Natural Heart Regeneration in a Neonatal Rat Myocardial Infarction Model

Figures

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  Figure 1. Neonatal rat myocardial infarction model. (A) A left anterior thoracotomy is performed to expose the heart. United States dime for scale. (B) A 6–0 polypropylene suture is passed below the left anterior descending (LAD) coronary artery (arrow). (C) The LAD artery is permanently ligated.

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  Figure 2. Histological assessment of the neonatal rat heart at 1 day after sham surgery or induction of myocardial infarction (MI) on postnatal day 1. (A) Explanted heart from the sham group. (B) Hematoxylin and eosin (H&E)-stained section from the sham group (n = 3), with (C) inset showing healthy myocardium, as well as a limited area of minor tissue injury from needle trauma during sham surgery (upper right corner). (D) Explanted heart from the MI group, showing a clear region of non-perfused myocardium below the coronary artery ligation suture (dotted line). (E) H&E-stained section from the MI group (n = 3), with (F) inset showing widespread areas of acute inflammatory infiltrate, severe ischemic necrosis, and tissue edema within the affected myocardium.

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  Figure 3. Troponin immunohistochemistry assessment of the neonatal rat heart at 1 day, 1 week, or 3 weeks after sham surgery or induction of myocardial infarction (MI) on postnatal day 1 (P1). (A) Sham heart at 1 day after P1 surgery (n = 3), with (B) inset. (C) MI heart at 1 day after P1 surgery (n = 3), with (D) inset showing significant loss of troponin+ cardiomyocytes. (E) Quantified percentage of left ventricular area devoid of troponin signal at 1 day after P1 surgery. (F) Sham heart at 1 week after P1 surgery (n = 3), with (G) inset. (H) MI heart at 1 week after P1 surgery (n = 3), with (I) inset showing minimal loss of troponin+ cardiomyocytes. (J) Quantified percentage of left ventricular area devoid of troponin signal at 1 week after P1 surgery. (K) Sham heart at 3 weeks after P1 surgery (n = 3), with (L) inset. (M) MI heart at 3 weeks after P1 surgery (n = 3), with (N) inset showing minimal loss of troponin+ cardiomyocytes. (O) Quantified percentage of left ventricular area devoid of troponin signal at 3 weeks after P1 surgery. *** indicates p < 0.001. Horizontal lines indicate mean. Vertical lines indicate standard deviation.

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  Figure 4. Collagen scar assessment of rat hearts at 3 weeks after sham surgery or induction of myocardial infarction (MI), performed in neonates on postnatal day 1 (P1) or in adults. Masson’s trichrome staining revealed minimal collagen scar after (A) P1 sham (n = 3) and (B) P1 MI surgery (n = 4), assessed at 3 levels below the level of suture placement. Significant collagen scar was detected after (D) adult MI (n = 3) compared to (C) adult sham surgery (n = 3). Collagen scar area relative to left ventricular area was quantified for neonates (E) and adults (F). Picrosirius red staining revealed minimal collagen scar after (G) P1 sham (n = 3) and (H) P1 MI surgery (n = 4), assessed at 3 levels below the level of suture placement. Significant collagen scar was detected after (J) adult MI (n = 3) compared to (I) adult sham surgery (n = 3). Collagen scar area relative to left ventricular area was quantified for neonates (K) and adults (L). * indicates p < 0.05. ** indicates p < 0.01. *** indicates p < 0.001. Horizontal lines indicate mean. Vertical lines indicate standard deviation.

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  Figure 5. Assessment of cardiomyocyte proliferation using 5-ethynyl-2′-deoxyuridine (EdU) at 3 weeks after sham surgery (n = 4) or induction of myocardial infarction (MI, n = 5) on postnatal day 1 (P1). EdU+/troponin+ cardiomyocytes were detected within the peri-infarct region after (A) P1 sham and (B) P1 MI surgery. Inset (C) focuses on one EdU+/troponin+ cardiomyocyte after P1 MI, marked by the yellow arrow. (D) Significantly more EdU+/troponin+ cardiomyocytes were detected within each region of interest (ROI) for the P1 MI group compared to the P1 sham group in both the peri-infarct and remote regions. ** indicates p < 0.01. Horizontal lines indicate mean. Vertical lines indicate standard deviation.

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  Figure 6. Assessment of cardiomyocyte proliferation using Ki-67 at 1 week after sham surgery (n = 3) or induction of myocardial infarction (MI, n = 3) on postnatal day 1 (P1). Ki-67+/troponin+ cardiomyocytes were detected within the peri-infarct region after (A) P1 sham and (B) P1 MI surgery. Inset (C) focuses on one Ki-67+/troponin+ cardiomyocyte after P1 MI, marked by the yellow arrow. (D) Significantly more Ki-67+/troponin+ cardiomyocytes were detected within each region of interest (ROI) for the P1 MI group compared to the P1 sham group in the peri-infarct region. * indicates p < 0.05. Horizontal lines indicate mean. Vertical lines indicate standard deviation.

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  Figure 7. Assessment of cardiomyocyte proliferation using aurora B kinase (AurB) at 1 week after sham surgery (n = 4) or induction of myocardial infarction (MI, n = 2) on postnatal day 1 (P1). AurB+/troponin+ cardiomyocytes were detected within the peri-infarct region after (A) P1 sham and (B) P1 MI surgery. Inset (C) focuses on one AurB+/troponin+ cardiomyocyte after P1 MI, marked by the yellow arrow. (D) Significantly more AurB+/troponin+ cardiomyocytes were detected within each region of interest (ROI) for the P1 MI group compared to the P1 sham group in the peri-infarct region. * indicates p < 0.05. Horizontal lines indicate mean. Vertical lines indicate standard deviation.

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  Figure 8. Time-course echocardiography was performed for neonatal rats at 1 day, 1 week, 2 weeks, and 3 weeks after sham surgery (n = 7) or induction of myocardial infarction (MI, n = 9) on postnatal day 1. Parameters studied include (A) heart rate, (B) left ventricular wall thickness in diastole (LVWTd), (C) left ventricular internal diameter in diastole (LVIDd) and systole (LVIDs), (D) ejection fraction, (E) stroke volume, and (F) cardiac output. * indicates p < 0.05. ** indicates p < 0.01. *** indicates p < 0.001. Large dots indicate mean. Small dots represent individual data points. Vertical lines indicate 95% confidence interval.