No functional TRPA1 in cardiomyocytes

Abstract

Aim: There is mounting evidence that TRPA1 has a role in cardiac physiology and pathophysiology. We aim to clarify the site of TRPA1 expression in the heart and in particular whether the channel is expressed in cardiomyocytes.

Methods: Due to the high calcium conductance of TRPA1, and marginal calcium changes being detectable, microfluorimetry in primary mouse cardiomyocytes, and in the cardiomyocyte cell lines H9c2 and HL-1, was applied. TRPA1 mRNA in mouse and human hearts, primary cardiomyocytes, and the cardiac cell lines were quantified. Dorsal root ganglia served as control for both methods.

Results: In addition to AITC, the more potent and specific TRPA1 agonists JT010 and PF-4840154 failed to elicit a TRPA1-mediated response in native and electrically paced primary cardiomyocytes, and the cardiomyocyte cell lines H9c2 and HL-1. There were only marginal levels of TRPA1 mRNA in cardiomyocytes and cardiac cell lines, also in conditions of cell differentiation or inflammation, which might occur in pathophysiological conditions. Similarly, TRPV1 agonist capsaicin did not activate primary mouse cardiomyocytes, did not alter electrically paced activity in these, and did not activate H9c2 cells or alter spontaneous activity of HL-1 cells. Human pluripotent stem cells differentiated to cardiomyocytes had no relevant TRPA1 mRNA levels. Also in human post-mortem heart samples, TRPA1 mRNA levels were substantially lower compared with the respective dorsal root ganglion.

Conclusion: The results do not question a role of TRPA1 in the heart but exclude a direct effect in cardiomyocytes.

Keywords: TRPA1; TRPV1; cardiomyocytes; heart; human; mouse.

FIGURE 1

TRPA1 or TRPV1 activation elicits no relevant calcium transients in primary murine cardiomyocytes. A‐C, Individual intracellular calcium level traces of freshly isolated primary mouse cardiomyocytes, measured by fura‐2 microfluorimetry. Traces are visualized in high transparency, therefore the overlay of multiple traces results in darker grey values; the median is visualized in black. Horizontal bars indicate the application periods of TRPA1 agonists PF‐4840154 2 µmol L^‐1 and AITC 100 µmol L^‐1, TRPV1 agonist capsaicin 1 µmol L^‐1, and KCl 60 mmol L^‐1. Right panels: Violin plots of the area under the curve (AUC) of the agonist and KCl application periods. A thick horizontal line indicates the median, while the grey areas represent the second and third quartile. D, Responses of mouse DRG neurons to the TRP channel agonists used in panel A‐C and to KCl. All panels contain cells of 5 dishes of at least three animals with experiments performed on independent days. The number of cells is indicated in the upper left corner of each panel

FIGURE 2

Electrically induced calcium transients in primary mouse cardiomyocytes are not affected by TRPA1 and TRPV1 modulation. A‐E, Intracellular calcium in freshly isolated cardiomyocytes. Electrical stimulation with rectangular pulses of 10 V and 1‐ms pulse duration (visualized by the inset) were delivered at a rate of 0.3 Hz as indicated by vertical lines. Traces show mean (black) ± SEM (grey). Horizontal bars indicate the application periods of control extracellular solution (same solution, but with switch to a separate perfusion channel), PF‐4840154 2 µmol L^‐1, AITC 100 µmol L^‐1, capsaicin 1 µmol L^‐1, A‐967079 10 µmol L^‐1, and KCl 60 mmol L^‐1, which was used to verify cell viability. The AUCs of the five stimuli indicated by the black triangles were compared between the five conditions after statistical adjustment for the following nuisance parameters: (i) the intracellular calcium concentration at the beginning of the quadratic polynomial as described in Figure S3, (ii) the AUC‐values of stimuli 3‐7 and (iii) the AUC during the application of KCl. Cells were isolated from two mice, cell numbers derived from at least two dishes per experimental day are indicated in the upper left corner of each panel. F, Least square AUC mean estimates of the five marked stimuli with 95% confidence intervals for each condition. These can be interpreted as the means if all cells had exactly the same values regarding all nuisance parameters

FIGURE 3

TRPA1 and TRPV1 agonists do not affect spontaneous activity of the cardiac cell line HL‐1. A, Experimental protocol with three HL‐1 cardiomyocytes exposed to PF‐4840154 2 µmol L^‐1 as specimen. B, Amplitudes of spontaneous calcium oscillation in HL‐1 cells. An interval of 180 s before and after AITC 100 µmol L^‐1 application was analysed. C, Number of spontaneous calcium peaks of HL‐1 cells in the 180s before and after application. D and E, Amplitudes and number of spontaneous calcium peaks before and after 30 s of PF‐4840154 2 µmol L^‐1 application. F and G, Amplitudes and number of spontaneous calcium peaks before and after 30 s of capsaicin 1 µmol L^‐1 application. Experiments are from 4 dishes per condition performed on 3 independent days, cell counts for stimulation with AITC, PF‐4840154 and capsaicin are 299, 279, and 277, respectively

FIGURE 4

No evidence for functional TRPA1 or TRPV1 expression in the rat myoblast cell line H9c2. A, Time course of intracellular calcium levels. H9c2 cells show no response to an addition of extracellular solution (negative control), but in response to depolarization by KCl 60 mmol L^‐1 (positive control) applied at the end of each measurement. Every trace reflects the measurement of a separate well. Right panel shows the calcium time course of HEK293t cells expressing mouse TRPA1 or mouse TRPV1, stimulated by the same substance as in the left panel. B and C, Time course of calcium traces in H9c2 and transfected HEK293t cells in response to AITC 100 µmol L^‐1 and PF‐4840154 2 µmol L^‐1. Right panels show responses of mouse TRPA1 or mouse TRPV1 as in panel A. D, Corresponding experiment in H9c2 and transfected HEK293t cells in response to capsaicin 1 µmol L^‐1

FIGURE 5

At best marginal TRPA1 or TRPV1 expression by quantitative PCR (qPCR) in cardiac tissue and cell lines. A, Isolated mouse cardiomyocytes and whole mouse hearts show higher Cq values for TRPA1 and TRPV1 than whole mouse dorsal root ganglions (DRGs). B, Cq values of H9c2 cardiomyoblasts show orders of magnitude lower TRPA1 and TRPV1 signal than rat DRGs. Differentiation by retinoic acid does not alter the expression. C, Exposure of H9c2 cells to inflammatory mediators also does not increase TRPA1 and TRPV1 expression. Control H9c2 cells for differentiation and inflammation were the same. D, Cq values of HL‐1 cardiac cell line, which similarly show orders of magnitude lower TRPA1 and TRPV1 expression than mouse DRGs, and inflammation does not alter the signal. E, Samples from seven areas of human hearts and human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC cardiomyocytes) show higher Cq values for TRPA1 than human DRGs. The three human donors are separated by circles, hexagons and squares. Human heart samples and DRGs were extracted less than 10 h post‐mortem. All runs had 43 cycles, curve fitting of the cycler extrapolates to 44 cycles, as indicated by a minor tick on the y‐axis. n.d. indicates that no cDNA was detected. In all panels every symbol corresponds to a biological sample (average of two technical replicates, which had a median difference of 0.14 cycles and an interquartile range of 0.07‐0.28 cycles; Figure S4)