Alternative Splicing of Pericentrin Contributes to Cell Cycle Control in Cardiomyocytes

Abstract

Induction of cardiomyocyte proliferation is a promising option to regenerate the heart. Thus, it is important to elucidate mechanisms that contribute to the cell cycle arrest of mammalian cardiomyocytes. Here, we assessed the contribution of the pericentrin (Pcnt) S isoform to cell cycle arrest in postnatal cardiomyocytes. Immunofluorescence staining of Pcnt isoforms combined with SiRNA-mediated depletion indicates that Pcnt S preferentially localizes to the nuclear envelope, while the Pcnt B isoform is enriched at centrosomes. This is further supported by the localization of ectopically expressed FLAG-tagged Pcnt S and Pcnt B in postnatal cardiomyocytes. Analysis of centriole configuration upon Pcnt depletion revealed that Pcnt B but not Pcnt S is required for centriole cohesion. Importantly, ectopic expression of Pcnt S induced centriole splitting in a heterologous system, ARPE-19 cells, and was sufficient to impair DNA synthesis in C2C12 myoblasts. Moreover, Pcnt S depletion enhanced serum-induced cell cycle re-entry in postnatal cardiomyocytes. Analysis of mitosis, binucleation rate, and cell number suggests that Pcnt S depletion enhances serum-induced progression of postnatal cardiomyocytes through the cell cycle resulting in cell division. Collectively, our data indicate that alternative splicing of Pcnt contributes to the establishment of cardiomyocyte cell cycle arrest shortly after birth.

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy

“…28–30 The transition of PCNT during cardiomyocyte differentiation involves a key isoform switch (from PCNT-B to PCNT-S), which contributes to postnatal cell cycle arrest through the preferential expression of PCNT-S in the perinuclear centrosome. 31…”

“…The precise role of RTTN in the cardiac centrosome is unknown, and it is unclear why the RTTN mutations described here only cause cardiac defects, in contrast to other published RTTN mutations, which cause central nervous system defects associated with centrosome amplification, 33,34,43 although there was at least one known patient carrying a RTTN mutation with a restrictive cardiomyopathy in addition to a co-occurring central nervous system defect. 34 Given the highly dynamic RTTN localization noted using SASY and the importance of alternative splicing in PCNT transition during cardiac differentiation, 31 we hypothesize that RTTN may likewise undergo substantial alternative splicing, resulting in tissue-specific isoforms with distinct roles in centrosome dynamics that may affect the development of specific organs. Although much attention has been paid to the association of centrosome reduction with cell cycle arrest, our study demonstrates that tissue-specific and developmentally programmed centrosome reorganization is essential for proper cardiomyocyte structure and function.…”

“…[28][29][30] The transition of PCNT during cardiomyocyte differentiation involves a key isoform switch (from PCNT-B to PCNT-S), which contributes to postnatal cell cycle arrest through the preferential expression of PCNT-S in the perinuclear centrosome. 31 Rotatin (encoded by RTTN) is known to be a centrosomal protein in both Drosophila (ana3) and humans, and has been shown previously to be involved in regulation of centrosome structure. 32,33 Because of this fact and the observed cardiomyocyte maturation defect in the iDCM-CMs, we then wondered whether developmentally programed centrosomal changes associated with cardiomyocyte maturation [28][29][30] were disrupted in the iDCM-CMs.…”

A Specialized Centrosome-Proteasome Axis Mediates Proteostasis and Influences Cardiac Stress through Txlnb

“…A recent publication implicates impaired centrosome reduction to cause infantile dilated cardiomyopathy, through loss-of-function mutations in rotatin (Rttn) 9 . Beyond this, much of the consequences of centrosomal reduction in cardiomyocytes have been focused on the reorganization of the MTOC, or terminal exit of cell-cycle proliferation [6][7][8] . Our results suggest that centrosome-related genes/processes are disrupted during heart failure, likely indicating functions beyond centrosome reduction or microtubule disorganization.…”

“…In addition, the specific pathways and players that regulate centrosome reduction in cardiomyocytes are incompletely understood but involve AKAP6/9 dependent recruitment and nuclear anchoring of pericentriolar proteins, 5,6 loss of CEP135 proteins, and alternative PCNT splicing. 7 Beyond this, failure of cardiomyocytes to undergo centrosome reduction leads to immature cardiomyocyte phenotypes with disorganized cytoskeleton and impaired contractility. 8,9 This suggests that specialized centrosomes play critical roles in maintaining cardiac homeostasis, yet the overall mechanisms and links to heart failure remain understudied.…”