P340Sarcoplasmic reticulum calcium release in response to caffeine is more organised in cardiomyocytes differentiated from iPSC cultured on structured substrates suggesting a matured phenotype

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Abstract

Purpose

The use of Induced Pluripotent Stem Cells derived Cardiomyocytes (iPSC-CM) as in vitro models of myocardial disease is frequently discussed; however their immature phenotype which is not representative of adult myocardium is a significant obstacle. We hypothesise that culture scaffolds which force the alignment of iPSC-CM will improve important determinants of cellular function such as Ca2+ cycling. Consequently we aim to characterise global calcium cycling and sarcoplasmic reticulum (SR) function.

Methods

iPSC-CM (Cellular Dynamics, Wisconsin) were seeded onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds (MS) fabricated using photolithography, or on unstructured PDMS membrane (UM). After two weeks in culture iPSC-CM were loaded with Fluo-4 AM. Confocal microscopy was used in line scanning mode. SR Ca2+ release was elicited using 50mM caffeine in Normal Tyrode (NT) or Na+/Ca2+ free solution (00) to assess Ca2+ extrusion.

Results

iPSC-CM cultured on MS had a shorter time to peak Ca2+ transient amplitude (tP) when stimulated at 1Hz (Mean + /-SEM: MS: 145 + /-6ms, n=37; UM: 200 + /-10ms, n=58; p=0.0002, Mann Whitney Test) and time to 50% transient decay (t50) (Mean + /-SEM: MS: 222 + /-8ms, n=37; UM: 258 + /-8ms, n=58; p=0.0065) but no change in time to 90% transient decay (t90). At 0.5Hz there was a shorter tP (Mean + /-SEM: MS: 199 + /-11ms, n=37; UM: 242 + /-10ms, n=64; p=0.0073) but no changes in t50 or t90. Similarly while iPSC-CM were beating spontaneously, there was a reduced tP (Mean + /-SEM: MS: 223 + /-19ms, n=18; UM: 280 + /-11ms, n=37; p=0.00121). The spontaneous beating rate was unchanged between groups. Coordinated caffeine induced SR Ca2+ release was elicited in 77% (n=177) of the MS but none of the UM (n=110) in NT (p < 0.0001), and in 70% (n=136) of the MS and 21%(n=63) of the UM in 00 (p < 0.0001). The amplitude (F/F0) of caffeine induced transients, measured in experiments where there was no spontaneous beating was larger in the UM group (MS: 3.059 + /-1.655, n=46; UM: 3.989 + /-1.760, n=13; p=0.0473)

Conclusions

We conclude that structured culture changes Ca2+ cycling and response to caffeine in iPSC-CM, our provisional data suggests that this may be due to SR maturity, in particular ryanodine receptor function, however further characterisation is required.

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