Regenerative Potential of Neonatal Porcine Hearts

Abstract

Background: Rodent hearts can regenerate myocardium lost to apical resection or myocardial infarction for up to 7 days after birth, but whether a similar window for myocardial regeneration also exists in large mammals is unknown.

Methods: Acute myocardial infarction (AMI) was surgically induced in neonatal pigs on postnatal days 1, 2, 3, 7, and 14 (ie, the P1, P2, P3, P7, and P14 groups, respectively). Cardiac systolic function was evaluated before AMI and at 30 days post-AMI via transthoracic echocardiography. Cardiomyocyte cell cycle activity was assessed via immunostaining for proliferation and mitosis markers, infarct size was evaluated histologically, and telomerase activity was measured by quantitative polymerase chain reaction.

Results: Systolic function at day 30 post-AMI was largely restored in P1 animals and partially restored in P2 animals, but significantly impaired when AMI was induced on postnatal day 3 or later. Hearts of P1 animals showed little evidence of scar formation or wall thinning on day 30 after AMI, with increased measures of cell-cycle activity seen 6 days after AMI (ie, postnatal day 7) compared with postnatal day 7 in noninfarcted hearts.

Conclusions: The neonatal porcine heart is capable of regeneration after AMI during the first 2 days of life. This phenomenon is associated with induction of cardiomyocyte proliferation and is lost when cardiomyocytes exit the cell cycle shortly after birth.

Keywords: cell cycle; heart; neonate; regeneration.

Figure 1.. Evaluation of cardiomyocyte regeneration in neonatal porcine hearts.

AMI was surgically induced in neonatal pigs on postnatal days 1, 2, 3, 7, and 14 (i.e., the P1, P2, P3, P7, and P14 groups, respectively). (A) B-Mode and M-Mode images were acquired via transthoracic echocardiography; representative images were displayed for animals in the P1 and P14 groups on day 30 after AMI induction. (B) Echocardiographic images of LV systolic thickening obtained before AMI induction and 30 days afterward were used to calculate the relative change in LV anterior wall systolic thickening/thining. *P<0.05 vs. P1, ^†P<0.05 vs. P2, ^‡P<0.05 vs. P3, ^§P<0.05 vs. P7. One-way ANOVA with the Holm-Sidak method. n=3–4 animals in each group. (C) Fractional shortening was evaluated before and 30 days after AMI induction. *P<0.05 vs. Pre-AMI (day 0) for the same experimental group; ^†P<0.05 vs. P1 at day 7 post-AMI; ^‡p<0.05 vs. P1 at day 30 post-AMI. Repeated measures two-way ANOVA. n=3–4 animals in each group. (D) LV heart sections from P1 and P14 animals were obtained on day 30 after AMI induction and stained with Sirius red and Fast-green to identify the scarred (red) and unscarred regions, respectively (left four panels) or observed macroscopically (right four panels) to evaluate wall thinning. LV anterior wall thickness (E) and Scar size (as indicated by % collagen deposition in F) were quantified. For (E): *P<0.05 vs. normal hearts of control animals; ^†p<0.05 vs. P1 AMI animals; ^‡P<0.05 vs. P2 AMI animals; for (F): *P<0.05 vs. P1 AMI animals; ^†p<0.05 vs. P2 AMI animals; One-way ANOVA with the Holm-Sidak method. n=3–4 animals in each group.

Figure 2.. Characterization of cell cycle activity and proliferation in neonatal porcine hearts.

(A-F) The expression of proliferation (Ki67), mitosis (PH3), and cytokinesis (Aurora B) markers was evaluated via immunofluorescent staining in paraformaldehyde-fixed heart sections; cardiomyocytes were visualized by staining for cTnT, and nuclei were labeled with anti-Nkx2.5 antibodies and counter-stained with DAPI. Cardiomyocytes that stained positively for each marker (Ki67, PH3, or Aurora B) were counted, normalized to the total number of cardiomyocytes (Nkx2.5-cTnT double-positive cells) and expressed as a percentage. A total of 3 sections of border zone myocardium (12 images) were counted. (A) Representative images are displayed for sections used to evaluate Ki67 expression on in the normal hearts of P1 and P14 animals (Bar = 20 µm) (B) Cardiomyocyte proliferation was quantified at the indicated day of sacrifice in normal hearts and in the AMI hearts of P1, P2, P3, P7, and P14 animals as the proportion of Nkx2.5-cTnT double-positive cells that also expressed Ki67. The data for days P30-P44 were pooled. *P<0.05 vs. P1 normal hearts; ^†P<0.05 vs. P2 normal hearts; ^‡P<0.05 vs. P3 normal hearts; ^§P<0.05 vs. P1 AMI hearts (harvested at P7); ^װP<0.05 vs. P2 AMI hearts (harvested at P8); **P<0.05 vs. P1 AMI hearts (harvested at P14). One-way ANOVA with the Holm-Sidak method. n=3–4 animals in each group. (C) Representative images are displayed for sections used to evaluate PH3 expression in the hearts of P1 and P14 normal animals (Bar = 20 µm). (D) Cardiomyocyte mitosis was quantified at the indicated day of sacrifice in normal hearts and in the AMI hearts of P1, P2, P3, P7, and P14 animals as the proportion of Nkx2.5-cTnT double-positive cells that also expressed PH3. The data for days P30-P44 were pooled. *P<0.05 vs. P1 normal hearts; ^†P<0.05 vs. P2 normal hearts; ^‡P<0.05 vs. P3 normal hearts; ^§P<0.05 vs. P1 AMI hearts (harvested at P7); ^װP<0.05 vs. P2 AMI hearts (harvested at P8); **P<0.05 vs. P1 AMI hearts (harvested at P14). One-way ANOVA with the Holm-Sidak method. n=3–4 animals in each group. (E) Representative images are displayed for sections used to evaluate Aurora B expression in the hearts of P1 and P14 normal animals (Bar = 20 µm). (F) Cardiomyocyte cytokinesis was quantified at the indicated day of sacrifice in normal hearts and in the AMI hearts of P1, P2, P3, P7, and P14 animals as the proportion of Nkx2.5-cTnT double-positive cells that also expressed PH3. The data for days P30-P44 were pooled. *P<0.05 vs. P1 normal hearts; ^†P<0.05 vs. P2 normal hearts; ^‡P<0.05 vs. P3 normal hearts; ^§P<0.05 vs. P1 AMI hearts (harvested at P7); ^װP<0.05 vs. P2 AMI hearts (harvested at P8); **P<0.05 vs. P1 AMI hearts (harvested at P14). One-way ANOVA with the Holm-Sidak method. n=3–4 animals in each group. (G) Telomerase activity was assessed in the myocardium of normal hearts on postnatal days 1, 2, 3, 7, 14, and 31 (P1, P2, P3, P7, P14, and P31, respectively). Control assessments were performed with (+) or without (−) telomerase which were included in the kit. *P<0.05 vs. P1; ^†P<0.05 vs. P2; ^‡P<0.05 vs. P3; ^§P<0.05 vs. P7. One-way ANOVA with the Holm-Sidak method. Experiments were repeated twice (n=3).