Heart regeneration in adult MRL mice

Abstract

The reaction of cardiac tissue to acute injury involves interacting cascades of cellular and molecular responses that encompass inflammation, hormonal signaling, extracellular matrix remodeling, and compensatory adaptation of myocytes. Myocardial regeneration is observed in amphibians, whereas scar formation characterizes cardiac ventricular wound healing in a variety of mammalian injury models. We have previously shown that the MRL mouse strain has an extraordinary capacity to heal surgical wounds, a complex trait that maps to at least seven genetic loci. Here, we extend these studies to cardiac wounds and demonstrate that a severe transmural, cryogenically induced infarction of the right ventricle heals extensively within 60 days, with the restoration of normal myocardium and function. Scarring is markedly reduced in MRL mice compared with C57BL/6 mice, consistent with both the reduced hydroxyproline levels seen after injury and an elevated cardiomyocyte mitotic index of 10-20% for the MRL compared with 1-3% for the C57BL/6. The myocardial response to injury observed in these mice resembles the regenerative process seen in amphibians.

Figure 1

Schematic representation of the location (A) and the trans-mural extent (B) of a typical cryogenically induced lesion on the right ventricular surface of the hearts of C57BL/6 and of MRL mice. The dashed line in A approximates the transverse sectional plane as shown in B and in Fig. 2. The boxed area in B shows the area of myocardium analyzed for Fig. 3.

Figure 2

Light micrographs of trichrome-stained, transversely sectioned cryoinjured hearts on days 5, 15, and 60 with the C57BL/6 (Left) and the MRL (Right) mice. (A) C57BL/6, 5 days postinjury, 40×. (B) MRL, 5 days postinjury, 40×. (C) C57BL/6, 15 days postinjury, 4× (Inset, 40×). (D) MRL, 15 days postinjury, 4× (Inset, 40×). (E) C57BL/6, 60 days postinjury, 4×. (F) MRL, 60 days post injury, 4×. The extent of injury in MRL and C57BL/6 hearts is indicated by arrows in C and D. By day 60, the C57BL/6 wound is entirely scar tissue. Conversely, the injured MRL myocardium seems normal with only a small amount of scar tissue on the epicardial surface. The right ventricular site of injury is indicated as RV in E and F.

Figure 3

BrdUrd labeling of myonuclei. The presence of BrdUrd as visualized by the deposition of 3,3′-diaminobenzidine precipitate within ventricular myocyte nuclei is shown. BrdUrd-positive nuclei can be seen in cardiomyocytes in the interventricular septal region (A, and blocked area at higher magnification as seen in C). The section seen in A can been seen upon double labeling with DAPI (B). The arrows in A and B show the same nucleus as a point of reference. BrdUrd-positive nuclei can also be seen in cardiomyocytes in the right ventricle of a representative MRL heart on day 60 near the wound site (arrows, D), but not in the left ventricle of the same MRL heart (E), where none of the labeled nuclei are myonuclear (arrow shows BrdUrd-labeled interstitial nuclei). Labeled nuclei in the C57BL/6 right ventricle of the heart at day 60 can be found in the connective tissue of the wound scar (arrow) and in the vasculature, but not in the myocardium to any apparent degree (F).

Figure 4

Labeling and indices of BrdUrd-positive myonuclei in individual hearts of C57BL/6 mice, n = 6 (Left) and MRL mice, n = 7 (Right), 60 days after cryoinjury. Labeling indices were obtained from double-labeled tissue in which nuclei were first stained with anti-BrdUrd antibody and then costained with DAPI. A total of 1,000 DAPI-positive nuclei were counted from each heart sample, and the mitotic index was calculated from the number of BrdUrd-labeled nuclei divided by the number of DAPI-positive nuclei × 100. Histograms were assembled from counts made from the area of the initial cryoinjury.

Figure 5

Confocal microscopic colocalization of BrdUrd and of sarcomeric α-actinin within the nuclei of MRL vetricular cardiomyocytes 15 days after injury. (A) The immunocytochemical detection sarcomeric α-actinin in cardiomyocytes by using a Cy5-conjugated secondary antibody whose fluorescent infrared emission was visualized in a false-red color. (B) The immunocytochemical detection of BrdUrd, by using an FITC-conjugated secondary antibody; (C) the combined images of A and B can be seen. The arrows indicate the BrdUrd-labeled nuclei.

Figure 6

Echocardiography of injured hearts. (A) An M-mode image from an MRL mouse 3 days after injury showing the ventricular chamber (RV, Upper; LV, Lower) dimensions throughout several cardiac cycles. The time points identified by the arrows allow the measurement of the end diastolic dimension (EDD, Left) and end systolic dimension (ESD, Right). The RV EDD is indicated by the upper left white bar. (B) The time course of right ventricular end diastolic diameter at base line, early after injury, 1 and 3 months after injury is presented. Individual lines show the response of individual MRL mice at each time point. The mean measurements are indicated (●), and the error bars are the standard deviation. The MRL mouse right ventricles dilate early in response to injury and recover by shrinking to their original size by 3 months.

Figure 7

Collagen expression in injured and healing myocardium. (A) Hydroxyproline content of ventricular myocardium as mg hydroxyproline/mg tissue protein can be seen on days 0, 15, and 60 after injury (n = 4–5 hearts per time point) for C57BL/6 (B0, B15, and B60; Left) and MRL (M0, M15, and M60; Right) mice. (B) Col1A1 RNA content in ventricular myocardium was determined by quantitative RT-PCR by using a light cycler on days 0, 5, and 15 after injury (n = 2 hearts/time point) for C57BL/6 (B0, B5, and B15) and for MRL (M0, M5, and M15) mice. Standard deviations are all within 10% of the means.