# Molecular Quiescence and Cardiomyocyte Maturation

> **NIH NIH R01** · HARVARD UNIVERSITY · 2020 · $422,500

## Abstract

Stem cell approaches to treat heart failure will require production of mature human cardiomyocytes
(CMs) to improve systolic heart function. However, CMs derived from embryonic or induced pluripotent stem
cells (iPSCs) remain functionally immature using current approaches. When delivered to adult large animal
models, these immature CMs result in potentially life-threatening ventricular arrhythmias. Successful
translation of cell therapies for cardiovascular disease will thus likely require defining molecular pathways to
mature human stem cell-derived CMs. Cellular quiescence is a temporary non-proliferating state that can last
for the lifetime of the organism. Cellular quiescence can be a diverse state with varying depth of quiescence
and other molecular conditions that fit within the overall quiescence concept. Based on new preliminary data
shown here, we seek to investigate whether cellular quiescence is required for CM maturation from human
iPSCs. During development, CMs undergo a shift from a proliferative state as a fetus, to a more mature but
quiescent state after birth. This shift is accompanied by a change in energy metabolism, with fetal CMs
deriving energy primarily through glycolysis, and adult CMs deriving energy primarily through fatty acid
oxidation. The mechanistic target of rapamycin (mTOR) signaling pathway plays a key role in nutrient sensing
and growth, and regulation of mTOR affects the metabolic shift from glycolysis to lipid metabolism. Cell cycle
arrest with transient mTOR inhibition may lead to cellular quiescence. We hypothesize that regulation of the
mTOR pathway is a key driver in CM maturation via driving cells to quiescence. The following Aims will test
mechanistic hypotheses to understand how the mTOR signaling pathway and the E2F family of transcription
factors can enhance CM maturation and test whether mTOR pathway manipulation in 3D systems also
enhances CM maturation.
 Specific Aim 1: To define the role of 4E-BP1 activation in Torin1-induced maturation of iPSC-derived
CMs. We will modulate 4E-BP1 at different stages of CM maturation and determine whether this mechanism
explains Torin1-induced CM maturation. We will evaluate electrophysiological properties, contractility,
metabolism, and gene and protein expression to characterize CM phenotype and maturation.
 Specific Aim 2: To define how quiescence depth by E2F affects maturation of iPSC-derived
cardiomyocytes. We will perform cell cycle analysis on differentiating or maturing CMs with or without cell cycle
inhibitors. We will evaluate whether overexpression of E2F1/2/3a or deletion of E2F3a-8 prevents CM
maturation.
 Specific Aim 3: To explore the role of transient inhibition of mTOR in the maturation of iPSC-derived
CMs in a 3D environment. Because mTOR signaling can differ in 2D versus 3D environments, we seek to test
whether mTOR inhibition increases contractility and enhances excitation-contraction coupling in CMs in 3D.

## Key facts

- **NIH application ID:** 9947271
- **Project number:** 1R01HL151684-01
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** RICHARD T LEE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $422,500
- **Award type:** 1
- **Project period:** 2020-04-05 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9947271

## Citation

> US National Institutes of Health, RePORTER application 9947271, Molecular Quiescence and Cardiomyocyte Maturation (1R01HL151684-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9947271. Licensed CC0.

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