are presented as mean ± SEM.
Figure 4
Figure 4. A/B. Representative LV tissue sections of control (A) and ISO damaged (B) hearts are shown
Cell loss, myocyte hypertrophy and replacement fibrosis can be seen in ISO-treated heart tissue. Collagen (Blue); cardiac tissue (Red). The percent blue area was quantified. The collagen content of the ventricles (C/D) and atria (E/F) increase after ISO injury: Masson’s Trichrome stained slides were used to measure changes in collagen content of all cardiac chambers. The assessment and analysis of the data was carried out in a blinded fashion. All chambers had significant increases in collagen content after ISO injury (Day 10), with small reductions during the next few weeks. Control-Day 10 (N=7); Day 17-Day 38 (N=6). *P<0.05; **P<0.01; ***P<0.001 versus Control. Data are presented as mean ± SEM.
Figure 4
Figure 4. A/B. Representative LV tissue sections of control (A) and ISO damaged (B) hearts are shown
Cell loss, myocyte hypertrophy and replacement fibrosis can be seen in ISO-treated heart tissue. Collagen (Blue); cardiac tissue (Red). The percent blue area was quantified. The collagen content of the ventricles (C/D) and atria (E/F) increase after ISO injury: Masson’s Trichrome stained slides were used to measure changes in collagen content of all cardiac chambers. The assessment and analysis of the data was carried out in a blinded fashion. All chambers had significant increases in collagen content after ISO injury (Day 10), with small reductions during the next few weeks. Control-Day 10 (N=7); Day 17-Day 38 (N=6). *P<0.05; **P<0.01; ***P<0.001 versus Control. Data are presented as mean ± SEM.
Figure 5
Figure 5. ISO injury increases the number of BrdU+ non-myocytes during injury and the number BrdU+ myocytes in the atria after injury
BrdU was infused for 7 days before euthanasia, except in pulse-chase studies where it was infused during injury (Pulse) and animals were euthanized at Day 38 (Chase). BrdU (Red); α-Actin (Green); DA PI (Blue). Few BrdU+ nuclei were observed in normal atria and most of these were non-myocyte nuclei (A/D/E). ISO induced injury caused a large increase in the number of BrdU+ nuclei (Day 10; B) but almost all of the BrdU+ nuclei were non-myocytes (D/E). The total number of BrdU+ nuclei decreased rapidly after removal of ISO minipumps, at Day 17 (C/D) and Day 38 (D). The number of BrdU+ myocyte nuclei increased at Day 17 (C/E) but was back to control levels by Day 38 (E). BrdU+ non-myocytes labeled during the injury phase (Pulse) persisted in the heart for 4 weeks (Day 38; Chase) and the largest number of BrdU+ myocytes were found when labeling was done at this time period (E). *P<0.05; **P<0.01; ***P<0.001 versus Control. Data are presented as mean ± SEM.
Figure 6
Figure 6. ISO injury increases the number of BrdU+ non-myocyte and myocyte nuclei in the ventricle
Experimental approaches were as described in Figure 6. Representative BrdU+ myocyte and non-myocyte nuclei (at Day 38) are shown in A and B. C: The number of BrdU+ nuclei was greatest during the injury phase (Day 10) and decreased rapidly afterwards. Nuclei labeled during injury (Pulse) were still present 4 weeks later (Day 38; Chase). D: The number of BrdU+ myocytes increased during the first week after injury (Day 17) and returned to control levels by Day 38. BrdU infusion during injury (Pulse), when few Brdu+ myocytes were found, resulted in BrdU labeling of myocyte nuclei at Day 38 (Chase). *P<0.05; **P<0.01; ***P<0.001 versus Control. Data are presented as mean ± SEM.
Figure 7
Figure 7. cKit+ cells in the heart proliferate during ISO induced injury
A: Representative cKit staining of a proliferative cell is shown. B: The % of cKit cells in the ventricle did not change during the time course of our study. C: A significant increase in the ratio of proliferative cKit cells to non-proliferative cKit cells was observed during the injury phase. D: cKit+/BrdU+ cells are a very small % of the cells in the ventricle, but this % increases during ISO injury. E–G: Similar results were observed in the left atrial sections. *P<0.05; **P<0.01; ***P<0.001 versus Control. Data are presented as mean ± SEM.
Figure 8
Figure 8. The % of cKit+/CD45- cells increases during ISO induced injury
A: Representative staining of tissue for cKit, CD45 and DAPI. Three cKit cells are present and one of these was also positive for CD45. B/C: Tissue sections from Control, Day 10 ISO-treated and Day 17 ISO-treated hearts were co-stained with cKit and CD45. Cells that stained positive for cKit only, CD45 only and with both cKit and CD45 were each counted. The percent of each cell type present (cKit+ only, CD45+ only, or cKit+/CD45+) relative to the group as a whole is plotted for Atrial (B) and Ventricular (C) sections. cKit (Red); CD45 (Green); DAPI (Blue). Control (N=3) Day 10 (N=4); Day 17 (N=3). *P<0.05; **P<0.01; ***P<0.001 versus Control. Data are presented as mean ± SEM.
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