The PIDDosome controls cardiomyocyte polyploidization during postnatal heart development

The mammalian adult heart is a post-mitotic organ characterized by mainly polyploid cardiomyocytes (CMs). The post-mitotic status of polyploid adult CMs poses a clear limit to heart regeneration. Thus, understanding how polyploid CMs acquire this status is relevant to heart regeneration therapies. Here, we aim to unveil whether the PIDDosome, a multi-protein complex known to limit scheduled and accidental polyploidization events by activating p53, implements a CM-specific genetic program that controls CM polyploidization during heart development. Flow cytometric DNA content analysis coupled with image-based 3D volumetric measurements of CM nuclei showed that PIDDosome knockout animals harbor significantly more polyploid CMs compared to WT animals. This increased CM ploidy levels do not interfere with both cardiac structure and functional output in steady-state and is a cell-autonomous phenotype. During heart development, ploidy analyses revealed that the PIDDosome starts to shape CM ploidy status at postnatal day 7 (P7), reaching a plateau of its action at P14. Moreover, PIDDosome activation in CM is dependent on PIDD1 localization to the extra mother centrioles via the distal appendage protein ANKRD26. Interestingly, in opposite to prior observations the PIDDosome limits CM polyploidization in a p53-independent manner that still involves p21, as confirmed in genetic and nuclear RNAseq analysis. Together these results identify a new molecular machinery that controls proliferation of polyploid CMs postnatally, adding a set of new players that control the tightly regulated program of the terminal CM differentiation occurring in the postnatal heart. Overall design: We performed bulk RNA-seq of both Casp2 deficient (XMLC2(+)Casp2(fl/fl)) and control (XMLC2(-)Casp2(fl/fl)) postnatal cardiomyocyte nuclei in mice. Cardiomyocytes were collected at postnatal days 1, 7 and 14. Four replicates were collected per postnatal day and genotype. To obtain sufficient RNA material, several hearts were pooled per replicate (P1: 15 hearts, P7: 6 hearts, P14: 2 hearts).