Perturb-seq uncovers pathological obstacles to direct cardiac reprogramming in vivo

Area of Science:

• Cardiovascular Biology
• Regenerative Medicine
• Molecular Biology

Background:

• Direct induction of cardiomyocytes from fibroblasts is a promising strategy for cardiac regeneration.
• In vivo transdifferentiation efficiency remains a significant challenge, particularly in pathological conditions like myocardial infarction.

Purpose of the Study:

• To systematically identify and rank barriers to in vivo cardiac reprogramming.
• To investigate the role of calreticulin (Calr) in inhibiting cardiomyocyte induction.
• To evaluate the therapeutic potential of Calr inhibition for cardiac repair.

Main Methods:

• Utilized a Perturb-seq platform to screen 140 potential barriers in a complex pathological environment.
• Analyzed shRNA distribution and single-cell RNA-seq trajectories to identify key inhibitors.
• Performed in vitro and in vivo experiments to assess the effects of Calr knockdown on cardiomyocyte induction and cardiac function post-myocardial infarction.

Main Results:

• Calreticulin (Calr) was identified as a major inhibitor of in vivo cardiac reprogramming.
• Calr knockdown significantly enhanced cardiomyocyte induction efficiency in vitro and in situ.
• Calr inhibition promoted synchronized calcium oscillations in induced cardiomyocytes and improved cardiac function while reducing fibrosis after myocardial infarction.
• Mechanistically, Calr knockdown activates calcium signaling, enhancing MEF2C activity to drive reprogramming.

Conclusions:

• Calreticulin is a critical regulator hindering in situ cardiomyocyte induction in pathological cardiac environments.
• Targeting Calr offers a promising therapeutic strategy for enhancing cardiac repair and regeneration after myocardial infarction.
• The study provides a framework and identifies key factors for improving in vivo cardiac reprogramming.