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Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5
Elisa Belian1, Michela Noseda1, Marta S Abreu Paiva1
1British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Imperial College London, London W14 0NN, United Kingdom.
Unlabelled:
Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized.
In Summary:
(1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.
Insights
Adding Myocardin (Myocd) to cardiac transcription factors (GMT) significantly enhances cardiac stem cell (CSC) reprogramming, inducing both cardiac genes and proteins for a more complete differentiation.
Area of Science:
- Cardiovascular Biology
- Stem Cell Biology
- Molecular Cardiology
Background:
- Adult cardiac stem cells (CSCs) possess endogenous cardiogenic transcription factors but lack Myocardin (Myocd).
- Myocd is a crucial co-activator for cardiac gene expression, and its absence may limit CSC reprogramming.
- Investigating Myocd's role is key to understanding and improving cardiac regeneration strategies.
Purpose of the Study:
- To investigate the role of Myocardin (Myocd) in the reprogramming of adult mouse Sca1+ side population cardiac stem cells (CSCs).
- To determine if supplementing the GATA4, MEF2C, and TBX5 (GMT) triad with Myocd improves CSC differentiation into cardiomyocytes.
- To assess the effectiveness of Myocd in combination with GMT for achieving a complete cardiac differentiation program.
Main Methods:
- Utilized doxycycline-inducible lentiviral vectors to express exogenous transcription factors (GMT and Myocd) in CSCs.
- Employed high-throughput quantitative RT-PCR to analyze the expression of 29 cardiac lineage markers.
- Assessed protein expression of key cardiac markers (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains) post-induction.
Main Results:
- The GATA4, MEF2C, and TBX5 (GMT) combination induced over half of the analyzed cardiac transcripts but failed to produce detectable cardiac proteins.
- Supplementation with Myocardin (Myocd) alongside GMT significantly enhanced reprogramming, crucially triggering the expression of all tested cardiac proteins.
- The combination of Myocd and TBX5 (Myocd+TBX) proved most effective, with homogeneous expression leading to up-regulation of 93% of cardiac transcripts and visualization of all five tested proteins.
Conclusions:
- GMT alone can induce cardiac gene expression in CSCs, but Myocd is essential for achieving complete cardiac protein synthesis and differentiation.
- Myocd plays a pivotal role in driving CSCs toward a cardiac muscle fate, validating this combinatorial reprogramming strategy.
- Homogeneous transduction with Myocd and TBX5 facilitates the identification and validation of CSCs undergoing complete cardiac differentiation.
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