Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

Recent interest in understanding cardiomyocyte cell-cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow abscission of daughter cardiomyocytes. Here we identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phosphoforms of α-adducin in-vitro and in-vivo identified Thr445/Thr480 phosphorylation of a short isoform of α adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. These results highlight an important mechanism for coordination of cytoskeletal morphological changes during cardiomyocyte mitosis.

Capturing Neonatal Cardiomyocyte – Endothelial interactions on-a-Chip

The neonatal heart has been the focus of numerous investigations due to its inherent regenerative potential. However, the interactions between neonatal cardiomyocytes (CMs) and endothelial cells (ECs) have been difficult to model and study due to the lack of an appropriate device. Here, we developed a method to culture primary neonatal CMs and ECs in a microchip and characterise their behavioural properties over a 14-day period. By implementing cell migration analyses coupled with immunostaining and confocal microscopy, we were able to identify and quantify sub-populations of migratory and non-migratory ECs. In CM–EC co-cultures, migrating ECs were found to move in higher numbers and longer distances compared to migrating CMs. In the presence of CMs, non-migrating ECs established connexin gap junctions and formed CM–EC cell aggregates, which were likely a priming event for endothelial organoid formation. This microfluidic device also enabled us to visualise the temporal sequence organoid formation and phenomena such as collective cell migration, CM–EC trans-differentiation and synchronisation of CM beating. This microchip based culture system has potential applications for tissue engineering and drug discovery.

Abstract 333: Cardiomyocyte Death and Fibrotic Scarring in the Infarcted Neonatal Mouse Heart

As one the leading causes of death in the United States, myocardial infarction (MI) occurs every 40 seconds, causing severe public health burden. Following MI, the loss of healthy cardiomyocytes leads to decreased contractility and eventually heart failure. Mature mammalian cardiomyocytes have a low turnover rate at only 0.5-2% per year, insufficient for repopulating damaged myocardium after MI. However, a contradictory discovery was made showing that the neonatal mammalian heart is regenerative, although this reparative ability is lost within days after birth. A great amount of effort is needed to understand the mechanisms underlying neonatal cardiomyocyte regeneration. In the current project, we attempt to profile different types of cell death in regenerating and non-regenerating mouse models following MI, in order to gain insights into a favorable type of cardiomyocyte death during regeneration. We induced MI in postnatal day 1 (P1, regenerative), and postnatal day 7 (P7, non-regenerative) mouse hearts by left anterior descending artery occlusion (LAD-O). The progressive scar formation was assessed using Masson’s Trichrome staining at multiple timepoints up to 14 days after MI. At each time point, we profile three major types of regulated cell death, apoptosis, necroptosis, and ferroptosis, using immunofluorescence staining. We also used AC16, a human cardiomyocyte cell line, to investigate the role of cell density in the regulation of ferroptosis. We found that the scar formation was most dynamic between 2 and 3 days after MI and that the course of scar formation varied greatly between P1 and P7 hearts. Immunofluorescence of different cell death markers reveal differentially progressed cell death between P1 and P7 hearts after MI. Our results indicate a different pattern of cardiomyocyte death in the regenerative P1 heart compared to the non-regenerative P7 heart, that could be more favorable for myocardial regeneration.