In the developing mouse heart, 23 of 50 tested genes altered cardiomyocyte maturation programs
In the developing mouse heart, 23 of 50 tested genes altered cardiomyocyte maturation programs
An article published in Nature Cardiovascular Research on September 7, 2026 describes PIP-seq, a method that links inactivation of a selected gene to changes in RNA in a cardiomyocyte, a heart muscle cell that contracts. In a pilot screen, 23 genes altered at least one maturation program; the authors described 21 of these as previously uncharacterized regulators.
After birth, cardiomyocytes grow, organize their sarcomeres (the protein structures that generate contraction), and change their energy metabolism and electrical properties. The authors tracked this transition on the day of birth and 7, 14, and 21 days later, profiling RNA from 67 thousand nuclei and mapping the positions of 402 thousand cells in heart tissue sections. The largest changes in gene activity occurred within the first 14 days. The effects of gene inactivation were therefore assessed on day 14.
To test the functions of candidate genes, the authors delivered a library of AAV9 viral vectors carrying genetic constructs into the hearts of newborn mice. Each construct carried two CRISPR guides, short RNAs that direct the Cas9 enzyme to the target gene. After tissue fixation, PIP-seq simultaneously identified the guide in each nucleus, evidence of editing at its target gene, and the RNA produced by the cell following the intervention. This linked inactivation of a specific gene to the cardiomyocyte's response within the heart in a single experiment.
The screen yielded 5 706 nuclei with an assigned guide. The genes Srf and Ppargc1a, which have established roles in maturation, served as positive controls and produced the expected disruptions to maturation programs. Inactivation of Rad21, a gene involved in DNA packaging, altered the activity of genes associated with contractile fiber assembly, electrical properties, and fatty acid metabolism.
The authors also inactivated Rreb1 in a separate experiment. By day 35 after birth, cardiomyocytes with inactivated Rreb1 were shorter, and their T-tubules (membrane channels that carry electrical signals into the cell) more often showed structural abnormalities. Mitochondria in these cells consumed less oxygen under maximal demand. Mice with inactivated Rreb1 had lower fractional shortening and ejection fraction, two ultrasound measures of cardiac contractile function. This separate validation linked the screening finding to changes in cell structure, cellular energy metabolism, and heart function.