Abstract Body: Background: Dilated cardiomyopathy (DCM) is a common form of human cardiomyopathy with complex genetic causes. Genome wide association studies (GWAS) have identified numerous non-coding single nucleotide polymorphisms (SNPs) associated with DCM, but their regulatory roles remain largely unknown. We hypothesize that these SNPs influence DCM pathogenesis by altering the transcription factors (TFs) binding and target gene expression.
Methods: We developed a computational algorithm to prioritize functional SNPs (fSNPs) based on their genomic loci and regulatory potential in cardiac cells. Electrophoretic mobility shift assays (EMSA) and mass spectrometry (MS) were applied to reveal the TFs binding biasedly to key fSNP loci. Moreover, CRISPR-based gene editing was employed to generate inducible TF depletion and fSNP knock-in (KI) human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Functional analyses, including sarcomere integrity, calcium handling, and autophagic stress markers, were conducted under normal and pathological conditions with maturation medium (MM) and isoproterenol (ISO) treatment.
Results: Our analysis identified 127 top-ranked fSNPs in regulatory genomic regions associated with DCM. Among them, rs17617337 emerged as a key protective candidate, residing in the BAG3 intron—a gene essential for cardiac structural and functional homeostasis. EMSA and MS studies revealed that the nuclear paraspeckle protein NONO preferentially binds the protective T allele of rs17617337, potentially regulating BAG3 expression. In the iPSC-CM models, NONO depletion using the dTAG system reduced BAG3 expression and disrupted sarcomere structure and contractile function. Under pathological stress, the increased LncRNA NEAT1 expression enhanced paraspeckle formation, serving as a functional scaffold for NONO’s regulatory role. The rs17617337 T allele facilitated NONO binding, and together with increased paraspeckles, synergistically preserved BAG3 expression, improved sarcomere organization, and maintained cardiac function in SNP-KI cells under cardiac stress.
Conclusion: Our findings uncover a protective mechanism in which rs17617337 and NONO regulate BAG3 expression and mitigate DCM risk under cardiac stress. This study underscores the potential of iPSC and CRISPR platforms for investigating the regulatory roles of SNPs, advancing post-GWAS mechanistic research, and guiding the development of personalized therapies for complex diseases.