Alterations in sarcomere function modify the hyperplastic to hypertrophic transition phase of mammalian cardiomyocyte development
Benjamin R. Nixon, Alexandra F. Williams, Michael S. Glennon, Alejandro E. de Feria, Sara C. Sebag, H. Scott Baldwin, Jason R. Becker
Benjamin R. Nixon, Alexandra F. Williams, Michael S. Glennon, Alejandro E. de Feria, Sara C. Sebag, H. Scott Baldwin, Jason R. Becker
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Research Article Cardiology Development

Alterations in sarcomere function modify the hyperplastic to hypertrophic transition phase of mammalian cardiomyocyte development

Abstract

It remains unclear how perturbations in cardiomyocyte sarcomere function alter postnatal heart development. We utilized murine models that allowed manipulation of cardiac myosin-binding protein C (MYBPC3) expression at critical stages of cardiac ontogeny to study the response of the postnatal heart to disrupted sarcomere function. We discovered that the hyperplastic to hypertrophic transition phase of mammalian heart development was altered in mice lacking MYBPC3 and this was the critical period for subsequent development of cardiomyopathy. Specifically, MYBPC3-null hearts developed evidence of increased cardiomyocyte endoreplication, which was accompanied by enhanced expression of cell cycle stimulatory cyclins and increased phosphorylation of retinoblastoma protein. Interestingly, this response was self-limited at later developmental time points by an upregulation of the cyclin-dependent kinase inhibitor p21. These results provide valuable insights into how alterations in sarcomere protein function modify postnatal heart development and highlight the potential for targeting cell cycle regulatory pathways to counteract cardiomyopathic stimuli.

Authors

Benjamin R. Nixon, Alexandra F. Williams, Michael S. Glennon, Alejandro E. de Feria, Sara C. Sebag, H. Scott Baldwin, Jason R. Becker

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Figure 5

Dysregulated cell cycle regulation results in increased cardiomyocyte endoreplication in MYBPC3-null hearts.

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Dysregulated cell cycle regulation results in increased cardiomyocyte en...
(A) Isolated cardiomyocytes (CMs) from 25-day-old control (Ctl) and MYBPC3-null (Null) hearts displaying mononucleated (1), binucleated (2), and multinucleated (≥3) CMs. CMs were stained with sarcomeric α-actinin (green), with nuclei labeled with DAPI (blue). Scale bars: 50 μm. (B and C) Quantification of P25 CM nucleation (B) and cell size (C) in isolated CMs from Ctl (n = 4) and Null (n = 4) hearts. A minimum of 300 CMs per heart were analyzed. CM nucleation represented as percentage of total CMs counted. (D–H) Measurement of gene expression of cyclin D1 (Ccnd1) (D), cyclin D2 (Ccnd2) (E), cyclin E2 (Ccne2) (F), cyclin A2 (Ccna2) (G), and cyclin B1 (Ccnb1) (H) at P2, P7, and P25 from Ctl (n = 3–4) and Null (n = 3–4) hearts. The genes of interest were normalized to Rpl32 expression. Fold changes are shown relative to P2 Ctl gene expression. (I) Western blot of phospho-retinoblastoma (phos-Rb) and total Rb protein levels from P7 and P25 Ctl and Null hearts. (J) Relative quantification of phos-Rb in Ctl (n = 3) and Null (n = 3) P7 hearts normalized to GAPDH. (K) Gene expression for p21 (Cdkn1a) at P2, P7, P25 from Ctl (n = 3) and Null (n = 3) hearts. These data were normalized to Rpl32 expression and fold changes are shown relative to P2 Ctl gene expression. (L) Western blot of p21 protein levels in Ctl and Null P7 and P25 hearts (top). Relative quantification of p21 in Ctl (n = 3) and Null (n = 3) P7 and P25 hearts normalized to GAPDH. All results are shown as mean ± SEM. Statistical analysis performed using an unpaired, 2-tailed Student’s t test. N.S., not significant.