# Cardiogenesis: Molecular Mechanisms

> **NIH NIH R01** · J. DAVID GLADSTONE INSTITUTES · 2023 · $803,250

## Abstract

PROJECT SUMMARY
Congenital heart disease is the result of abnormal development of discrete sub-types of cardiogenic cells during
early embryogenesis, and is the most common of all human birth defects. The recently developed ability to
analyze the transcriptional and epigenetic state of thousands of individual cells at a time makes it tractable to
discern the consequences of genetic alterations on small subsets of cells that could lead to congenital heart
defects (CHDs). Nearly 30% of all CHDs involve the developing valvuloseptal region, but the mechanisms
underlying valvuloseptal development are incompletely understood. Trisomy 21, the cause of Down Syndrome,
offers an opportunity to investigate the genetic basis of valvuloseptal defects, as 50% of Down Syndrome children
have CHD, and it is the most common known genetic cause of atrioventricular (AV) septal defects and other
defects of septation. However, which of the 241 genes on Ch21 are responsible for causing cardiac defects
when present in three copies remains unclear. A minimal critical region containing 148 duplicated syntenic genes
sufficient for recapitulating cardiac defects in mice has been established (Dp1Tyb). Using our single cell RNAseq
atlas of mouse cardiogenesis, we found only 66 of the 148 genes present in the Dp1Tyb region were expressed
during cardiac development, and we defined cardiac cell types with greatest transcriptional and epigenetic
changes in the Dp1Tyb mouse model. Furthermore, we differentiated a human trisomy 21 iPS cell line and its
isogenic disomic iPS control toward the cardiac lineage, and observed substantial single cell transcriptional and
epigenetic dysregulation in trisomic AV myocardial cells. We engineered the disomic human iPS cell line with a
CRISPR-based gene activation system, introduced guide RNAs to moderately activate the 66 candidate genes
on Ch 21, and used a machine learning approach to identify thirteen Ch21 genes that shifted the transcriptional
signature in the AV myocardium to be more similar to that observed in trisomy 21 cells. Among these were 5
transcription factors, including BACH1 and HMGN1. In parallel, scATACseq analysis of trisomy 21 AV myocardial
cells revealed a dramatic increase in motifs for the transcriptional repressor BACH1 among more closed
chromatin regions. Based on these data, we hypothesize that the cardiac defects of Trisomy 21 arise due to the
presence of three copies of one or more of the 13 genes identified in our pooled CRISPR screen, and that
increased dosage of one or more transcription factors, such as BACH1 or HMGN1, alters AV myocardium to
contribute to such defects. To test this, we propose to: 1) identify trisomy 21 gene(s) duplicated in Down
Syndrome that are necessary and sufficient for modulation of iPS-derived AV myocardium; 2) determine the
mechanism by which transcription factors from our screen affect transcriptional and epigenetic signatures in AV
myocardium; and 3) determine the in vivo dose depend...

## Key facts

- **NIH application ID:** 10734722
- **Project number:** 2R01HL057181-28
- **Recipient organization:** J. DAVID GLADSTONE INSTITUTES
- **Principal Investigator:** DEEPAK SRIVASTAVA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $803,250
- **Award type:** 2
- **Project period:** 1997-01-10 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10734722

## Citation

> US National Institutes of Health, RePORTER application 10734722, Cardiogenesis: Molecular Mechanisms (2R01HL057181-28). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10734722. Licensed CC0.

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