MicroRNA205: A Key Regulator of Cardiomyocyte Transition from Proliferative to Hypertrophic Growth in the Neonatal Heart

Abstract

The mammalian myocardium grows rapidly during early development due to cardiomyocyte proliferation, which later transitions to cell hypertrophy to sustain the heart's postnatal growth. Although this cell transition in the postnatal heart is consistently preserved in mammalian biology, little is known about the regulatory mechanisms that link proliferation suppression with hypertrophy induction. We reasoned that the production of a micro-RNA(s) could serve as a key bridge to permit changes in gene expression that control the changed cell fate of postnatal cardiomyocytes. We used sequential expression analysis to identify miR205 as a micro-RNA that was uniquely expressed at the cessation of cardiomyocyte growth. Cardiomyocyte-specific miR205 deletion animals showed a 35% increase in heart mass by 3 months of age, with commensurate changes in cell cycle and Hippo pathway activity, confirming miR205's potential role in controlling cardiomyocyte proliferation. In contrast, overexpression of miR205 in newborn hearts had little effect on heart size or function, indicating a complex, probably redundant regulatory system. These findings highlight miR205's role in controlling the shift from cardiomyocyte proliferation to hypertrophic development in the postnatal period.

Keywords: Hippo pathway; hypertrophy; postnatal heart development.

Figure 1

Identification of miR205. (A) Total mRNA extracted for temporal miRNA expression analysis. (B) Heatmap analysis of temporal miRNA expression. (C) RT-qPCR was performed to confirm the miR205 trend observed in the microarray. Mean and SEM, n = 3 per time point. (D) In situ hybridization showing miR205 localization to the epicardium in 5 day old mouse hearts.

Figure 2

Conditional loss of miR205 in the neonatal heart results in an increased number of cardiomyocytes due to delayed cell cycle exit. (A) Significant reduction of miR205 expression (RT-qPCR) in 5-day-old miR205 null hearts. (B) Increased heart weight to body weight (HW:BW) of 14-day-old miR205 null hearts. (C) Western blot analysis and (D,E) immunohistochemical analysis of cell cycle markers reveal delayed cell cycle exit in miR205 null hearts. Mean and SEM, n = 3 per group per timepoint. (F) Persistent Hippo pathway activity (Western blot analysis) in miR205 null hearts. (G) Increased cardiomyocyte density of 14-day-old miR205 null hearts. Cellular boundaries were marked by WGA staining (green), and nuclei were labelled with DAPI (blue). Bar = 100 μm. Mean and SEM, n = 3 per group per timepoint. For all p-values, significance was tested using Student’s t-test.

Figure 3

Cardiac overexpression of miR205 leads to dysregulated Hippo pathway in neonatal hearts. (A) Generation of a doxycycline inducible cardiac-specific miR205 expression mouse model. Dotted line represents time of birth. Created by BioRender.com (accessed on 12 July 2023) Western blot analysis of (B) PTEN and cell cycle markers and (C) Hippo pathway.

Figure 4

Ectopic expression of miR205 in neonatal hearts has no impact on cardiomyocyte number. (A) Immunochemical analysis of cell cycle markers phospho-Histone 3 (pH3) and Ki67 in 14-day-old hearts. Bar = 300 μm. (B) Cell density of 14-day-old hearts. Cellular boundaries were marked by WGA staining (green), and nuclei were labelled with DAPI (blue). Bar = 100 μm. Mean and SEM, n = 3 per group per timepoint.

Figure 5

Proposed model of miR205′s relationship to the Hippo (hyperplastic growth) pathway and IP3K/Akt/mTOR (hypertrophic growth) pathway in neonatal cardiomyocytes. miR205, through its interaction with YAP and PTEN, facilitates the transition from Hippo pathway-mediated cardiomyocyte hyperplasia to the PI3K/Akt/mTOR regulated cardiomyocyte hypertrophy. Created with BioRender.com (accessed on 8 January 2024).