Sex Chromosome-Informed High-Throughput Screening of Anti-Hypertrophic Drugs Using hESC-Derived Cardiomyocytes

Cardiac hypertrophy is a major contributor to heart failure, with sex differences influencing disease progression and therapeutic responses. Utilizing XX (H9) and XY (HUES7) human embryonic stem cell (hESC) lines, we established a stepwise differentiation and replating protocol that generates high-purity cardiomyocytes (CMs), optimized for quantitative phenotyping and high-throughput screening (HTS) within a sex-informed context. XX-derived CMs exhibited earlier contractile onset, larger cytoplasmic areas, and greater calcium transients than their XY counterparts. Cultured in 384-well plates, these CMs consistently displayed basal hypertrophy-like morphology, enabling compound screening without external hypertrophic stimuli. We screened 3,520 compounds, including 3,113 FDA-approved drugs, using high-content imaging (HCI) to quantify atrial natriuretic peptide (ANP) expression and cellular morphology. Several candidates, including Agomelatine and Bepridil hydrochloride, showed sex-independent antihypertrophic efficacy. This scalable hESC-based platform provides a standardized, accessible alternative to patient-derived induced pluripotent stem cells (iPSCs), supporting reproducible, sex-aware drug discovery for cardiac hypertrophy and related pathologies.