Myocardial infarction (MI) remains a leading cause of mortality and heart failure (HF) worldwide. Despite advances in diagnosis and clinical intervention, approximately 20–30% of MI patients develop HF within one year. This progression is primarily driven by cardiac tissue damage during ischemia/reperfusion (I/R), characterized by cardiomyocyte (CM) loss and progressive infarct expansion. Consequently, there is an urgent need for novel therapeutic strategies to prevent CM death during the I/R phase.
In this study, we utilized human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate the proton-sensing G protein-coupled receptor GPR68 as a potential cardioprotective target. Analysis of Bulk RNA sequencing data from mouse and porcine MI models revealed a significant GPR68 upregulation in the infarct and border zones of ischemic hearts. Consistently, increased expression was also observed in publicly available human datasets from patients with ischemic cardiomyopathy.
To investigate the functional role of GPR68, we generated GPR68 knockout (KO) iPSC lines using CRISPR/Cas9 gene editing and validated successful gene disruption by Sanger sequencing and qPCR. Human iPSC-CMs were differentiated from GPR68-KO and their isogenic wild-type (WT) control lines with high purity. Loss of GPR68 did not affect cardiac differentiation efficiency. Furthermore, maturation of iPSC-CMs was enhanced using a fatty acid-based medium combined with electrical stimulation. Under simulated I/R conditions, GPR68-KO iPSC-CMs exhibited significantly increased cell death compared to WT controls, as assessed by live/dead staining. Treatment with the GPR68 positive allosteric modulator Ogerin during the ischemic phase significantly reduced cell death in WT iPSC-CMs. In contrast, this protective effect was markedly attenuated in GPR68-KO cells, as confirmed by both live/dead staining and lactate dehydrogenase release assays.
To model oxidative stress during reperfusion, WT and KO iPSC-CMs, as well as primary mouse CMs, were exposed to hydrogen peroxide. Ogerin treatment significantly improved cell viability in WT iPSC-CMs and mouse CMs but failed to confer protection in GPR68-KO iPSC-CMs. Mechanistically, Ogerin stabilized the AMP/ATP ratio, an important indicator of energetic stress, by reducing AMP accumulation during ischemia. These findings suggest the involvement of the AMPK-mTOR signaling axis in mediating the observed cardioprotective effects.
Furthermore, bioluminescence resonance energy transfer (BRET) assays in transiently transfected HEK cells demonstrated that Ogerin enhanced pH-induced GPR68 coupling to Gq proteins, while recruitment of Gs proteins and β-arrestin3 was comparatively less pronounced.
Taken together, our findings demonstrate that pharmacological activation of GPR68 by Ogerin protects human iPSC-CMs from ischemia- and oxidative stress-induced cell death. This cardioprotective effect may be mediated through regulation of AMP levels and modulation of the AMPK-mTOR signaling, in addition to altered downstream signaling, particularly via enhanced Gq coupling. Future studies will focus on elucidating the mechanisms underlying Ogerin-induced changes in G protein signaling and determining how GPR68 activation influences AMP level and AMPK-mTOR pathway. Targeting GPR68 during I/R may therefore represent a promising therapeutic strategy to limit infarct size and reduce the incidence of post-infarction heart failure.
