G-quadruplexes (G4s) are four-stranded DNA secondary structures, which are involved in a diverse range of biological processes including maintaining the stability of telomeres, mediating genome recombination, and regulating gene transcription. To explore the feasibility and potential of multiple-G4s-targeting strategy at the pathway level in the treatment of cardiovascular diseases, we have investigated the significance of promoter G4s in genes at the pathway level by bioinformatics, with particular focus on those pathways related to cardiac functions. Using human cTnI as a model system, we have carried out biophysical characterizations of G4s found in the promoters of this gene and its interaction with synthetic G4-binding ligands. In vitro studies of regulatory functions of a G4 (-80G4, centred at -80 bp) near transcription start site and the minisatellite G4 (MNSG4, between -528 bp and -319 bp) suggest that the -80G4, situated in a region with high location significance, may operate as a gatekeeper in regulating downstream luciferase expression in HEK293 cells; the MNSG4, on the other hand, acts as a fine-tuner in this regulation process. It is likely that both types of G4s orchestrate the gene expression of cardiac troponin I in the heart.