The type of injury dictates the mode of repair in neonatal and adult heart - PubMed (original) (raw)
The type of injury dictates the mode of repair in neonatal and adult heart
Tal Konfino et al. J Am Heart Assoc. 2015.
Abstract
Background: The neonatal heart possesses the unique power to regenerate in response to resection of the left ventricular apex. We sought to determine whether the type of injury affects the mode of repair and regeneration.
Methods and results: Apical resection, or permanent left anterior descending coronary artery ligation, was induced in neonatal 1-day-old mice. Echocardiography was used to confirm and monitor cardiac injury and remodeling. Histological and immunohistochemical examinations of the resected and infarcted neonatal hearts revealed inflammation and granulation tissue formation. From day 3, early regeneration was identified at the injured sites and was characterized by dedifferentiation and proliferation of cardiomyocytes around the injured areas. The young cardiomyocytes infiltrated the granulation tissue and replaced it with a new myocardium. The ability of neonatal cardiomyocytes to proliferate was confirmed in neonatal heart organ cultures. Notably, myocardial infarction in neonatal mouse produced incomplete regeneration with a residual small infarct and, sometimes, aneurysm at 28 days after myocardial infarction. We then repeated the same experiments in the adult heart. Remarkably, myocardial infarction in the adult mouse heart produced a typical thin scar, whereas apical resection revealed an abnormal, epicardial, hemorrhagic scar 21 days after injury.
Conclusions: Our findings suggest that the type of injury, resection, or infarction affects the mode of repair in both neonatal and adult mouse hearts. Identifying the differences in the mechanisms or repair of these 2 types of injuries could help to develop novel regenerative therapies relevant to human patients.
Keywords: cardiomyocytes; fibrosis; inflammation; macrophages; myocardial infarction; myocardial regeneration.
© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.
Figures
Figure 1.
Myocardial regeneration after apical resection in neonatal mice. Newborn mice (1 day old) were anesthetized and cooled down on ice, and the chest was opened by left thoracotomy. Iridectomy scissors were used to resect the heart apex until the LV chamber was exposed. A, At 1 day after resection, H&E staining revealed blood clot formation at the resection area (arrow). Scale bar: 500 μm. B, At 3 days after resection, H&E staining showed inflammation and granulation tissue at the injured area (arrow). The dashed line marks the resection plane. Scale bar: 2 mm. C, At 21 days after resection, Masson's trichrome staining showed that robust myocardial regeneration replaced the inflammation and granulation tissue, with minimal epicardial fibrosis (arrow). Scale bar: 2 mm. D, At 21 days after resection, Masson's trichrome staining of another neonatal heart demonstrated minimal fibrosis (arrow). Scale bar: 2 mm. E, Monocytes and macrophages (dark brown) penetrated the blood clot that covered the injured area 1 day after resection (arrows). Scale bar: 200 μm. F, Robust infiltration of monocytes and macrophages (dark brown) into the apex 3 days after myocardial infarction. Scale bar: 500 μm. G, Sharp decrease in monocyte and macrophage number at the regenerating apex and in other parts at 5 days after resection. Scale bar: 2 mm. H&E indicates hematoxylin and eosin; LV, left ventricle.
Figure 2.
Dedifferentiation and proliferation of cardiomyocytes at the injured area 3 days after resection. A, Cardiac actin staining reveals that cardiomyocytes (brown) infiltrated the injured tissue and replaced the granulation tissue. Nuclei are stained blue with hematoxylin. Scale bar: 200 μm. B, Higher magnification of the penetrating cardiomyocytes at the border zone demonstrates cardiomyocyte dedifferentiation, characterized by sarcomeric disorganization, appearance of intercellular spaces, rearrangement of sarcomers toward the cell wall (arrow), and nuclei division (arrow). The manifestation of dedifferentiating, double‐nuclei cardiomyocytes is clearly different from the adjacent, single‐nuclei, sarcomere‐full cardiomyocytes. Scale bar: 50 μm. C, Higher magnification of different area at the border zone shows double‐nuclei cardiomyocytes with disorganization of sarcomeres. Again, the look of these divided cardiomyoctes is different from adjacent single‐cell cardiomyocytes. Scale bar: 50 μm. D, pHH3 immunostaining for dividing nuclei showed intensive mitotic activity at day 3 after resection. Scale bar: 100 μm. E, At day 5 after resection, pHH3 immunostaining (brown) revealed dividing cardiomyocytes. Note typical sarcomeric striation in cells with stained nuclei (arrows). Scale bar: 100 μm. F, At days 3 and 5 after resection, the amount of nuclei division was 2‐fold higher in the injured neonatal heart compared with sham‐operated hearts. Differences between pHH3 stain for mitosis were compared by 2‐way ANOVA, with Bonferroni's multiple comparison post‐test. Differences were considered significant at P<0.05. pHH3 indicates phosphorylated histone H3.
Figure 3.
Beating outgrowth of cells from neonatal heart in organ culture expressed dedifferentiation markers. One‐day‐old mouse hearts were harvested, sliced, and placed on a coated tissue culture dish (100 mm) using cardiomyocyte growth media. Cells budding from the cultured heart were stained with specific antibodies. A, Beating outgrowth of neonatal heart at day 14 in culture. Scale bar: 200 μm. B, Cardiomyocyte budding from cultured heart at day 35 in culture. Cardiomyocytes were stained green with cardiac actin, nuclei were stained blue with DAPI. Scale bar: 100 μm. C, The percentage of outgrowth cells that expressed the stem cell marker c‐Kit was near 25% after 35 days in culture. C‐kit expressing cells in red, nuclei stained blue with DAPI. These findings suggest resident cardiac stem‐cell proliferation or cardiomyocyte dedifferentiation. Scale bar: 200 μm. DAPI indicates 4′,6‐diamidino‐2‐phenylindole.
Figure 4.
Incomplete myocardial regeneration after MI in neonatal heart. To determine the effect of ischemic injury on myocardial regeneration, newborn mice (1 day old) were anesthetized and cooled down on ice. The chest was opened by left thoracotomy. An 8‐0 prolene suture was used to permanently occlude the left anterior descending artery. A, At 1 day after MI, H&E staining revealed cell necrosis and initial inflammatory response at the infarcted myocardium. Scale bar: 500 μm. B, At 3 days after MI, H&E staining demonstrated extensive inflammation around the necrotic anteroapical areas (arrows). Scale bar: 2 mm. C, At day 3 after MI, mac‐3 staining revealed robust accumulation of monocytes and macrophages (brown staining) at the necrotic myocardium. Scale bar: 200 μm. D, Masson's trichrome staining showed a small thin scar at 28 days after MI (arrow). Scale bar: 2 mm. E, Cardiac actin staining of the border zone 3 days after MI revealed less evidence of proliferating cardiomyocytes or sarcomere disassembly. Cardiomyocytes were stained brown with cardiac actin, nuclei were stained blue with hematoxylin. Scale bar: 100 μm. F, Cardiac actin staining of sham‐operated heart 3 days after procedure revealed intact myocardium without inflammation or cardiomyocyte dedifferentiation. Scale bar: 100 μm. G, pHH3 staining demonstrated mitotic nuclei at day 3 after MI (arrows). Scale bar: 100 μm. H, At days 3 and 5 after MI, the number of mitotic events in the myocardium was similar to sham‐operated hearts. Differences between pHH3 stain for mitosis were compared by 2‐way ANOVA, with Bonferroni's multiple comparison post‐test. Differences were considered significant at P<0.05. I, Transthoracic echocardiography was performed after MI or sham MI using a special small animal echocardiography system equipped with a 22‐ to 55‐MHz linear‐array transducer. Images of 2D echocardiogram 28 days after MI in 1‐day‐old mouse demonstrated apical aneurysm (arrow) and LV dilatation. J, Images of 2D echocardiogram of a sham‐operated mouse of the same age showed normal LV size and function. K, LVEF was slightly lower after apical resection and MI at 28 days after injury. Differences between echocardiography measurements were compared by 1‐way ANOVA, with Bonferroni's multiple comparison post‐test. Differences were considered significant at P<0.05. L, Mouse at 6 days after MI (arrow) next to sham‐operated mice at the same age. Note growth retardation of the mouse subjected to MI, suggesting the impact of significant myocardial injury on development. 2D indicates 2‐dimensional; H&E, hematoxylin and eosin; LV, left ventricle; LVEF, left ventricle ejection fraction; MI, myocardial infarction; pHH3, phosphorylated histone H3.
Figure 5.
No regeneration and scar formation is noted in adult heart after apical resection or MI. ICR 12‐week‐old female mice were anesthetized, intubated, and ventilated. Chests were shaved and opened by left thoracotomy. Iridectomy scissors were used to carefully resect the heart apex to a smaller extent than neonatal resection because of severe bleeding and high mortality rate. A, At day 21 after resection in 12‐week old mouse, Masson's trichrome revealed an apical, hemorrhagic scar at the resected area (arrow). Dashed line marks the resection plane. Scale bar: 2 mm. B, At day 21 after resection in adult mouse, H&E staining revealed active healing with inflammation and hematoma encapsulated within the apical scar. Scale bar: 500 μm. C, In contrast, MI induced a typical thin, transmural scar at day 21 after MI. Scale bar: 2 mm. H&E indicates hematoxylin and eosin; LV, left ventricle; MI, myocardial infarction.
References
- Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R, Nadal‐Ginard B, Silvestri F, Leri A, Beltrami CA, Anversa P. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001; 344:1750-1757. - PubMed
- Braunwald E, Pfeffer MA. Ventricular enlargement and remodeling following acute myocardial infarction: mechanisms and management. Am J Cardiol. 1991; 68:1D-6D. - PubMed
- Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990; 81:1161-1172. - PubMed
Publication types
MeSH terms
LinkOut - more resources
Full Text Sources
Medical
Miscellaneous