Figure 5.
Flag-Rad adult murine cardiomyocytes treated with β-adrenergic agonists show modulated ICa,Lthat phenocopies untreated Flag-RadΔCT and reduced t-tubular expression. (A) Flag-Rad cardiomyocytes with 300 nM β-adrenergic receptor agonist ISO show an increase of ICa,L. Black traces (baseline). Cyan (Flag-Rad) and pink (Flag-RadΔCT) show sweeps after the addition of β-adrenergic receptor agonist. Ramp protocol (above current sweeps) repeated at 3 s inter-pulse interval. (B) Diary plot of fold change of mean peak ICa,L recorded during continuous recordings (sweeps) relative to initial peak current of sweep 1. (C) The fold change in ICa,L after application of ISO (after steady state was reached). ICa,L of Flag-Rad cardiomyocytes significantly increased 2.9-fold from a mean −2.8 (95% CI, −3.5, −2.2) to −8.2 (95% CI, −10.0, −6.4) pA/pF (linear mixed model, ISO treatment P = 0.024, F = 51.001, N = 3 mice, n = 13 cells). ICa,L of Flag-RadΔCT cardiomyocytes significantly changed 0.7-fold from mean −11.1 (95% CI, −13.1, −9.1) to −7.5 pA/pF (linear mixed model, ISO treatment P = 0.018, F = 6.491, N = 3 mice, n = 12 cells). (D) Confocal micrographs of adult cardiomyocytes treated with 1 µM β-adrenergic receptor agonist ISO from Flag-Rad or Flag-RadΔCT mice. Scale bar, 5 μm. Fluorescence intensity profiles of regions of interest from the representative images and their FFT power spectrums. (E) FFT data of untreated cardiomyocytes (data from Fig. 2) were analyzed together with ISO-treated cells to consider for both genotype and drug treatment as fixed factors with a linear mixed model (nesting cells into mice and accounting for cells untreated or treated originated from the same mouse). Data of untreated cells shown in Fig. 2 is repeated for clarity. The drug treatment main effect (P = 0.032, F = 5.147) was significant and, as reported in Fig. 2, the genotype main effect was significant (P = 0.002, F = 16.502); the interaction term was not (P = 0.255, F = 1.360). The FFT peak power median of Flag-Rad treated with β-adrenergic receptor agonist 15.4 (IQR: 5.8-42.9; N = 6 mice, n = 30 cells, 64 images) was significantly reduced 62% of untreated Flag-Rad 40.9 (IQR: 20.4–87.6) but still significantly 115 and 140% higher than untreated Flag-RadΔCT 7.1 (IQR: 2.6–20.6) and ISO-treated Flag-RadΔCT 6.4 (IQR: 4.1–9.5; N = 6 mice, 33 cells, 62 images). The P values of pairwise comparisons were corrected for multiple comparisons using Holm adjustment of estimated marginal means. 76% of Flag-Rad and 66% of Flag-Rad + ISO whereas 22 and 19% of Flag-RadΔCT ± ISO had a fundamental peak with power >10. Blinded researchers classified 79 and 60% of Flag-Rad ± ISO and 7 and 0% of Flag-RadΔCT ± ISO cells as having organized t-tubular expression in at least one region of the cell. Two to four technical replicate images’ FFT power were averaged per cell. The total sampling size was 12 mice, 143 cells, and 311 images.

Flag-Rad adult murine cardiomyocytes treated with β-adrenergic agonists show modulated I Ca,L that phenocopies untreated Flag-RadΔCT and reduced t-tubular expression. (A) Flag-Rad cardiomyocytes with 300 nM β-adrenergic receptor agonist ISO show an increase of ICa,L. Black traces (baseline). Cyan (Flag-Rad) and pink (Flag-RadΔCT) show sweeps after the addition of β-adrenergic receptor agonist. Ramp protocol (above current sweeps) repeated at 3 s inter-pulse interval. (B) Diary plot of fold change of mean peak ICa,L recorded during continuous recordings (sweeps) relative to initial peak current of sweep 1. (C) The fold change in ICa,L after application of ISO (after steady state was reached). ICa,L of Flag-Rad cardiomyocytes significantly increased 2.9-fold from a mean −2.8 (95% CI, −3.5, −2.2) to −8.2 (95% CI, −10.0, −6.4) pA/pF (linear mixed model, ISO treatment P = 0.024, F = 51.001, N = 3 mice, n = 13 cells). ICa,L of Flag-RadΔCT cardiomyocytes significantly changed 0.7-fold from mean −11.1 (95% CI, −13.1, −9.1) to −7.5 pA/pF (linear mixed model, ISO treatment P = 0.018, F = 6.491, N = 3 mice, n = 12 cells). (D) Confocal micrographs of adult cardiomyocytes treated with 1 µM β-adrenergic receptor agonist ISO from Flag-Rad or Flag-RadΔCT mice. Scale bar, 5 μm. Fluorescence intensity profiles of regions of interest from the representative images and their FFT power spectrums. (E) FFT data of untreated cardiomyocytes (data from Fig. 2) were analyzed together with ISO-treated cells to consider for both genotype and drug treatment as fixed factors with a linear mixed model (nesting cells into mice and accounting for cells untreated or treated originated from the same mouse). Data of untreated cells shown in Fig. 2 is repeated for clarity. The drug treatment main effect (P = 0.032, F = 5.147) was significant and, as reported in Fig. 2, the genotype main effect was significant (P = 0.002, F = 16.502); the interaction term was not (P = 0.255, F = 1.360). The FFT peak power median of Flag-Rad treated with β-adrenergic receptor agonist 15.4 (IQR: 5.8-42.9; N = 6 mice, n = 30 cells, 64 images) was significantly reduced 62% of untreated Flag-Rad 40.9 (IQR: 20.4–87.6) but still significantly 115 and 140% higher than untreated Flag-RadΔCT 7.1 (IQR: 2.6–20.6) and ISO-treated Flag-RadΔCT 6.4 (IQR: 4.1–9.5; N = 6 mice, 33 cells, 62 images). The P values of pairwise comparisons were corrected for multiple comparisons using Holm adjustment of estimated marginal means. 76% of Flag-Rad and 66% of Flag-Rad + ISO whereas 22 and 19% of Flag-RadΔCT ± ISO had a fundamental peak with power >10. Blinded researchers classified 79 and 60% of Flag-Rad ± ISO and 7 and 0% of Flag-RadΔCT ± ISO cells as having organized t-tubular expression in at least one region of the cell. Two to four technical replicate images’ FFT power were averaged per cell. The total sampling size was 12 mice, 143 cells, and 311 images.

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