Cryoinjury: a model of myocardial regeneration

Cardiovascular Pathology 17 (2008) 23 – 31

Original Article

Cryoinjury: a model of myocardial regeneration Machteld J. van Amerongen4, Martin C. Harmsen, Arjen H. Petersen, Eliane R. Popa, Marja J.A. van Luyn Department of Pathology and Laboratory Medicine, University Medical Center Groningen, University of Groningen, Medical Biology Section, 9713 GZ, Groningen, The Netherlands Received 31 August 2006; received in revised form 30 November 2006; accepted 12 March 2007

Abstract Introduction: Although traditionally adult cardiomyocytes are thought to be unable to divide, recent observations provide evidence for cardiomyocyte proliferation after myocardial injury. Myocardial cryoinjury has been shown to be followed by neovascularization. We hypothesize that, in addition to neovascularization, cardiomyocyte proliferation after myocardial cryoinjury contributes to regeneration. Method: Cryolesions were applied to the left ventricle of mouse hearts. Inflammatory cell infiltration (F4/80, neutrophils), neovascularization (CD31), and cardiomyocyte proliferation (5-bromo-2-deoxyuridine, Ki-67, mitotic spindle) were determined at different time points (2–70 days) after cryoinjury. Results: Between Days 7 and 14 after injury, a 150- and 280-fold increase in number of proliferating cardiomyocytes was observed, as compared to controls. At the same time, numerous proliferating capillaries were found in between the proliferating cardiomyocytes. Presence of high numbers of macrophages in the cryolesion preceded and coincided with this proliferation. The area of cryolesion decreased significantly between Days 7 (23F5%) and 14 (8F2%) after cryoinjury. Moreover, regeneration of viable, nonhypertrophied myocardium was observed. After 14 days, cardiomyocyte proliferation decreased to numbers observed in controls, and concomitantly, the number of macrophages strongly decreased. Conclusion: Our data show that adult cardiomyocytes proliferate in sufficiently high numbers to effectuate myocardial regeneration after left ventricular cryoinjury in mice. D 2008 Published by Elsevier Inc. Keywords: Myocardial cryoinjury; Cardiomyocyte proliferation; Myocardial regeneration; Neovascularization; Inflammation

1. Introduction Until recently, the dogma prevailed that cardiomyocytes in the adult mammalian heart were terminally differentiated cells and, as a consequence, were unable to proliferate [1,2]. Therefore, the mammalian heart was considered to be unable to regenerate after injury. Recent observations, however, show that cardiomyocyte proliferation can occur under physiologic and pathological conditions of the heart [3–5], although the level of proliferation observed is insufficient for adequate regeneration of diseased myocardium [6,7]. In a number of (cardio)vascular pathological conditions, such as myocardial infarction and vascular trauma, physiologic and 4 Corresponding author. Department of Pathology and Laboratory Medicine, University Medical Center Groningen, Medical Biology Section, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands. Tel.: +31 0 50 363 2417; fax: +31 0 50 361 9911. E-mail address: [email protected] (M.J. van Amerongen). 1054-8807/08/$ – see front matter D 2008 Published by Elsevier Inc. doi:10.1016/j.carpath.2007.03.002

therapeutic neovascularization is stimulated by inflammatory factors in the injured tissue [8–12]. It is conceivable that also cardiomyocyte proliferation may be stimulated by inflammatory factors. Before these factors can be identified, the circumstances under which extensive cardiomyocyte proliferation occurs need to be understood. In this study, cryoinjury was used as a model for myocardial injury of the left ventricle (LV) wall in mice because cryoinjury produces a uniform, highly reproducible area of necrosis, followed by an extensive inflammatory reaction [13]. Clinically, cardiac cryolesions are used as an ablative technique in the surgical treatment of arrhythmias [14,15]. We hypothesized that cardiomyocyte proliferation after myocardial cryoinjury contributes to regeneration. The time course of cardiomyocyte proliferation, inflammatory cell infiltration, and neovascularization was determined at different time points (2–70 days) after myocardial cryoinjury. We show that adult cardiomyocytes proliferate in

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sufficiently high numbers to effectuate myocardial regeneration after LV cryoinjury in mice. 2. Methods 2.1. Animals Twelve-week-old male C57BL/6JOlaHsd mice (Harlan Nederland, Horst, The Netherlands) were housed individually under conventional conditions. Mice received pelleted diet (RMH-B 10mm, Arie Blok, Woerden, The Netherlands) and water ad libitum. All procedures performed on mice were approved by the local committee for care and use of laboratory animals and were performed according to strict governmental and international guidelines on animal experimentation.

of the cryolesion. The cryolesion area was measured and expressed as percentage of the total ventricular circumference, without the luminal area. Final infarct size was calculated as the average of all slices from each heart. The wall thickness of the noninjured myocardium at the endocardial site of the cryolesion and the diameter of LV cardiomyocytes was measured using planimetry software (Leica Qwin version 2.7). Cardiomyocyte diameter was measured across the nucleus in 50 LV cardiomyocytes that were cut transversally. All measurements were done by two investigators in a blinded manner independently. 2.4. Histology For all histological evaluations, 3 transversal serial sections per heart were evaluated by light microscopy by two investigators in a blinded manner independently.

2.2. Operation procedure Under general isoflurane (2.5%), N2O (2%) anesthesia mice were shaved and disinfected with chloral hexidine. The mice were intubated and ventilated with the anesthetic mixture using a mechanical ventilator (Hugo Sachs Elektronik, March-Hugstetten, Germany). At this point, the analgesic drug buprenorfine (0.03 mg/kg) was given subcutaneously. The heart was exposed through a left lateral thoracotomy. Cryoinjury was inflicted by applying a round 3-mm-diameter metal probe cooled to 1968C with liquid nitrogen to the LV wall for 10 s. After the frozen myocardium had thawed, the procedure was repeated two times to the same area, as previously described [16]. The cryoinjured area was macroscopically identified as a firm white disk-shaped region. The intercostal space and skin were closed with sutures. Mice received 100% oxygen until wakening, after which, they were extubated. Sham-operated control mice (n=5) underwent the same procedure, but a nonfrozen probe was used. One group of mice (n=5) was not operated. Mice received drinking water containing 1 mg/ml 5bromo-2-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) ad libitum 3 days before being euthanized. Mice euthanized on day 2 after cryoinjury received BrdU containing drinking water 1 day before the operation. Two (n=10), 4 (n=11), 7 (n=13), 14 (n=13), 28 (n=12), and 70 (n=8) days after cryoinjury, mice were euthanized by cervical dislocation under general anesthesia, and the hearts were excised immediately. Hearts were snap-frozen in liquid nitrogen and stored at 808C or fixed in 2% paraformaldehyde (MERCK, Darmstadt, Germany) containing 0.1 M phosphate buffer (pH 7.4) for 2 h. 2.3. Characterization of cryoinjury

2.4.1. Immunohistochemical staining for macrophages, neutrophils, endothelial cells, and cardiomyocytes Cryosections (5 Am) were cut, fixed with acetone and preincubated in phosphate-buffered saline (PBS) containing 10% serum of the species in which the secondary antibody was produced. Endogenous peroxidase (PO) was blocked by incubation with 0.1% H2O2 in PBS for 10 min. Slides were incubated for 1 h with monoclonal rat antimouse F4/80 (Serotec, Oxford, UK) for macrophages, monoclonal rat antimouse neutrophils (Serotec), monoclonal rat antimouse CD31 (Pharmingen, San Diego, CA, USA) for endothelial cells, and polyclonal goat antihuman troponin T-C (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for cardiomyocytes, all diluted 1:100 in PBS with 1% bovine serum albumin (BSA). Following incubation with F4/80, slides were incubated with rabbit antirat IgG conjugated to biotin (DAKO, Glostrup, Denmark), diluted 1:100 for 30 min. Next, the slides were incubated with appropriate antibodies conjugated to PO [Streptavidin, rabbit antiRat IgG or Rabbit antiGoat IgG (DAKO)] diluted in PBS for 30 min. Colour development was performed with 3-amino-9-ethyl-carbazole (AEC, Sigma, Steinheim, Germany) as substrate dissolved in N,N-dimethylformamide (MERCK) and 0.5 M acetate buffer (pH 4.9). Slides were counterstained with Mayer’s hematoxylin (Fluka Chemie, Buchs, Switzerland) and mounted in Kaiser’s glycerin (MERCK). All incubation steps were followed by appropriate washing steps. Macrophage quantification was performed by counting the number of F4/80 positive cells in photographs (400) taken in 3 areas in the cryolesion. Average numbers were calculated using data from all three slices. Macrophage density was expressed as cells per square millimeter. 2.5. Quantitation of cardiomyocyte proliferation

Masson’s trichrome stain, which stains collagen blue and myocardial cells red, and troponin T-C staining were used to measure the size of the cryolesion using computerized planimetry (Leica Qwin version 2.7). Three transversal serial sections (5 Am) of each heart were cut in the center

2.5.1. Immunofluorescent double staining for Ki-67 and troponin T-C Transversal sections (5 Am) of the snap-frozen hearts underwent the same procedure as described above. After

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incubation with monoclonal polyclonal rabbit antihuman Ki-67 (Novo Castra Laboratories, Newcastle upon Tyne, UK) at 1:1000 dilution for 1 h, slides were incubated with polyclonal goat antihuman troponin T-C (Santa Cruz

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Biotechnology) at 1:10 dilution for 1 h, followed by incubation with donkey antigoat conjugated to FITC (Chemicon International, Temecula, CA, USA) at 1:100 dilution for 30 min. Nonspecific protein binding was

Fig. 1. Light micrographs of transversally sectioned cryoinjured mouse hearts stained with troponin T-C antibody (left) and Masson’s trichrome (middle and right) on Days 2, 4, 7, 14, and 28. The cryolesion is indicated by arrows. Two days after cryoinjury, myocardial cell debris is present throughout the cryolesion (C). On Day 4 after cryoinjury, a complete lack of cardiomyocytes and extensive infiltration of inflammatory cells can be observed in the cryolesion (F). The cryolesion size decreases significantly between Days 7 and 14 (G, H, J, K). After 14 days, only a small amount of scar tissue remains on the epicardial surface (J–O). Blood vessels in the cryolesion are indicated by asterisks in L and O. Original magnification 400 (C, F, I, L, O); others, 10.

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blocked by incubation with PBS containing 10% goat serum for 30 min. Next, slides were incubated with goat antirabbit IgG conjugated to biotin (DAKO, Glostrup, Denmark) at 1:100 dilution for 30 min, followed by incubation with streptavidin conjugated to Cy-3 (Zymed, San Francisco, CA, USA) at 1:200 dilution for 30 min. All incubation steps were followed by appropriate washing steps. Slides were mounted in citifluor and evaluated with a fluorescence microscope. The percentage of Ki-67-positive cardiomyocytes was assessed by counting 700 cardiomyocyte nuclei in the border zone around the cryolesion in a random manner and determining the percentage of Ki-67-positive nuclei.

two-tailed unpaired Student’s t test. A difference was considered statistically significant when Pb.05.

3. Results 3.1. Characterization of the cryolesion No differences were observed in morphology of the myocardium of sham-operated and control mice; therefore, they were analyzed as one control group. Application of the liquid nitrogen cooled probe caused a well-defined disc-

2.5.2. Immunohistochemical staining for BrdU The paraformaldehyde-fixed hearts were dehydrated in graded series of acetone and embedded in Technovit 8100 (T-8100, Heraeus Kulzer, Wehrheim, Germany). We used a plastic embedding procedure for the detection of BrdU because this yields the best morphology and allows cutting sections of a thickness of 2 Am [17]. Sections were pretreated with 0.1% trypsin in 0.1 M Tris buffer (pH 7.8), containing 0.1% CaCl2 at 378C for 15 min. DNA was denatured by incubation with 1 M HCl at 608C for 30 min, followed by incubation with 0.05% pepsin (Roche Diagnostics, Mannheim, Germany) in 0.35 M HCl at 378C for 15 min. Next, slides were incubated with monoclonal antiBrdU (Sigma, St. Louis, MO, USA) in PBS with 1% BSA diluted at 1:100 for 1 h, followed by incubation with goat antimouse IgG conjugated to biotin (DAKO) diluted at 1:100 for 30 min. Subsequently, slides were incubated with streptavidin conjugated to PO (DAKO) diluted 1:100 in PBS for 30 min. Colour development was performed with 3.3-diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO, USA). All incubation steps were followed by appropriate washing steps. Slides were counterstained with toluidine blue and mounted in Permount (Fisher Chemicals, Fair Lawn, NJ, USA). The percentage of BrdU-positive cardiomyocytes was assessed by counting 700 cardiomyocyte nuclei in the border zone around the cryolesion in a random manner and determining the percentage of BrdU-positive nuclei. The number of proliferating endothelial cells was quantified by counting the number of BrdU-positive endothelial cells in photographs (400) taken in 4 areas at the endocardial site of the cryolesion, directly below the LV lumen; the area in which proliferating cardiomyocytes were mainly observed. Average numbers per square millimeter were calculated. 2.6. Statistical analysis All data represented are expressed as meanFSEM. The data were analyzed using computer statistical software (GraphPad Prism, version 3.00, GraphPad Software). Interrater agreement was evaluated by paired-samples t testing. The statistic significance of differences in the findings was evaluated by a one-way analysis of variance, followed by a

Fig. 2. Regeneration of the cryoinjured heart. The graphs show the cryolesion size (A), the thickness of the noninjured myocardium at the endocardial site of the cryolesion (B), and the cardiomyocyte diameter in time (C) after cryoinjury. *Pb.05; **Pb.01; ***Pb.001 vs. Day 7. Bars represent mean and S.E.M.

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Fig. 3. Cardiomyocyte proliferation in the cryoinjured heart. (A) Immunofluoresencent staining for Ki-67 (red) and troponin T-C (green) 14 days after cryoinjury showing a troponin T-C positive cardiomyocyte that concomitantly expresses Ki-67. (B) Cardiomyocyte proliferation in time as defined by Ki-67 and troponin T-C double positivity. (C and D) Control heart stained with the BrdU antibody. BrdU-positive nuclei are present in between the cardiomyocytes (selection indicated by arrowheads). (E and F) cryoinjured heart stained with the BrdU antibody 14 days after injury. BrdU-positive cardiomyocyte nuclei (arrows) are found at the endocardial site of the cryolesion, directly below the LV lumen. The LV lumen is indicated as LV in (C) and (E). Other BrdU-positive nuclei are present in between the cardiomyocytes (selection indicated by arrowheads). (G) Mitotic BrdU-positive cardiomyocyte (arrow). (H) Cardiomyocyte proliferation in time as defined by BrdU positivity. The linings in the figure in (B) and (H) indicate the evaluated area and the endocardial site within the evaluated area where proliferating cardiomyocytes were observed. Original magnification 200 (C and E); others, 1000. *Pb.05; ***Pb.001 vs. control. Bars represent mean and S.E.M.

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shaped cryolesion. The location of the cryolesion on the LV wall was highly reproducible. Two days after cryoinjury, myocardial cellular debris was present throughout the cryolesion (Fig. 1C). On day 4 after cryoinjury, a complete lack of cardiomyocytes and extensive infiltration of inflammatory cells were observed in the cryolesion (Fig. 1D–F). The area of cryolesion decreased significantly between days 7 (23F5%) and 14 (8F2%) after cryoinjury ( Pb.05) (Figs. 1G, H, J, and K and 2A). At these same time points, the wall thickness of the myocardium at the endocardial site of the cryolesion, containing viable cardiomyocytes, increased significantly (Day 7 vs. day 14, Pb.001) (Figs. 1G, H, J, and K and 2B). As of Day 28, the thickness of myocardium containing viable cardiomyocytes was similar to controls (Fig. 2B). Only a small amount of

scar tissue remained on the epicardial surface (Fig. 1J–O). Furthermore, there was no change in diameter of the individual cardiomyocytes, indicating that no cardiomyocyte hypertrophy had occurred ( P N.5) (Fig. 2C). 3.2. Cardiomyocyte proliferation The decrease in cryolesion size and the simultaneous increase in healthy myocardium suggested a significant proliferation of cardiomyocytes in response to cryoinjury. To determine cardiomyocyte proliferation, we used antibodies directed against Ki-67 and BrdU. Ki-67 is a nuclear protein expressed in cycling cells in late G1, S, and G2 phases and mitosis. Expression of Ki-67 wanes rapidly during G1 and is absent in resting cells (G0) [18]. The

Fig. 4. Neovascularization after cryoinjury. Light micrographs of transversally sectioned control hearts (A and B) and hearts 7 days after cryoinjury (C and D) stained with antibodies for CD31 (A and C) and BrdU (B and D). (A and B) The control hearts show a rich, microvascular network (arrowheads), containing sporadic BrdU-positive endothelial cells (arrows). (C and D) Numerous BrdU-positive capillaries (arrows) are present on Day 7 after cryoinjury. (E) Quantitative analysis of BrdU-positive endothelial cells at the endocardial site of the cryolesion. Significant differences compared to sham-operated controls are indicated as *Pb.05. Bars represent meanFS.E.M. Original magnification 1000.

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thymidine analog BrdU is incorporated in the DNA in the S phase of the cell cycle. In the myocardium of control mice, only sporadically Ki-67-positive cardiomyocytes (0.025%) were found (Fig. 3B). As compared to these controls, cryoinjury led to a 50-fold (1.2%) increase in the number of Ki-67-positive cardiomyocytes on Days 7 and 14, respectively, as determined by immunofluorescent doublestaining for Ki-67 and troponin T-C ( Pb.001) (Fig. 3A and B). The number of Ki-67-positive cardiomyocytes significantly decreased on Days 28 and 70 after cryoinjury. The time course of BrdU incorporation (Fig. 3H) was strikingly similar to Ki-67 expression (Fig. 3B) in cardiomyocytes. Cryoinjury led to a 150- (4.5%) and 280-fold (8.5%) increase in the number of BrdU-positive cardiomyocytes on Days 7 and 14, respectively, compared to controls ( Pb.05 and Pb.001) (Fig. 3C–F and H). At Days 7 and 14, BrdU-positive mitotic spindles were observed in cardiomyocytes as well (Fig. 3G). Remarkably, BrdU- and Ki-67-positive cardiomyocytes were infrequently found at the border of the cryolesion. Instead, these proliferating cardiomyocytes were found at the endocardial site of the cryolesion, directly below the LV lumen (Fig. 3C and E). Other BrdU- or Ki-67-positive cells that were not troponin T-C-positive were mainly observed in the cryolesion and were identified as endothelial cells or inflammatory cells (macrophages and neutrophils) based on their morphology (data not shown). 3.3. Neovascularization The control hearts showed a rich microvascular network, containing low numbers of BrdU-positive endothelial cells (Fig. 4A, B and E). Seven days after cryoinjury, a significantly higher number of BrdU-positive endothelial cells was found at the site of cardiomyocyte proliferation, as compared to controls ( Pb.05; Fig. 4C–E). Numerous Ki-67positive capillaries were observed on Day 7 as well. After Day 7, no significant differences were found in the number of BrdU-positive endothelial cells, as compared to controls. The cryolesion exhibited a low capillary density after 4 days and later time points, compared to controls, whereas the number of pre- and post capillary vessels, i.e., arterioles and venules, increased (data not shown). 3.4. Time course of inflammatory cell infiltration 3.4.1. Macrophages In the LV myocardium of control mice, low numbers of macrophages were present (density 116.7F28.3 cells/mm2) (Fig. 5). The cryolesion showed a progressive increase in macrophage accumulation, which preceded and coincided with the time course of cardiomyocyte proliferation (compare Fig. 3B and H, Figs. 3–5). The macrophage density in the cryolesion increased, compared to control myocardium 2 days after cryoinjury (density 576.7F58.3 cells/mm2, Pb.01 vs. control). Macrophage density peaked

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Fig. 5. Course of the macrophage infiltration after cryoinjury. The cryolesion shows a progressive increase and decrease in macrophage density, with peak numbers present on Days 4 and 7. *Pb.05; ***Pb.001 vs. control. Bars represent mean and SEM.

on Days 4 and 7 (density 1416.7F113.3 cells/mm2 and 1678.3F193.3 cells/mm2, respectively, Pb.001 vs. vs. control). Fourteen days after cryoinjury, macrophage density in the cryolesion had decreased markedly (density 611.7F146.7 cells/mm2, Pb.001 vs. control). Subsequently, as fibroblasts and collagen accumulated in the residual cryolesion, macrophage density further decreased and was comparable to controls on Days 28 and 70. 3.4.2. Neutrophils and mast cells The LV myocardium of control mice contained few neutrophils. Neutrophils rapidly infiltrated the cryolesion and were present in high numbers on Days 2 and 4 after cryoinjury. The number of neutrophils in the cryolesion decreased markedly on day 7 after cryoinjury and neutrophils were only sporadically observed at later time points. Mast cell infiltration was not found at any time point studied. The morphology of the spared and the regenerated LV myocardium remained comparable to controls throughout the whole period studied and only differed in the formation of capillaries in the regenerated myocardium, as described above.

4. Discussion In this study, we have demonstrated that adult cardiomyocytes proliferate to an extent that is sufficient to effectuate myocardial regeneration after extensive cryoinjury of the LV myocardium in mice. Interestingly, the highest levels of macrophages preceded and coincided with the highest levels of cardiomyocyte proliferation, suggesting a functional role of these cells in myocardial regeneration. Both markers, Ki-67 and BrdU, confirmed the high level of cardiomyocyte proliferation at Days 7 and 14 after cryoinjury. Although the existing proliferation markers do not detect cytokinesis but only demonstrate cell cycle activation and progression, the absence of cardiomyocyte hypertrophy and, moreover, the increase in

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viable myocardium to control levels, confirms that cardiomyocytes had proliferated. A sufficient supply of oxygen and nutrients is critical for the regenerating injured myocardium to sustain cell metabolism and function. Our data demonstrate formation of new capillary vessels between the proliferating cardiomyocytes. This is in agreement with Schuster at al. [19], who demonstrated that increased myocardial neovascularization by bone marrow angioblasts results in cardiomyocyte proliferation. So far, no approaches are known to effectively induce cardiomyocytes to re-enter the cell cycle. This study reveals the circumstances under which cardiomyocyte proliferation occurs and provides a model to identify the mitogenic factors that induce cardiomyocyte proliferation. Identification of these factors may lead to interventions that increase the number of cardiomyocytes in the myocardium and improve in heart function after injury. Peak levels of proliferating cardiomyocytes were present on Days 7 and 14 after cryoinjury, indicating that the responsible mitogens are present at these time points. Furthermore, proliferating cardiomyocytes were detected almost exclusively at the subendocardial site of the cryolesion, directly below the LV lumen. This suggests that either the local concentration of cardiogenic factors is important for cardiomyocyte proliferation or that, depending on their localization within the LV, cardiomyocytes differ in their proliferative capacity. We hypothesize that macrophages are responsible for secretion of cardiogenic factors, either directly or through an indirect, paracrine mechanism. This is supported by the fact that peak numbers of macrophages preceded and coincided with cardiomyocyte proliferation. Furthermore, recently, we have shown that inhibition of macrophage infiltration after cryoinjury and, thus, modulation of the inflammatory response is associated with decreased repair of the infarct site and severe myocardial remodeling, suggesting a link between macrophages and cardiomyocyte proliferation/ myocardial regeneration (Van Amerongen et al, AJP 2006, in press). Also, in a number of (cardio)vascular pathological conditions, such as myocardial infarction and vascular trauma, physiologic and therapeutic neovascularization is stimulated by soluble factors, which are secreted by macrophages after tissue injury [8,9,11,12]. Specific types of cell death, e.g., necrosis after cryoinjury vs. apoptosis after ischemic injury [20], may trigger different types of wound healing, which might explain the low or absent cardiomyocyte proliferation observed after ischemic myocardial injury [6,7]. The cryoinjury model has been used previously in mice; however, these studies do not describe a spatial–temporal study of the cryolesion size and do not analyze cardiomyocyte proliferation. Leferovich et al. [21] did not observe myocardial regeneration in C57BL/6 mice. However, they created myocardial injury on the right ventricle. The

underlying mechanism of the LV cardiomyocyte proliferation observed in this study may not be applicable to right ventricular cardiomyocytes, as right and LV cardiomyocytes are of different embryological origin [22]. Furthermore, the BrdU exposure, immediately after infliction of cryoinjury up till 60 days, might have resulted in lower BrdU labeling of cardiomyocytes (1–3%) because long-term exposure to BrdU can seriously impair the potential of cells to proliferate [23]. In conclusion, a high level of cardiomyocyte proliferation, sufficient to effectuate myocardial regeneration, is demonstrated, and therefore, the cryoinjury in mice may be used as a model to identify the mitogenic factors that drive cardiomyocyte proliferation in injured myocardium. Future research should reveal the role of the inflammatory reaction, especially of macrophages and its soluble mediators, in cardiomyocyte proliferation and cardiac regeneration. If similar mechanisms govern the re-entry of cardiomyocytes into the cell cycle in man, therapeutic regeneration of the injured myocardium may become feasible.

Acknowledgment H.M. Salomons, M.Sc., is kindly acknowledged for helping with the statistical analysis.

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