(G-250) The Effects of Osteopontin (OPN) on Human iPSC-derived Cardiomyocyte Function

Cardiovascular disease including myocardial infarction (MI) is the leading cause of death in the Western world. Following MI, myocardial inflammation plays a crucial role in the pathogenesis of MI and macrophages are among the key cells activated during the initial phases of the host response regulating the healing process. Our previous study demonstrated that macrophages play an important role in cardiomyocyte function, yet the underlying mechanism remained unclear [1]. Thus, the focus of this study was to investigate one of the macrophage-derived factors, OPN on cardiomyocyte function. OPN is constitutively expressed and secreted by macrophages and while OPN level remains low in the healthy adult heart, it is significantly upregulated upon injury. Since OPN has been shown to be involved in inflammatory and reparative response post-MI [2, 3], it is hypothesized that OPN is one of the potential key factors affecting cardiac cell function.

The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were first co-cultured with polarized macrophage subsets for 24 hours. The co-culture took place in a well with two distinct populations, physically separated by a distance of 500 μm This setup allowed for interaction between the cell types through paracrine signaling and soluble factors without direct cell-to-cell contact. The function of CM was then examined using a Triple Transient Measurement system which measures action potential, calcium, and contraction signals simultaneously [4]. The expression of cardiac specific markers involved in sarcomere assembly (TNNT2, TNNI3, MYL2, MYH6, MYH7, ACTN2), in cardiac action potential (SCN5A, CACNA1C), and in calcium-handling (NCX1, SERCA2A, CASQ2) were examined by RT-PCR. To further determine whether OPN leads to the same functional changes in CM, recombinant mammalian derived human OPN (500 ng/ml, R&D Systems) was added to the CM culture for 24 hours and the expression of the above mentioned cardiac specific genes was examined.

Similar to our previous study using mouse embryonic stem cell derived CM [1], co-culture with different subtypes of macrophages resulted in functional changes in hiPSC-CM. Specifically, CM co-cultured with anti-inflammatory macrophages exhibited a prolonged contraction time (RT90), action potential duration at 90% repolarization (APD90), and calcium slow decay time at 90% repolarization (CSD RT90) compared to monocultured CM. Moreover, co-culture of hiPSC-CM with anti-inflammatory macrophages led to a significant upregulation of calcium voltage-gated channel subunit alpha1C (CACNA1C) and sodium voltage-gated channel alpha subunit (SCN5A) genes, which are known regulators of ion influx. Notably, these results correlated with the OPN secretion as OPN was significantly higher in the co-culture of anti-inflammatory macrophages and CM compared to all the other groups. This correlation between OPN and gene expression profile implies that OPN may be a crucial mediator in the communication between macrophages and CM. To further investigate the role of OPN, exogenous OPN was added into hiPSC-CM culture. Interestingly, a significant upregulation of CACNA1C and SCN5A genes was observed, mirroring the pattern observed in the co-culture experiments. This further supports that macrophage derived factors play a significant role in CM function especially in electrophysiological and Ca²⁺ handling properties of CM.  

 Our study sheds new light on the interactions between macrophages and CM during post-MI and highlights the potential role of OPN as a critical macrophage-derived factor in modulating cardiomyocyte function. Identifying such key macrophage-derived factors involved in the cardiac healing process is critical for further understanding the pathogenesis of post-MI and uncovering new therapeutic targets.

1.         Hitscherich, P.G., et al., The effects of macrophages on cardiomyocyte calcium‐handling function using in vitro culture models. Physiological reports, 2019. 7(13): p. e14137.

2.         Zhao, X., et al., Impairment of myocardial angiogenic response in the absence of osteopontin. Microcirculation, 2007. 14(3): p. 233-240.

3.         Shirakawa, K. and M. Sano, Osteopontin in cardiovascular diseases. Biomolecules, 2021. 11(7): p. 1047.

4.         van Meer, B.J., et al., Simultaneous measurement of excitation-contraction coupling parameters identifies mechanisms underlying contractile responses of hiPSC-derived cardiomyocytes. Nat Commun, 2019. 10(1): p. 4325.