Temporal effects of hypoxia on beating parameters and signaling events in iPS-derived human cardiomyocytes

A. Williams¹, C. Tobin-O'Brien¹, K. E. O'Sullivan², L. MacDonagh¹, E. Breen¹, D. J. Buggy³, J. P. Hurley⁴, H. C. Gallagher¹. ¹School of Medicine, University College Dublin, Dublin, Ireland, ²Mater Misericordiae University Hospital, Dublin, Ireland, ³University Department of Anaesthesia, Mater Misericordiae University Hospital, Dublin, Ireland, ⁴Mater Private Hospital, Dublin, Ireland,

Introduction: Recently, iPS-derived human cardiomyocytes have been validated as an authentic model for the prediction of in vivo functional cardiotoxicity. To date, there have been no studies in these cells examining effects of hypoxia or ischemia, despite the high clinical burden associated with ischemic heart disease. Here, we used commercially available Cor.4U iPS-derived cardiomyocytes (Axiogenesis) to assess signalling pathways and functional parameters involved in the hypoxic response.

Method: Cardiomyocytes were grown until beating synchronously, followed by exposure to physiological normoxia (10% O₂) or hypoxia (1% O₂) for time-periods of up to 16h. Direct video microscopy, combined with image analysis software (Cellogy, Pulse), was used to determine effects on beating rate/strength. Effects of insulin-like growth factor 1 (100ng/μl), ruxolitinib (1μM and 100nM), AG490 (10μM), and vEGF (100ng/μl) on cardiomyocyte beating parameters were assessed. A slide-based array (Cell Signaling) was employed to elucidate intracellular signaling pathways modulated by hypoxia and a membrane-based array (Raybiotech) was used to measure effects of hypoxia on secreted cardiokines.

Results: Beating rate started to decline within 2h of hypoxia exposure and was reduced by 26.82±1.82% after 12h (p<0.01, n=3) and further decreased to 48.69±10.69 % of normal after 16h (p<0.01, n=3). Induction of hypoxia inducible factor alpha (HIF-α) within 1h (p<0.001, n=3) and increased LDH release after 8h (p<0.01, n=3) confirmed that cells were under hypoxic stress and that hypoxia was cardiotoxic. In normoxic conditions, no functional or gross toxicity was observed with the beating rate remaining at 90.3 ± 4.7 % (n=3) after 16h. Exposure to 100ng/ul insulin-like growth factor 1 (IGF-1), which activates JAK/STAT and other signalling pathways, protected cardiomyocytes against the effects of hypoxia on beating, inducing a sustained increase of 24.3 ± 4.3 % (p<0.01,n=3) above the beating rate observed under hypoxic conditions. Other JAK/STAT activators and inhibitors (vEGF and AG490) did not affect cardiomyocyte beating rate, however, a reduction in beating strength of up to 50% was observed following exposure to ruxolitinib (p<0.01, n=3) and AG490 (p<0.01,n=3) in both hypoxia and normoxia. Semi-quantification of arrays (n=3) indicated that PRAS40, Bad and Amp Kinase α were temporally upregulated by hypoxia. Hypoxia (16h) also induced the release of the cytokines GROα, RANTES and MCP-1.

Conclusions: These results highlight effects of hypoxia on cardiomyocyte functionality and associated signaling pathways involved in the hypoxic response. Further investigations will help generate new therapeutic strategies aimed at minimizing the damage to the cardiomyocyte induced by hypoxia and ischemia/reperfusion injury.