Optimized protocol for direct cardiac reprogramming in mice using Ascl1 and Mef2c

Area of Science:

• Cardiovascular Research
• Stem Cell Biology
• Molecular Cardiology

Background:

• Direct cardiac reprogramming offers a potential strategy for cardiac repair by generating cardiomyocyte-like cells (iCMs) without intermediate progenitor stages.
• Fibroblast-to-cardiomyocyte conversion is a key focus in regenerative medicine for treating heart disease.

Purpose of the Study:

• To present a detailed protocol for direct cardiac reprogramming of fibroblasts into iCMs in mice.
• To demonstrate the efficiency and characteristics of iCMs generated using the Ascl1 and Mef2c factors.

Main Methods:

• Isolation of primary neonatal mouse cardiac fibroblasts.
• Preparation of retroviruses encoding the reprogramming factors Ascl1 and Mef2c.
• Execution of direct cardiac reprogramming protocol in vitro.

Main Results:

• Successful generation of cardiomyocyte-like cells (iCMs) from cardiac fibroblasts.
• Characterization of iCMs showing cardiomyocyte-like sarcomere structure.
• Confirmation of iCMs exhibiting cardiomyocyte-specific gene expression and functional calcium flux.

Conclusions:

• The presented protocol enables efficient direct cardiac reprogramming using Ascl1 and Mef2c.
• The generated iCMs possess key functional and structural properties of native cardiomyocytes.
• This method provides a valuable tool for studying cardiac reprogramming and potential therapeutic applications.