Initiating Events in Direct Cardiomyocyte Reprogramming

Abstract

SUMMARY Direct reprogramming of fibroblasts into cardiomyocyte-like cells (iCM) holds great potential for heart regeneration and disease modeling and may lead to future therapeutic applications. Currently, application of this technology is limited by our lack of understanding of the molecular mechanisms that drive direct iCM reprogramming. Using a quantitative mass spectrometry-based proteomic approach, we identified the temporal global changes in protein abundance that occur during initial phases of iCM reprogramming. Collectively, our results show systematic and temporally distinct alterations in levels of specific functional classes of proteins during the initiating steps of reprogramming including extracellular matrix proteins, translation factors, and chromatin-binding proteins. We have constructed protein relational networks associated with the initial transition of a fibroblast into an iCM. These findings demonstrate the presence of an orchestrated series of temporal steps associated with dynamic changes in protein abundance in a defined group of protein pathways during the initiating events of direct reprogramming.

Unique Transcription Factor Functions Regulate Epigenetic and Transcriptional Dynamics During Cardiac Reprogramming

“…The initial stages of reprogramming (early iCMs) are represented by clusters 0 and 5, identified by activation of genes such as Nid2 and Tnnt2 and incomplete repression of fibroblast-associated genes such as Col8a1 and Ptgs2 (Figure 1E, Figure S1B). This signature was present within 48 hours of GMT transduction, in agreement with previous reports (Liu et al, 2017;Sauls et al, 2018). Cluster 2 represents late iCMs that express cardiomyocyte-related genes such as Nppa, Tnni3, Sln, and Myl7, and have downregulated the fibroblast gene program (Figure 1E, Figure S1B).…”

Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies

“…Progression along those stages strongly correlated with GMT expression but was inversely related to cell proliferation. A study by Sauls et al (12) complements those findings with population-based global proteomic data that revealed temporal changes in the expression of ECM, chromatin, translation, and nucleic acid binding proteins during reprogramming initiation.…”

Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

“…Digested samples were concentrated by speed vac to one half the original volume prior to labeling and adjusted to 20% acetonitrile (ACN). All three biological replicates were labeled concurrently with a 10-plex TMT kit (Thermo Fisher Scientific) as in (Sauls et al, 2018). The TMT reagents (0.8mg per channel) were dissolved in 42µL of anhydrous ACN and 14µL of this was added to each sample following the scheme in Figure 1A and allowed to react at RT for 1h.…”