Abstract
Loss of cardiomyocyte proliferative capacity after birth is a major obstacle for therapeutic heart regeneration in adult mammals. We and others have recently shown the importance of hypoxic in utero environments for active foetal cardiomyocyte proliferation. Here, we report the unexpected expression of novex-3, the short splice variant of the giant sarcomeric protein connectin (titin), in the cardiomyocyte nucleus specifically during the hypoxic foetal stage in mice. This nuclear localisation appeared to be regulated by the N-terminal region of novex-3, which contains the nuclear localisation signal. Importantly, the nuclear expression of novex-3 in hypoxic foetal cardiomyocytes was repressed at the postnatal stage following the onset of breathing and the resulting elevation of oxygen tension, whereas the sarcomeric expression remained unchanged. Novex-3 knockdown in foetal cardiomyocytes repressed cell cycle-promoting genes and proliferation, whereas novex-3 overexpression enhanced proliferation. Mechanical analysis by atomic force microscopy and microneedle-based tensile tests demonstrated that novex-3 expression in hypoxic foetal cardiomyocytes contributes to the elasticity/compliance of the nucleus at interphase and facilitates proliferation, by promoting phosphorylation-induced disassembly of multimer structures of nuclear lamins. We propose that novex-3 has a previously unrecognised role in promoting cardiomyocyte proliferation specifically at the hypoxic foetal stage.
Highlights
While foetal cardiomyocytes in mammals actively proliferate in utero to form the primitive heart, they stop dividing soon after birth
In C. elegans embryos, two connectin homologues, TTN-1/Ce-titin (2.2 MDa) and kettin (500–550 KDa), were detected at the interphase nuclear envelope[7], and kettin was localised to mitotic spindles during mitosis
The molecular basis for the nuclear localisation of connectin was demonstrated by the discovery of a nuclear localisation signal (NLS) located at the N-terminus between the Z2 and Z repeats (200-PAKKTKT)[17]; this NLS is shared by all connectin isoforms
Summary
While foetal cardiomyocytes (fCMs) in mammals actively proliferate in utero to form the primitive heart, they stop dividing soon after birth. Connectin ( known as titin; Ttn) is the largest protein discovered to date (3~4 MDa) and spans from the Z-disk, I-band and A-band to the M-band region of the sarcomere of cardiac and skeletal muscle[4,5] It has 4 major isoforms, N2A, N2B, N2BA and foetal cardiac connectin, as well as numerous isoforms, including shorter fragments that are produced through complex alternative splicing pathways. In Drosophila, mutations in D-titin cause chromosome under-condensation, chromosome breakage, loss of diploidy and premature sister chromatid separation in neuroblasts, as well as defects in muscle development[11] These nuclear functions have all been reported to date in non-muscle cells
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