Absence of regeneration in the MRL/MpJ mouse heart following infarction or cryoinjury

Abstract

Background: Myocardial infarcts in mammals heal by scar formation rather than formation of new muscle tissue. The MRL/MpJ [Murphy Roths large (MRL) derived by the Murphy group of the Jackson Laboratory (MpJ)] mouse, however, has been reported to exhibit minimal scarring and subsequent cardiac regeneration after cryoinjury of the right ventricle. Other groups have reported that permanent and temporary ligation of the coronary artery resulted in scarring without regeneration.

Methods: To clarify these contradictory results, we studied the temporal evolution of infarcts in MRL/MpJ and C57BL/6 control mice from 1 to 90 days post injury and the effects of intrathoracic cryoinjury to 28 days.

Results: After infarction, the conversion from necrotic myocardium to granulation tissue and then to scar proceeded identically in the two groups. Infarct DNA synthesis, measured by incorporation of a 5-bromo-2-deoxyuridine pulse, peaked at 4 days in both strains and did not differ between strains at any time point. Endothelial cell and total vascular density in the both the infarcted and noninfarcted cardiac tissue did not differ between groups at any time. Histological analysis of directly cryoinjured right and left ventricular myocardium showed indistinguishable wound healing in both strains, and final scar size was identical in each group.

Conclusions: These studies demonstrate that both myocardial infarcts and cryoinjuries in MRL/MpJ mice heal by typical scar formation rather than muscle regeneration, in a manner very similar to C57BL/6 controls. We conclude that the MRL mouse is not a model for myocardial regeneration.

Figure 1

Infarct repair in MRL/MpJ and C57BL/6 mouse hearts. Midlevel cross-sections of C57BL/6 and MRL/MpJ mouse hearts from sham operated mice and 1, 4, 14 and 90 days after myocardial infarction stained by hematoxylin and eosin (A) and sirius red and fast green (B) show a progression from necrosis at day 1 (C) to granulation tissue at day 4 (D) to scar and compensatory septal hypertrophy (days 14 and 90). Each feature is comparable between strains. (Bars = 1 mm and 50 μm) (E) Infarct size as a percent of the left ventricular circumference is similar in both strains at all times.

Figure 2

DNA synthesis determined by BrdU incorporation (brown nuclear staining) in the infarcts of (A) MRL/MpJ and (B) C57BL/6 at 4 days do not co-localize with sarcometic actin immunostaining (red). Arrows indicate BrdU positive nuclei (bar = 50 μm). (C) Total BrdU incorporation rates between 1 and 14 days is equivalent in both strains.

Figure 3

Endothelial density determined by CD31 staining (brown cytoplasmic staining) 7 days after myocardial infarction in the injury border region of the (A) C57BL/6 and (B) MRL/MpJ mouse strain (Bar = 50 μm). The insets show vessel structure (Inset Bar = 10 μm). Example endothelial cells are indicated by (+) and CD31-negative cells are marked with (−). CD31 staining in the infarcted region of C57BL/6 and MRL/MpJ mouse strains indicates similar vessel density between the strains. No significant differences exist in endothelial cell density between the two strains in (C) the uninjured myocardium and (F) the infarct scar to 90 days. Vessel density measurements (G) are also equivalent between the two strains.

Figure 4

Cardiac scar morphology by hematoxylin and eosin and picrosirius red/fast green staining in response to a 10 second direct cryoinjury is equivalent in the MRL/MpJ and C57BL/6 mouse 28 days (A) and 7 days (B) after injury. (Bar = 1mm) Extent of cryoinjury includes the right ventricle in each intervention, indicating lack of regeneration in that area. (C) Scar volume measured from serial sirius red stained sections is equivalent between each strain at both 1 and 4 weeks. (D) Each strain showed both sharp and jagged transitions at the infarct border zones owing to the muscle fiber orientation at that area of the tissue section. (Bar = 50 μm.) (E) No proliferating cardiomyocytes were found 7 days after cryoinjury as measured by immunostaining for BrdU (brown nuclei) and sarcomeric actin (red cytoplasm). (Bars = 50 and 20 μm.)