*3.3. Calcium Handling*

Differences in the calcium handling properties between hiPSC-CMs cultured on PET 5 textiles and coverslips were analyzed after 12 days of culture (the age of the cells was 33–39 days after initiation of differentiation). Cells exhibiting normal calcium transients were distinguished from those exhibiting arrhythmias and analyzed separately. The structure of the PET 5 did not hinder the Ca2+ imaging. There was no significant difference in the Ca2+ peak duration between CMs cultured on PET 5 (*n* = 160) or the control (*n* = 40) plates (582 ± 229 ms and 590 ± 202 ms, respectively; Table 4). However, there were statistically significant differences in rise and decay times, which were 112 ± 49 ms and 295 ± 131 for PET 5 samples and 90 ± 41 ms and 324 ± 96 ms for control samples (*p* < 0.05), respectively. This indicates that the release of calcium was slower while the uptake of calcium was faster for CMs grown on PET 5 compared to the control. Additionally, the amplitude of the peaks was significantly lower in CMs

grown on PET 5 compared to controls (0.048 ± 0.037 ΔF/F0 and 0.067 ± 0.030 ΔF/F0 for PET and control samples, respectively; *p* < 0.05), indicating that control cells released more calcium during contraction cycles. The beating frequency was significantly higher in CMs grown on PET 5 compared to controls (0.93 ± 0.52 Hz and 0.75 ± 0.34 Hz, respectively; *p* < 0.05).

**Table 4.** Functionality of the hiPS-CMs cultured on PET 5 analyzed using Ca2+ imaging. The structure of the PET 5 textile did not hinder the Ca2+ imaging and the calcium handling properties were assessable from the hiPSC-CMs cultured on PET 5 textiles. Culturing on the PET 5 textiles slightly altered the calcium handling properties of the hiPSC-CMs; however, no significant changes were observed.


The response of hiPS-CMs cultured on PET 5 to adrenaline and its effect on calcium handling properties was studied (*n* = 43) (Table 5 and Figure 4). There were statistically significant differences in the peak parameters between baseline and adrenaline measurements (*p* < 0.05). The peak duration decreased by 4.8% (648 ± 101 ms at baseline vs. 614 ± 87 after adrenaline). The peak amplitude decreased by 61.4% (0.0360 ± 0.0183 ΔF/F0 at baseline vs. 0.0310 ± 0.0139 ΔF/F0 after adrenaline). The rise time increased by 6.1% (115 ± 31 ms at baseline vs. 122 ± 34 ms after adrenaline). The decay time decreased by 7.6% (328 ± 73 ms at baseline vs. 303 ± 63 ms after adrenaline). The beating frequency increased by 21.2% (0.709 ± 0.254 Hz at baseline vs. 0.859 ± 0.242 Hz after adrenaline) as expected.

**Table 5.** Adrenaline significantly increased beating frequency and decreased peak duration in CMs grown on PET 5 textiles.


**Figure 4.** Adrenaline significantly increased beating frequency and decreased peak duration (**a**) when compared to the baseline (**b**) in hiPS-CMs grown on PET 5 textiles.

### *3.4. Expression of Cardiac-Specific Genes*

The expression of cardiac specific genes was analyzed using qRT-PCR. Data from PET 5 (*n* = 6) and control coverslip (*n* = 6) samples collected at day 11 (the age of the cells was 32–38 days after di fferentiation initiation) were calibrated with the day 1 (*n* = 4) samples. Two biological replicates were analyzed from each sample, and all the samples were run as triplicates. TATA-box binding protein (*TBP*), eukaryotic translation elongation factor 1 alpha 1 (*EEF1A1*), and glyceraldehyde-3-phosphate dehydrogenase (*GAPDH*) were used as endogenous control genes for normalization. Overall, high variation was observed between the experiments. The expression levels of the genes coding for the contractile proteins, such as Troponin T (*TNNT2*), myosin binding protein C (*MYBPC*), and cardiac alpha actinin (*ACTN2*) had an increasing trend after the 11 days of culturing on both the PET 5 and control hiPS-CMs. However, only expression of TNNT2 was significantly higher in the hiPSC-CMs cultured on PET 5 compared to controls on glass coverslips (*p* < 0.05, Figure 5). The expression levels of the genes coding for cardiac ion channels were similar for the CMs cultured on glass coverslips and PET 5 (Figure S2).

**Figure 5.** Expression levels of genes coding for the cardiac specific structural proteins: (**a**) α-actinin 2 (*ACTN2*), (**b**) myosin heavy chain 6 (*MYH6*), (**c**) myosin regulatory light chain 2 (*MYL2*), (**d**) slow skeletal and cardiac type troponin C1 (*TNNC1*), (**e**) myosin binding protein C, cardiac (*MYBPC3*), (**f**) myosin heavy chain 7 (*MYH7*), (**g**) myosin regulatory light chain 7 (*MYL7)*, (**h**) cardiac type troponin T2 (*TNNT2*), (**i**) titin (*TTN*), (**j**) tropomyosin (*TPM1*), and (**k**) myosin regulatory light chain 9 (*MYL9*). Only the expression level of (h) *TNNT2* was significantly higher in hiPSC-CMs cultured on gelatin coated PET 5 textiles compared to the control sample (*p* < 0.05), marked with \*.
