Modeling ischemic heart disease: only matured human iPS-derived cardiomyocytes are sensitive to ischemic injury
Authors: Peters M C , Van Adrichem I , Doevendans P A M , Chamuleau S A J , Neef K , Sluijter J P G 
Published: October 2021

Abstract

Background
Ischemic heart disease remains the leading cause of death worldwide due to irreversible loss of cardiomyocytes (CMs) during ischemic injury. To test potential factors that can stimulate cardiac repair, murine and porcine animal models or in vitro assays are often used. However, clinical translation remains ineffective as the CMs in the human heart differ signifantly from those in animal hearts1. Human induced pluripotent stem cell (hiPSC) derived CMs have been considered a very promising tool but their immature nature remains a roadblock for human cardiac disease modeling. A media to improve maturation of hiPSC-CMs has recently been developed2.

Purpose
To test whether we can model human myocardial ischemia, we assess the effect of metabolic maturation on ischemia susceptibility of hiPSC-CMs.

Methods
hiPSCs were differentiated into CMs and matured either in RPMI/B27 media or in maturation media (MM)2. Ischemia was induced by exposure to 5% or 1% oxygen for 4 to 24 hours in one of 5 types of media (DMEM/10%KOSR; RPMI/B27; RPMI pur/B27; RPMI pur/lactate; starvation MM) to assess the effect of media nutrient content on ischemia susceptibility.

Results
CMs matured in RPMI/B27 media did not show a significant increase in cell death independent of media glucose or serum content during ischemia (ratio dead/live RPMI pur/lactate: 0,02±0,004 [24 hours 21% O2]; 0,11±0,04 [24 hours 1% O2], p&gt;0,05). CMs matured in MM did show an increase in cell death with increasing duration of ischemia for both 5% and 1% O2 (0,58±0,06 [0h]; 1,1±0,09 [24h 1%]). Furthermore, only metabolically matured CMs showed increased TUNEL+ CMs (7,5±1,3% [control]; 42,2±11,0%, p&lt;0,001 [24 hours 1% O2]) and decreased viability measured by flow cytometry (90,8±0,71% [control]; 76,3±2,2% [24 hours 1% O2], p&lt;0,01) compared to immature CMs (84,5±2,0% [control]; 73,7±3,4% [24 hours 1% O2], p&gt;0,05). Although cardiomyocytes matured in MM showed stable α-actinin expression after 24 hours 1% oxygen (92,7±3,35 [24 hour 21%]; 93,8±1,7 [24 hour 1%]), both incubation in 5% and 1% oxygen led to decreased myofibrillar organization as seen by α-actinin and cardiac troponin T (cTnT) immunoreactivity. Assessment of metabolites in the media showed increased lactate production after 24 hours incubation in 1% oxygen but not in 5% oxygen (from 11,8±0,31mM [24 hour 21%], 11,7±0,3mM [24 hour 5%] to 19,9±1,4 mM [24 hour 1%], p&lt;0,001) indicating that even though 5% oxygen levels were sufficient to induce ischemic damage, further lowering oxygen saturation does have an additional effect on the cardiomyocyte metabolic response to ischemia.

Conclusion
Taken together, we have shown improvement of metabolic maturation of hiPSC-CMs increases the susceptibility to ischemia and therefore their use to mimic ischemic heart disease. It would be of interest to further investigate the underlying metabolic mechanisms of cardiac ischemia.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): MMCP is supported by a Netherlands CardioVascular ResearchInitiative (CVON) grant