The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells

Abstract

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have been widely proposed as in vitro models of myocardial physiology and disease. A significant obstacle, however, is their immature phenotype. We hypothesised that Ca(2+) cycling of iPSC-CM is influenced by culture conditions and can be manipulated to obtain a more mature cellular behaviour. To test this hypothesis we seeded iPSC-CM onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds fabricated using photolithography, or onto unstructured PDMS membrane. After two weeks in culture, the structure and function of iPSC-CM were studied. PDMS microgrooved culture substrates brought about cellular alignment (p < 0.0001) and more organised sarcomere. The Ca(2+) cycling properties of iPSC-CM cultured on these substrates were significantly altered with a shorter time to peak amplitude (p = 0.0002 at 1 Hz), and more organised sarcoplasmic reticulum (SR) Ca(2+) release in response to caffeine (p < 0.0001), suggesting improved SR Ca(2+) cycling. These changes were not associated with modifications in gene expression. Whilst structured tissue culture may make iPSC-CM more representative of adult myocardium, further construct development and characterisation is required to optimise iPSC-CM as a model of adult myocardium.

Fig. 1

Schematic demonstrating the fabrication of microgrooved tissue culture substrates (not drawn to scale).

Fig. 2

Representative immunofluorescence of iPSC-CM cultured on unstructured PDMS (A) and microgrooved PDMS (B), Red – sarcomeric α-actin, Blue – DAPI, scale bar 20 μm. Quantification of cell alignment iPSC-CM on structured and unstructured constructs (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Time to peak of the Ca²⁺ transient (tP), 50% decay (t50), 90% decay (t90), and fluorescence amplitude (fp/f0) of iPSC-CM cultured on unstructured PDMS and microgrooved constructs field-stimulated at 1 Hz, 0.5 Hz, and beating spontaneously.

Fig. 4

Time to peak of the Ca²⁺ transient (tP), 50% decay (t50), 90% decay (t90), and fluorescence amplitude (fp/f0) of spontaneously beating and non-spontaneously beating iPSC-CM cultured on structured and control substrates field-stimulated at 0.5 Hz.

Fig. 5

Representative traces showing response to the application of 50 mm caffeine solution of isolated adult rat ventricular cells illustrating “mature caffeine response” (A), NRVM illustrating “immature caffeine response” (B), iPSC-CM cultured on structured PDMS (C), and iPSC-CM cultured on unstructured PDMS (D). Proportion of experiments that elicited an organised response to caffeine when cells were superfused in NT (E). Proportion of experiments that elicited an organised response to caffeine when cells were superfused in Na⁺ and Ca²⁺ free solution (F).

Fig. 6

Spontaneous APD measured using sharp microelectrodes (A), spontaneous beating rate (B), and APD corrected for spontaneous beating rate (C). Panels D and E suggest that Bazett's correction (curved line) does not adequately describe the relationship between APD and beating rate.

Fig. 7

(A) Immunohistochemistry of iPSC-CM cultured non-structured PDMS, Red – PLN, Green – Cx43, Blue – DAPI, scale bar 20 μm. (B) Red – Cav1.2 channel, Green – RyR, Blue – DAPI, scale bar 20 μm. (C) Immunohistochemistry of iPSC-CM cultured structured PDMS, Red – PLN, Green – Cx43, Blue – DAPI, scale bar 20 μm. (D) Red – Cav1.2 channel, Green – RyR, Blue – DAPI, scale bar 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Comparison of expression of genes encoding ultrastructural proteins in cardiomyocytes (α-MHC, β-MHC, MLC2V, cTNT, BIN1, CAV3) when normalised to GAPDH and expressed relative to adult heart tissue in iPSC-CM cultured on structured and control substrates, fibroblasts, adult heart and foetal heart tissue.

Fig. 9

Comparison of expression of genes encoding proteins important for Ca²⁺ cycling in cardiomyocytes (IP3R, RyR, SERCA2a, CASQ2, CALR, JPH2, PLN, Ca_v3.1, Ca_v1.2, NCX and TRDN) when normalised to GAPDH and expressed relative to adult heart tissue in iPSC-CM cultured on structured and control substrate, fibroblasts, adult heart and foetal heart tissue.