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. 2018 Feb 13;22(7):1913-1922.
doi: 10.1016/j.celrep.2018.01.047.

Initiating Events in Direct Cardiomyocyte Reprogramming

Kimberly Sauls  1 , Todd M Greco  2 , Li Wang  3 , Meng Zou  1 , Michelle Villasmil  4 , Li Qian  3 , Ileana M Cristea  2 , Frank L Conlon  5
Affiliations

Initiating Events in Direct Cardiomyocyte Reprogramming

Kimberly Sauls et al. Cell Rep. .

Abstract

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.

Keywords: cardiac; direct reprogramming; heart; iCM; induced cardiomyocytes; quantitative mass spectrometry.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Induced Cardiomyocyte Proteomic Analysis
(A) First, retroviruses packaged with either a pMXs-DsRed (Red) construct or a pMXs-MGT (green) construct, were generated. Freshly generated virus was used to transduce MEFs (
Figure 2
Figure 2. Extracellular Matrix Protein Networks Are Predominantly Upregulated at Both 48 and 72 hr Post-reprogramming
(A) Gene set enrichment analysis of TMT protein ratios found that proteins annotated to the PANTHER protein class extracellular matrix protein (n = 70) were on average significantly upregulated at both 48 (blue circles, p < 8.43E-6) and 72 hr (orange circles, p < 6.3E-4) compared to the distribution of all proteins (orange and blue lines). (B and C) The functional connectivity and relative abundance of the proteins annotated to extracellular matrix protein networks was visualized by STRING analysis. Node color indicates average relative increase (yellow) or decrease (blue) in protein abundance at 48 and 72 hr post-transduction. Black node color indicates the protein was not detected at that time point. Extracellular matrix (ECM) protein abundance was similarly increased at both time points, although on average the magnitude was greater at 48 hr.
Figure 3
Figure 3. Translation Factor Networks Are Strongly Downregulated at 48 hr Post-reprogramming but Then Rebound at 72 hr
(A) Gene set enrichment analysis of TMT protein ratios found proteins annotated to the PANTHER protein translation factors (n = 46) were on average significantly downregulated at 48 hr (blue circles, p < 1.79E-08) but not at 72 hr (orange circles) compared to the distribution of all proteins (orange and blue lines). (B and C) The functional connectivity and relative abundance of the proteins annotated to translational protein networks was visualized by STRING analysis. Node color indicates average relative increase (yellow) or decrease (blue) in protein abundance at 48 and 72 hr post-transduction. Individual protein abundances were strongly decreased at 48 hr, but less so at 72 hr when all proteins showed <50% change.
Figure 4
Figure 4. Chromatin-Associated Protein Networks Are Predominantly Downregulated at 72 hr Post-reprogramming
(A)Gene set enrichment analysis of TMT protein ratios found proteins annotated to the PANTHER protein class chromatin/chromatin-binding proteins (n = 38) were on average significantly downregulated at 72 hr (orange circles, p < 4.5E-10) but not at 48 hr (blue circles) compared to the distribution of all proteins (orange and blue lines). (B and C) The functional connectivity and relative abundance of the proteins annotated to chromatin protein networks was visualized by STRING analysis. Node color indicates average relative increase (yellow) or decrease (blue) in protein abundance at 48 and 72 hr post-transduction. Black node color indicates the protein was not detected at that time point.
Figure 5
Figure 5. Temporal Changes in ECM, Translation, and Chromatin Binding Protein Networks
(A–D) Western blot analysis was performed on nuclear fractions of MEFs transduced with either MGT or DsRed and were probed with antibodies against representative proteins in each class. (A) Nid2 an extracellular matrix protein and (B) Agrin at 72 hr post-infection (C) SMARCC1 and (D) HMGB1, chromatin-associated proteins, at 48 post-transduction. Densitometry quantification reveals changes in levels of proteins examined are consistent with the TMT proteomic results. These changes were significant for both Nid2, Agrin, and SMARCC1 (p < 0.05). (E) Schematic overview of infection and summary of the changes in relative protein abundances at 48 and 72 hr post-transduction. Data are represented as mean ± SEM.
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