Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Dev Biol. 2014 Dec 31;399(1):91–99. doi: 10.1016/j.ydbio.2014.12.018

Differential Regenerative Capacity of Neonatal Mouse Hearts after Cryoinjury

Ali Darehzereshki 1,2,#, Nicole Rubin 1,2,#, Laurent Gamba 1,2, Jieun Kim 1,2, James Fraser 1,2, Ying Huang 1,2, Joshua Billings 1,2, Robabeh Mohammadzadeh 3, John Wood 1,2,4, David Warburton 2,4, Vesa Kaartinen 5, Ching-Ling Lien 1,2,6,*
PMCID: PMC4339535  NIHMSID: NIHMS653522  PMID: 25555840

Abstract

Neonatal mouse hearts fully regenerate after ventricular resection similar to adult zebrafish. We established cryoinjury models to determine if different types and varying degrees of severity in cardiac injuries trigger different responses in neonatal mouse hearts. In contrast to ventricular resection, neonatal mouse hearts fail to regenerate and show severe impairment of cardiac function post transmural cryoinjury. However, neonatal hearts fully recover after non-transmural cryoinjury. Interestingly, cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjuries. Epicardial activation and new coronary vessel formation occur after cryoinjury. The profibrotic marker PAI-1 is highly expressed after transmural but not non-transmural cryoinjuries, which may contribute to the differential scarring. Our results suggest that regenerative medicine strategies for heart injuries should vary depending on the nature of the injury.

Keywords: neonatal mouse heart regeneration, cryoinjury, cardiomyocyte proliferation, epicardium, neovascularization

Introduction

In contrast to mammals, zebrafish and newts possess remarkable regenerative abilities (Choi and Poss, 2012; Lien et al., 2012; Poss, 2007; Poss et al., 2003; Poss et al., 2002; Raya et al., 2003). After 20% resection of the ventricle, zebrafish fully regenerate lost heart tissue (Poss et al., 2002; Raya et al., 2003). Newborn (1 day old) mouse hearts have been shown to have regenerative capacity similar to adult zebrafish; however, this capacity is rapidly lost after postnatal day (P) 7 (Porrello et al., 2011). Zebrafish myocardium regenerates by proliferation of existing cardiomyocytes (Jopling et al., 2010; Kikuchi et al., 2010). Murine cardiomyocytes can also proliferate during fetal and neonatal development but the proliferative capacity rapidly decreases after postnatal day 5 (Soonpaa et al., 1996). The decrease in cardiomyocyte proliferation correlates to the time period when regeneration capacity diminishes (Porrello et al., 2011; Porrello et al., 2013). Approaches to extend the regenerative capacity of neonatal hearts to juvenile or even adult hearts could offer new treatment options.

It remains to be determined if hearts respond to different types and severities of injuries differently. Ventricular resection, or heart amputation, removes heart tissue with very little dead tissue remaining and is quite different from the pathogenesis of myocardial infarction. Cryoinjury models have been established to mimic the pathogenesis of post myocardial infarction and to study heart regeneration in zebrafish (Chablais et al., 2011; Gonzalez-Rosa et al., 2011; Schnabel et al., 2011). Similar regenerative responses such as cardiomyocyte proliferation and epicardial activation occur in zebrafish in response to both ventricular resection and cryoinjury (Chablais et al., 2011; Gonzalez-Rosa et al., 2011; Schnabel et al., 2011). However, different scarring responses and collagen deposition were reported during the regenerative process by different groups. In one report by Chablais et al., a fibrotic scar starts to form at 7 days post cryoinjury (dpc) and is resolved by 30-60 dpc (Chablais et al., 2011). In the report by Gonzalez-Rosa et al. (2011), the fibrotic scar formation reaches the maximum level at 21 dpc and is not resolved until 130 dpc. The additional time necessary for regeneration in zebrafish hearts following cryoinjury is thought to be due to the time needed for collagen scar resolution (Gonzalez-Rosa et al., 2011). In the report by Schnabel et al, minimal collagen scar but mainly fibrin clot forms during the regeneration process (Schnabel et al., 2011). Comparing these reports suggests that different severities of cryoinjuries induce different regenerative responses.

Based on pathology and MRI analysis, acute MI can be classified into two types: transmural (full wall thickness) or non-transmural (partial thickness) infarctions. Early evaluation of the transmural extent of necrosis is important for reperfusion therapy (Perazzolo Marra et al., 2011; Wu et al., 2013). To determine if neonatal mouse hearts can regenerate after cryoinjury and how transmurality affects the regenerative responses, we developed injury models to produce transmural (severe) and non-transmural (mild) injuries to the hearts. Here, we show that transmural cryoinjury produces significantly more damage to the ventricular wall than non-transmural cryoinjury and ventricular resection. Histological analysis and echocardiography indicated that neonatal mouse hearts do not regenerate after transmural cryoinjury partially due to a lack of increased cardiomyocyte proliferation. The epicardium was activated and extensive neovascularization occurred after cryoinjuries. Plasminogen activator inhibitor 1 (PAI-1) was highly expressed in the endocardium and myocardium closer to the lumen after transmural but not non-transmural cryoinjuries, which may have contributed to the differential scarring we observed. More detailed characterization of the regenerative response of neonatal mouse hearts in response to different types of injuries enhances our understanding of the pathophysiology of the myocardial injury response, which is essential for the development of efficient regenerative approaches for treatment of coronary heart diseases.

Materials and methods

Mouse maintenance and cryoinjury of neonatal mouse hearts

Animals were housed at the Children’s Hospital Los Angeles (CHLA) animal facility. All protocols related to animal work were approved by CHLA IACUC. Wild type ICR/CD-1 strain pregnant mice were ordered from Charles River Laboratories, MA around E13-E15 and housed at the CHLA animal facility until delivery. Surgical procedures were performed as previously described (Porrello et al., 2011) with some modifications in order to create a cold probe injury on the left ventricular wall. Briefly, 1-2 day old neonatal mice (P1-2; ICR/CD-1 strain, Charles River Laboratories, Wilmington, MA, USA) were anesthetized under hypothermic conditions. Left lateral thoracotomies were performed and hearts were exposed via an incision at intercostal space 6-7. Meanwhile, a 2 mm metal probe was chilled in liquid nitrogen for 30 seconds. Non-transmural (mild) and transmural (severe) cryoinjuries were created by application of the pre-chilled probe on the left ventricular anterolateral wall for 1 and 5 seconds, respectively. Sham-operated hearts were only exposed for 1 or 5 seconds without probe application. The thoracic wall incision was closed with a figure-of-eight suture using Prolene 7-0 (8701H, Ethicon, Somervile, NJ USA). The skin incision was repaired by topical tissue adhesive (Gluture, Abbott Laboratories, IL, USA). After surgery, mice recovered on a warm bed for 5-10 minutes. The survival rate was 90-100% correlating with the severity of the injury.

Tissue harvest

To collect hearts at different time points, neonatal mice younger than postnatal day (P) 10 were euthanized by exposure of CO2 (gradually fill the chamber with CO2) followed by decapitation. For mice older than p10, CO2 exposure (gradually fill the chamber with CO2) followed by cervical dislocation was used for euthanasia. Hearts were embedded in paraffin and sectioned transversely from the apex to the base at 7μm.

H&E, immuno and AFOG staining

Paraffin sections from hearts one day after surgery (1 dp) were stained with hematoxylin and eosin (VWR, Radnor, PA, USA) according to standard protocols to evaluate the extent of injury. The end time point heart sections (21, 60 and 120 dpi) were stained for acid fuchsin orange G-stain (AFOG) in order to determine the extent of collagen deposition. After deparaffinization in toluene, the sections were washed with ethanol and PBS, then incubated in preheated Bouin's fixative (Electron Microscopy Sciences, PA, USA; 2.5 h at 56°C, 1 h at room temperature), washed in tap water, incubated in 1% phosphomolybdic acid (Sigma-Aldrich, St. Louis, MO, USA; 5 min), rinsed with distilled water, and stained with AFOG staining solution (3 g of Acid Fuchsin, 2 g of Orange G, 1 g of Anilin Blue dissolved in 200 ml of acidified distilled water, pH 1.09 HCl, 5 min). Stained sections were rinsed with distilled water (2 min), dehydrated with ethanol and toluene, and mounted. In AFOG staining, the scar tissue is stained blue and muscle tissue appears orange/brown. For immunostaining, sections are incubated with the following primary antibodies: Wilm's Tumor protein (WT-1; 1:100, rabbit, Abcam), PECAM (1:50, Neomarkers), phospho-histone H3 (PHH3; 1:150, mouse, Millipore), myocyte enhancer factor-2 (MEF-2; 1:200, rabbit, Santa Cruz Biotechnology), plasminogen activator inhibitor 1 (PAI-1, 1:100, rabbit, Molecular Innovations) overnight. Apoptosis was evaluated by immunohistochemistry for caspase-3 antibody (1:300; rabbit, Cell Signaling).

Echocardiography

Mice were anesthetized under 1.5%-2 isoflurane in 100% oxygen at 60 days after surgery. Echocardiography was performed using a Visual Sonics Vevo 770 Ultrasound (VisualSonics Inc., Toronto, ON, Canada) equipped with a 30-MHz transducer. For each animal, two dimensional parasternal long axis and short axis views were obtained, and M Mode tracing at the level of papillary muscle was recorded.

PAI-1 Enzyme-Linked Immunosorbent Assay (ELISA)

PAI-1 antigen levels after cardiac injury were quantified using protein lysates from mouse hearts with Mouse PAI-1 Total Antigen Assay (Molecular Innovations).

Quantification and Statistics

Data are presented as mean ± SEM. Multiple group comparison was performed by one-way ANOVA followed by Bonferroni test for comparisons of means using GraphPad Prism 5 (GraphPad Software, Inc.). Comparison between two groups was done by the two-tailed Student's t test. *P<0.05 and ** P<0.01 were considered statistically significant. Quantification of scar size as a ratio of scar area to the left ventricle area and MEF2/PHH3, Caspase-3, PECAM, and WT-1 staining were conducted manually using Image J software by one observer who was blind to the experimental groups. Cardiomyocyte proliferation was quantified at both the injury and remote areas in each heart with a minimum of eight fields representative of the scar/border zones and remote area.

Results

Neonatal mouse hearts show differential regenerative capacity after cryoinjuries of different severities

To determine if different severities of heart injuries can trigger different regenerative and repair responses, we turned to the neonatal mouse hearts in which we can better control the severities of induced cryoinjuries. We developed a cold probe injury model of two different severities (non-transmural vs. transmural) by placing the cryoprobe on the anterolateral cardiac wall for two different lengths of time (see Methods for details). Compared to sham operated hearts (Fig. 1A and D), both non-transmural and transmural cryoinjuries produced necrotic myocardial tissue. Transmural cryoinjury produced damage and necrosis of full wall thickness (Fig. 1C and F) while non-transmural cryoinjury created damage up to nearly half of the ventricular wall thickness (Fig. 1B and E). To determine the level of apoptosis induced by the different types of injuries, sections of the hearts on 1 day post amputation (1dpa), non-transmural cryoinjury (1dpmc), transmural cryoinjury (1dpsc) or sham operation (1dps) were stained and quantified for cleaved caspase-3 (Suppl. Fig. 1A-E). Severe transmural injury produced the most apoptosis (Suppl Fig. 1D and E) while sham operated hearts had the least cell death (Suppl Fig. 1A and E). These results indicate that cryoinjury is an injury model producing consistent injuries that mimic the pathogenesis of myocardial infarction.

Fig. 1. Partial and full thickness damage induced by non-transmural (mild) and transmural (severe) cryoinjuries.

Fig. 1

Open in a new tab

H&E staining of cross sections of neonatal mouse hearts one day after sham operations (A and D, 1dps), non-transmural mild cryoinjury (B and E, 1dpmc) and transmural severe cryoinjury (C and F, 1dpsc). Dashed lines mark non-transmural injury and demarcate the injured area in transmural injury. RV, right ventricle; LV, left ventricle. b, border zone; s, injury area ; r, remote area. Scale bars: 500 μm (upper panel), and 50 μm (lower panel). n ≥ 4.

To determine if neonatal mouse hearts regenerate after cryoinjury, we performed AFOG staining at 21, 60 and 120 dpsc to visualize scar tissue. Compared to normal histology in sham operated hearts (Fig. 2A), AFOG staining revealed minimal scar formation at 21 days post non-transmural (mild) cryoinjury (dpmc) [Fig. 2B, 2.5±1.9 % Scar/Left Ventricle (LV) area, Fig. 2J] while extensive full thickness scarring, wall thinning, and dilated left ventricles were observed at 21 days post transmural (severe) cryoinjury (dpsc) (Fig. 2C, 13.7±2.3 % Scar/LV area, Fig. 2J). This result is very different from what was reported after ventricular resection (Porrello et al., 2011) and ligation of the left anterior descending (LAD) coronary artery (Porrello et al., 2013) in P1 neonatal mice. However, it has been shown that adult zebrafish hearts take a longer time to fully regenerate after cryoinjury (Chablais et al., 2011; Gonzalez-Rosa et al., 2011; Schnabel et al., 2011). Therefore, we examined the hearts at 60 (Fig. 2D-F) and 120 dpsc (Fig. 2G-I). Minimal scar tissue was found in the hearts following non-transmural cryoinjury (Fig. 2E and H, 1.0±1.2% and 0.6±0.3% Scar/LV area, respectively, Fig. 2J); however, extensive scarring, wall thinning and dilation persisted in hearts after transmural injury at either 60 or 120 dpsc (Fig. 2F and I, 7.3±1.2% and 6.6±1.9% Scar/LV area, respectively, Fig. 2J). Quantification of the scar area indicates the scar size did not change significantly; however, the Scar/LV ratio decreased significantly after transmural injury (13.7±2.3 vs. 7.3±1.2; P<0.05) from 21 to 60 dpsc due to the increase of heart size (Fig. 2J).

Fig. 2. Neonatal mouse hearts do not regenerate after transmural cryoinjury.

Fig. 2

Open in a new tab

AFOG staining of neonatal mouse hearts at 21 (A-C), 60 (D-F) and 120 (G-I) days after sham operation (A, D, G), non-transmural (B, E, H) and transmural (C, F, I) cryoinjuries. AFOG stains collagen blue and normal tissues brown. n ≥ 3. RV, right ventricle; LV, left ventricle; dps, days post sham operation; dpmc, days post mild non-transmural cryoinjury; dpsc, days post severe transmural cryoinjury. Scale bars: (A-I) 1000 μm. (J) Quantification of scar size as a ratio to LV area. * indicates significant differences of Scar size/LV size from 21 to 60 dpsc (P<0.05). (K-M) Echocardiography performed at 60 days after sham, non-transmural and transmural cryoinjury. Compared to normal end diastolic (red dashed line) and end systolic (green dashed line) dimensions in sham-operated non-transmurally injured hearts, hearts with transmural cryoinjury showed significant increase in both dimensions. n ≥ 4.

To assess the functional recovery of the neonatal mouse hearts after cryoinjury, echocardiography (Fig. 2K-M) was performed at 60 days after injury. Similar to sham operated hearts (Fig. 2K), a normal systolic function was observed at 60 dpmc after non-transmural injury (Fig. 2L); whereas in transmurally cryoinjured hearts, systolic dysfunction was apparent by a significant reduction in ejection fraction and fractional shortening, as well as a marked increase in end diastolic and end systolic diameters (Fig. 2M and Suppl Table 1). These results suggest that neonatal mouse hearts can fully recover and restore normal functions after non-transmural cryoinjury, but fail to regenerate after transmural cryoinjury.

Cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjury while epicardial activation and neovascularization occur

Despite the failure in regeneration after transmural cryoinjury, we examined if neonatal mouse hearts could still respond to the insult by increasing cardiomyocyte proliferation. To investigate cardiomyocyte mitosis, double staining of phospho-histone-3 (pHH3) and the cardiomyocyte marker myocyte enhancer factor-2 (MEF2) was performed at 7 days after surgery, a time point when cardiomyocyte proliferation peaks. We observed more pHH3 positive cardiomyocytes around the wound area in the transmural injury groups (Fig. 3C and F) than in non-transmural injury group (Fig. 3B and E); however, it was not significantly different compared to sham operated hearts (Fig. 3A and D) in either the border zone (Fig. 3G) or the remote area (Fig. 3H). The results of these experiments indicated that cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjuries; therefore it was not sufficient for the hearts to regenerate after transmural cryoinjury. Nonetheless, cardiomyocytes still proliferate after cryoinjuries at the normal rate of growing neonatal hearts. The postnatal growth maintained enough cardiac functions after non-transmural injury (Fig. 2L) and perhaps precluded the need to form a fibrotic scar.

Fig. 3. Cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjury.

Fig. 3

Open in a new tab

(A-H) Cardiomyocyte mitosis at 7 days post cryoinjuries. Representative immunostaining images of whole heart cross-sections at the mid-ventricle level 7 days after sham (A, D), non-transmural (B, E) and transmural (C, F) cryoinjuries. Dashed lines delineate the injury borders. (D-F): High magnification images of showing co-localization of pHH3 (green) with MEF2 (red) positive cardiomyocytes (arrowheads) or non-cardiomyocytes (arrows). (G and H) Quantification of pHH3 and MEF2 positive cardiomyocytes in the border zone and the remote area reveals no significant difference among different groups. n ≥ 3.

It has been shown that neovascularization occurs after ventricular resection and coronary artery ligation in neonatal mice (Porrello et al., 2011; Porrello et al., 2013). To determine if neovascularization occurs after cryoinjury, we performed PECAM staining at 21 days after injury. Quantification of blood vessel lumens per field revealed significantly increased neovascularization at the border zone after non-transmural (Fig. 4B) and transmural (Fig. 4C) cryoinjuries compared to sham operated (Fig. 4A) hearts (Figure 4D; 11.3±2.5 and 23.8±8.6, respectively, compared to 5.3±0.6; P<0.05).

Fig. 4. Neovascularization and epicardial activation and occur in neonatal mouse hearts after cryoinjury.

Fig. 4

Open in a new tab

(A-C) Neovascularization occurs after non-transmural and transmural cryoinjuries. PECAM immunostaining (red) of heart sections at 21 days after surgery with markedly increased number of vascular lumens around the wound area after non-transmural (B) and transmural (C) cryoinjuries compared to sham-operated (A) hearts. Scale bars: 50 μm. n ≥ 3. (D) Quantification of neovascularization as blood vessel (PECAM+) lumens/field. P<0.05. (E-G’) Epicardium is similarly activated after non-transmural and transmural cryoinjuries. (E-G): Epicardium is activated in neonatal mouse hearts after cryoinjuries. WT-1 immunostaining (red) of heart sections at 7 days after surgery showed marked thickening of the epicardium after non-transmural (F) and transmural (G) cryoinjuries. (E’-G’): Co-immunostaining of WT1 and PCNA was performed at 7 days after surgery. Arrows indicate cells co-positive for WT-1 and PCNA while arrowheads indicate WT-1 negative and PCNA positive cells. WT1: red; PCNA: green; DAPI: blue; dps, days post sham operation; dpmc, days post mild non-transmural cryoinjury; dpsc, days post severe transmural cryoinjury. Scale bar: 20 μm. n ≥ 3. (H) Quantification of PCNA positive WT1+ epicardial cells. (** P<0.01).

Epicardium is known to undergo an epithelial-to-mesenchymal transition (EMT) to give rise to perivascular cells in adult fish and mouse hearts (Kikuchi et al., 2011; Porrello et al., 2011; Zhou et al., 2011). A similar response was also observed in the epicardium of neonatal mouse hearts after amputation. We also observed significantly thickened epicardium by staining for the epicardial marker WT-1 at 7 days after non-transmural (Fig. 4F, F’ and H) and transmural (Fig. 4G, G’ and H) cryoinjuries. To determine whether the expansion of WT-1 positive epicardial cells is due to proliferation of the epicardium, double immunostaining of WT-1 and proliferating cell nuclear antigen (PCNA) was performed at 7 days after injury. Whereas sham-operated hearts showed a low number of PCNA-positive epicardial cells (3.09±1.8%) (Fig. 4E’, H), hearts with either non-transmural (Fig. 4F’ and H) and transmural (Fig. 4G’ and H) cryoinjuries demonstrated a marked increase in the number of double-stained nuclei (Fig. 4H; 24.19±12.5 and 26.15±11.5%, respectively; P<0.01). These proliferating epicardium-derived cells (EPDCs) are also present in the sub-epicardial space and myocardium suggesting that EMT of epicardial cells occurred after non-transmural (Fig. 4F and F’) and transmural (Fig. 4G and 4G’) cryoinjuries.

Plasminogen activator inhibitor 1 is highly elevated after transmural cryoinjury

We next examined protein samples collected 3 and 7 days after injuries for the presence of fibrotic or inflammatory factors. A number of fibrotic factors were highly enriched in the transmurally-injured hearts compared to the non-transmural injury group, including PAI-1 (plasminogen activator inhibitor 1). PAI-1 is a secreted serine protease inhibitor that blocks the conversion of plasminogen to plasmin and plays an essential role in fibrogenesis in various injury-induced fibrotic tissues (reviewed in Ghosh and Vaughan, 2012). A mouse PAI-1 total antigen assay detected 10.2 fold increase in PAI-1 at 3 days after transmural cryoinjury compared to sham and amputated hearts (Fig. 5A). The non-transmural cryoinjury group, however, only showed a 3.1 fold increase in PAI-1 compared to sham operation and amputation (Fig. 5A). By 7 days, PAI-1 levels decreased significantly in all groups except the hearts after transmural cryoinjury (Fig. 5A), suggesting a role of PAI-1 in fibrosis following transmural cryoinjury. To determine the location of PAI-1 expression, we performed immunostaining on heart sections collected at 3 days post non-transmural and transmural injuries. Interestingly, we detected strong expression of PAI-1 in the endocardium and myocardium closer to the lumen around the border zone and septum in the hearts after transmural injury but not after non-transmural injury (Fig. 5B). There was no expression detected in the epicardium (Fig. 5B), although the epicardium sustained direct damage in both non-transmural and transmural cryoinjuries. This differential expression pattern of PAI-1 might contribute to different levels of fibrosis after non-transmural and transmural cryoinjuries.

Fig. 5. PAI-1 expression is upregulated after transmural cryoinjury.

Fig. 5

Open in a new tab

(A) ELISA assay of mouse PAI-1 total antigen at 3 and 7 days post sham operation (dps), ventricular amputation (dpa), non-transmural cryoinjury (dpms) and transmural cryoinjury (dpsc). (B) Immunostaining of PAI-1. Transmural injury on the neonatal heart induces strong PAI-1 expression (red) in the myocardium in the border zone close to the lumen and endocardium of the left ventricle and septum. Non-transmural injury on the neonatal heart does not induce PAI-1 expression (3 dpmc). PAI-1, red, DAPI, blue, A, atrium; B, blood; BZ, border zone; EN, endocardium; EP, epicardium; I, injury area; LV, left ventricle; S, septum. Scale bar 100μm, 25μm (inset). n ≥ 3.

Discussion

The present study indicates that cryoinjury on 1-day old neonatal mouse hearts induces a differential regenerative response depending on the severities of the injuries. Hearts with non-transmural injuries appear to be able to heal with minimal or no scarring. By contrast, hearts undergoing transmural damage fail to regenerate, which results in a full thickness scar and cardiac dysfunction. We compared the differences between cardiomyocyte proliferation, neovascularization, and epicardial activation after non-transmural and transmural cryoinjuries to elucidate the mechanisms underlying the differential regenerative capacities (summarized in Fig. 6).

Fig. 6. Model of differential regenerative responses in neonatal hearts after non-transmural and transmural injuries.

Fig. 6

Open in a new tab

After non-transmural cryoinjury (marked in blue of half wall thickness) and transmural cryoinjury (marked in blue of full wall thickness), cardiomyocyte proliferation does not increase compared to sham operated hearts. However, cardiomyocytes are proliferating in the border zone or in the non-injured area as the neonatal hearts are growing. Significant epicardial activation and proliferation are observed after both non-transmural and transmural injuries. Neovascularization (PECAM+ blood vessels) also occurs after both non-transmural and transmural cryoinjuries. PAI-1 expression is observed in the endocardium and myocardium closer to the heart lumen after transmural but not in non-transmural cryoinjuries. It is likely that either damage to the endocardium or larger injury size triggers more fibrosis after transmural than non-transmural cryoinjuries

Adult mammalian hearts have very limited regenerative capacity due to a lack of proliferation in adult cardiomyocytes. Neonatal cardiomyocytes maintain a proliferative capacity that strongly correlates with the regenerative capacity of neonatal hearts after ventricular resection or ligation of the coronary artery (Porrello et al., 2011; Porrello et al., 2013). However, in contrast to the proliferative capacity of adult cardiomyocytes in zebrafish, cardiomyocytes in neonatal mice quickly lose their proliferative capacity within the first week of life. If cardiomyocyte proliferation is the major mechanism for heart regeneration, but neonatal mouse hearts do not increase cardiomyocyte proliferation after cryoinjury efficiently within the proliferative window, then we anticipate that they will not regenerate. We did not observe a statistically significant increase in cardiomyocyte proliferation in either the border or remote areas after transmural or non-transmural cryoinjuries. Indeed, a solid collagen scar was observed after transmural cryoinjury-induced myocardial infarction in neonatal hearts. Regardless of whether the hearts are undamaged or have undergone any type of injury, cardiomyocytes still undergo proliferation required for postnatal myocardial growth. The minimal scar formed following non-transmural injury reflects the adequate growth of myocardial tissue, which is sufficient to maintain cardiac functions and thus precludes the physiological need for scar formation.

Interestingly, a recent report has raised questions and discussion regarding the regenerative capacity of neonatal mouse hearts after ventricular resection (Andersen et al., 2014a; Andersen et al., 2014b; Sadek et al., 2014). Based on our results, the discrepancy might be partially explained by differences in the severities of the injuries created. We also observed that cryoinjury produces injury sizes that are more consistent than ventricular resection. In nature, it is well documented that different types and severities of injury trigger differential regenerative/repair responses. For instance, hydra use different mechanisms to regenerate head in response to decapitation or mid gastric section (Galliot and Chera, 2010). On this note, different regenerative responses have also been observed for zebrafish hearts after cryoinjuries. A collagen scar forms after a potentially more severe cryoinjury in adult zebrafish hearts at 21 dpc while cardiomyocyte proliferation peaks early at 3 dpc (Gonzalez-Rosa et al., 2011). Additional time (more than 2-3 months) was shown to be required to resolve scars for adult zebrafish hearts to regenerate after cryoinjury (Gonzalez-Rosa et al., 2011) compared to less severe cryoinjury models (Chablais et al., 2011; Gonzalez-Rosa et al., 2011; Schnabel et al., 2011). Our result suggests that perhaps cardiomyocyte proliferation alone is not sufficient for hearts to regenerate after severe/ transmural cryoinjury.

Jesty et al. reported that neonatal mouse hearts can partially regenerate after cryoinjury-induced myocardial infarction through c-kit positive progenitor cells (Jesty et al., 2012). Regression of fibrosis, the appearance of interspersed cardiomyocyte clusters at the fibrotic regions at 21 dpc and marked regeneration with minimum scar tissue formation were observed at 94 dpc (Jesty et al., 2012). In this study no information on the severity of the injury was given, making the comparison of their results with ours difficult. We observed that the hearts did not scar extensively and displayed minimal cell death after non-transmural cryoinjury. In contrast, we examined the hearts up to 120 days after transmural cryoinjury and still observed no signs of regeneration. The differences might be explained by a variation in responses due to differing severity of cryoinjuries. In accord with this, another group (Muller-Ehmsen et al., 2012) explored the role of infarct transmurality on the therapeutic effect of bone marrow mononuclear cells after myocardial infarction and showed scar size reduction and improved myocardial function only in patients with non-transmural myocardial infarcts but not transmural lesions.

We found differential PAI-1 expression in hearts 3 and 7 days after non-transmural and transmural cryoinjuries. Increased PAI-1 expression is induced in cardiomyocytes in a mouse myocardial infarction model induced by coronary artery ligation (Takeshita et al., 2004). Interestingly, we found that PAI-1 is highly expressed in the endocardium and myocardium closer to the lumen in the border area after transmural cryoinjury, which damages the endocardium in addition to myocardium and epicardium. Non-transmural and transmural cryoinjury directly damages epicardium but we did not detect PAI-1 expression in the epicardium. These data suggest that the fibrosis after transmural cryoinjury might be triggered by damage to endocardium. Elevated PAI-1 prevents proteolytic activity of the urokinase/tissue type plasminogen activator (uPA/tPA)/plasmin/MMP system and contributes to decreased collagen degradation and fibrogenesis (Ghosh and Vaughan, 2012). Although PAI-1 deficient mice often die due to cardiac wall rupture within 7 days after coronary artery ligation (Askari et al., 2003), they are protected from cardiac fibrosis (Takeshita et al., 2004). Paradoxically, lack of PAI-1 is associated with spontaneously developed cardiac fibrosis in aged mice (Moriwaki et al., 2004). In addition to resident tissue fibroblasts, fibroblasts have been reported to originate from endothelial cells through endothelial to mesenchymal transition (EndMT) processes in adult mouse hearts (Zeisberg and Kalluri, 2010; Zeisberg et al., 2007). PAI-1 has been shown to be involved in EMT and EndMT (Ghosh et al., 2010; Ghosh and Vaughan, 2012; Zhang et al., 2007). Consistent with these reports of PAI-1 functions in cardiac fibrosis, our data suggest that it is important to regulate temporal and spatial inhibition of PAI as an approach to decrease cardiac fibrosis.

An alternative model to explain our results is simply that transmural injury creates greater damage and triggers more activation and proliferation of resident fibroblasts than non-transmural injury and results in fibrosis in neonatal mouse hearts. Increased PAI-1 expression might reflect enhanced activation of signaling pathways such as TGFβ after transmural cryoinjury. Interestingly, a recent report suggests that neither epicardial EMT nor endothelial EndMT mediates pressure overload-induced cardiac fibrosis in adult mice heart (Moore-Morris et al., 2014). Instead, resident fibroblasts are activated and proliferate to contribute to fibrosis (Moore-Morris et al., 2014). Currently we cannot distinguish between whether resident fibroblasts are spared from cryoinjury or whether they have migrated in from the border zone.

Our study of regenerative and repair responses of neonatal mouse hearts to non-transmural and transmural cryoinjuries provided a unique opportunity to evaluate cardiac regeneration strategies. The fact that neonatal mouse hearts do not scar after non-transmural cryoinjury suggest that although cardiomyocyte proliferation is not increased, proliferation in the border and remote area is sufficient for the hearts to regenerate. In contrast, cardiomyocyte proliferation alone might not be sufficient after severe transmural cryoinjury and a combinatorial approach to prevent scar formation and increase cardiomyocyte proliferation might be needed. These results suggest that different cardiac regeneration strategies should be considered based on the nature of heart injuries.

Supplementary Material

Acknowledgements

We thank Drs. M. Chao, H. Sucov and T. Tuan for critical reading of the manuscript. We also thank Dr. E. Fernandez for imaging support, Drs. T. Hsiai, Y. DeClerck and V. Placencio for technical assistance, and Drs. H. Sadek, B. Kuhn, T. Tuan and M. Harrison for helpful discussion. This work was supported by the National Heart, Lung and Blood Institute Grant (R01HL096121 to C.-L. L.), The Saban Research Career Development Award (C.-L. L.) and California Institute of Regenerative Medicine (CIRM) postdoctoral fellowship (TG2-01168 to J. K. and L. G).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Andersen DC, Ganesalingam S, Jensen CH, Sheikh SP. Do Neonatal Mouse Hearts Regenerate following Heart Apex Resection? Stem cell reports. 2014a;2:406–413. doi: 10.1016/j.stemcr.2014.02.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersen DC, Jensen CH, Sheikh SP. Response to Sadek et al. and Kotlikoff et al. Stem cell reports. 2014b;3:3–4. doi: 10.1016/j.stemcr.2014.06.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Askari AT, Brennan ML, Zhou X, Drinko J, Morehead A, Thomas JD, Topol EJ, Hazen SL, Penn MS. Myeloperoxidase and plasminogen activator inhibitor 1 play a central role in ventricular remodeling after myocardial infarction. J Exp Med. 2003;197:615–624. doi: 10.1084/jem.20021426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chablais F, Veit J, Rainer G, Jazwinska A. The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev Biol. 2011;11:21. doi: 10.1186/1471-213X-11-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Choi WY, Poss KD. Cardiac regeneration. Current topics in developmental biology. 2012;100:319–344. doi: 10.1016/B978-0-12-387786-4.00010-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Galliot B, Chera S. The Hydra model: disclosing an apoptosis-driven generator of Wnt-based regeneration. Trends in cell biology. 2010;20:514–523. doi: 10.1016/j.tcb.2010.05.006. [DOI] [PubMed] [Google Scholar]
  7. Ghosh AK, Bradham WS, Gleaves LA, De Taeye B, Murphy SB, Covington JW, Vaughan DE. Genetic deficiency of plasminogen activator inhibitor-1 promotes cardiac fibrosis in aged mice: involvement of constitutive transforming growth factor-beta signaling and endothelial-to-mesenchymal transition. Circulation. 2010;122:1200–1209. doi: 10.1161/CIRCULATIONAHA.110.955245. [DOI] [PubMed] [Google Scholar]
  8. Ghosh AK, Vaughan DE. PAI-1 in tissue fibrosis. Journal of cellular physiology. 2012;227:493–507. doi: 10.1002/jcp.22783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gonzalez-Rosa JM, Martin V, Peralta M, Torres M, Mercader N. Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development (Cambridge, England) 2011;138:1663–1674. doi: 10.1242/dev.060897. [DOI] [PubMed] [Google Scholar]
  10. Jesty SA, Steffey MA, Lee FK, Breitbach M, Hesse M, Reining S, Lee JC, Doran RM, Nikitin AY, Fleischmann BK, Kotlikoff MI. c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:13380–13385. doi: 10.1073/pnas.1208114109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jopling C, Sleep E, Raya M, Marti M, Raya A, Belmonte JC. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature. 2010;464:606–609. doi: 10.1038/nature08899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kikuchi K, Gupta V, Wang J, Holdway JE, Wills AA, Fang Y, Poss KD. tcf21+ epicardial cells adopt non-myocardial fates during zebrafish heart development and regeneration. Development (Cambridge, England) 2011;138:2895–2902. doi: 10.1242/dev.067041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, Egnaczyk GF, Evans T, Macrae CA, Stainier DY, Poss KD. Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature. 2010;464:601–605. doi: 10.1038/nature08804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lien CL, Harrison MR, Tuan TL, Starnes VA. Heart repair and regeneration: Recent insights from zebrafish studies. Wound Repair Regen. 2012;20:638–646. doi: 10.1111/j.1524-475X.2012.00814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moore-Morris T, Guimaraes-Camboa N, Banerjee I, Zambon AC, Kisseleva T, Velayoudon A, Stallcup WB, Gu Y, Dalton ND, Cedenilla M, Gomez-Amaro R, Zhou B, Brenner DA, Peterson KL, Chen J, Evans SM. Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis. J Clin Invest. 2014;124:2921–2934. doi: 10.1172/JCI74783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moriwaki H, Stempien-Otero A, Kremen M, Cozen AE, Dichek DA. Overexpression of urokinase by macrophages or deficiency of plasminogen activator inhibitor type 1 causes cardiac fibrosis in mice. Circulation research. 2004;95:637–644. doi: 10.1161/01.RES.0000141427.61023.f4. [DOI] [PubMed] [Google Scholar]
  17. Muller-Ehmsen J, Tossios P, Schmidt M, Scheid C, Unal N, Bovenschulte H, Hackenbroch M, Krug B, Gossmann A, Mehlhorn U, Schwinger RH, Erdmann E. Transmurality of scar influences the effect of a hybrid-intervention with autologous bone marrow cell injection and aortocoronary bypass surgery (MNC/CABG) in patients after myocardial infarction. Int J Cardiol. 2012;156:303–308. doi: 10.1016/j.ijcard.2010.11.010. [DOI] [PubMed] [Google Scholar]
  18. Perazzolo Marra M, Lima JA, Iliceto S. MRI in acute myocardial infarction. Eur Heart J. 2011;32:284–293. doi: 10.1093/eurheartj/ehq409. [DOI] [PubMed] [Google Scholar]
  19. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN, Sadek HA. Transient regenerative potential of the neonatal mouse heart. Science (New York, N.Y. 2011;331:1078–1080. doi: 10.1126/science.1200708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Porrello ER, Mahmoud AI, Simpson E, Johnson BA, Grinsfelder D, Canseco D, Mammen PP, Rothermel BA, Olson EN, Sadek HA. Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:187–192. doi: 10.1073/pnas.1208863110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Poss KD. Getting to the heart of regeneration in zebrafish. Seminars in cell & developmental biology. 2007;18:36–45. doi: 10.1016/j.semcdb.2006.11.009. [DOI] [PubMed] [Google Scholar]
  22. Poss KD, Keating MT, Nechiporuk A. Tales of regeneration in zebrafish. Dev Dyn. 2003;226:202–210. doi: 10.1002/dvdy.10220. [DOI] [PubMed] [Google Scholar]
  23. Poss KD, Wilson LG, Keating MT. Heart regeneration in zebrafish. Science (New York, N.Y. 2002;298:2188–2190. doi: 10.1126/science.1077857. [DOI] [PubMed] [Google Scholar]
  24. Raya A, Koth CM, Buscher D, Kawakami Y, Itoh T, Raya RM, Sternik G, Tsai HJ, Rodriguez-Esteban C, Izpisua-Belmonte JC. Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proceedings of the National Academy of Sciences of the United States of America 100 Suppl. 2003;1:11889–11895. doi: 10.1073/pnas.1834204100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sadek HA, Martin JF, Takeuchi JK, Leor J, Nei Y, Giacca M, Lee RT. Multi-Investigator Letter on Reproducibility of Neonatal Heart Regeneration following Apical Resection. Stem cell reports. 2014;3:1. doi: 10.1016/j.stemcr.2014.06.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schnabel K, Wu CC, Kurth T, Weidinger G. Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS ONE. 2011;6:e18503. doi: 10.1371/journal.pone.0018503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Soonpaa MH, Kim KK, Pajak L, Franklin M, Field LJ. Cardiomyocyte DNA synthesis and binucleation during murine development. Am J Physiol. 1996;271:H2183–2189. doi: 10.1152/ajpheart.1996.271.5.H2183. [DOI] [PubMed] [Google Scholar]
  28. Takeshita K, Hayashi M, Iino S, Kondo T, Inden Y, Iwase M, Kojima T, Hirai M, Ito M, Loskutoff DJ, Saito H, Murohara T, Yamamoto K. Increased expression of plasminogen activator inhibitor-1 in cardiomyocytes contributes to cardiac fibrosis after myocardial infarction. The American journal of pathology. 2004;164:449–456. doi: 10.1016/S0002-9440(10)63135-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wu Z, Shu X, Fan B, Dong L, Pan C, Chen S. Differentiation of transmural and nontransmural infarction using speckle tracking imaging to assess endocardial and epicardial torsion after revascularization. Int J Cardiovasc Imaging. 2013;29:63–70. doi: 10.1007/s10554-012-0050-4. [DOI] [PubMed] [Google Scholar]
  30. Zeisberg EM, Kalluri R. Origins of cardiac fibroblasts. Circulation research. 2010;107:1304–1312. doi: 10.1161/CIRCRESAHA.110.231910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, Gustafsson E, Chandraker A, Yuan X, Pu WT, Roberts AB, Neilson EG, Sayegh MH, Izumo S, Kalluri R. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med. 2007;13:952–961. doi: 10.1038/nm1613. [DOI] [PubMed] [Google Scholar]
  32. Zhang G, Kernan KA, Collins SJ, Cai X, Lopez-Guisa JM, Degen JL, Shvil Y, Eddy AA. Plasmin(ogen) promotes renal interstitial fibrosis by promoting epithelial-to-mesenchymal transition: role of plasmin-activated signals. J Am Soc Nephrol. 2007;18:846–859. doi: 10.1681/ASN.2006080886. [DOI] [PubMed] [Google Scholar]
  33. Zhou B, Honor LB, He H, Ma Q, Oh JH, Butterfield C, Lin RZ, Melero-Martin JM, Dolmatova E, Duffy HS, Gise A, Zhou P, Hu YW, Wang G, Zhang B, Wang L, Hall JL, Moses MA, McGowan FX, Pu WT. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. J Clin Invest. 2011;121:1894–1904. doi: 10.1172/JCI45529. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS653522-supplement.psd (6MB, psd)

RESOURCES