Identification and validation of hypoxia-responsive signature pathways in human cardiomyocytes

Abstract

The present study was designed to investigate the effect of hypoxia (1% O₂) for 24 h in human AC16 cells by analyzing alterations in the expression of cardiac markers and signature pathways using immunocytochemistry and next-generation sequencing respectively. The Gene set enrichment analysis and Cytoscape software were used for data analysis and visualization respectively. Sequencing data validation and functional characterization were done using flow cytometry, qRT-PCR, an antibody array, and immunoblotting. The result revealed that the expression levels of troponins decreased; however, the expression levels of VEGF-A and HIF-alpha increased under hypoxia compared with unexposed control. A total of 2120 genes corresponding to 457 gene sets were significantly altered, 153 of which were significantly upregulated and 304 of which were downregulated in hypoxic cardiomyocytes. The significantly altered gene sets corresponded to key cellular and molecular pathways, such as cardiac hypertrophy, transcription factors, microRNAs, mitochondrial abnormalities, RNA processing, cell cycle, and biological oxidation pathways. Thus, this analysis revealed multiple pathways associated with hypoxia which provides valuable insights into the molecular mechanisms underlying human cardiomyocytes, identifying potential targets for addressing cardiac illnesses induced by hypoxia.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04271-z.

Keywords: Cardiac markers; Cardiomyocytes; Cytoscape; GSEA; Hypoxia; Next-generation sequencing.