TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway

Figures & data

Figure 1. Allyl isothiocyanate (AITC) increases [Ca^2±]_I and shortening in CMs. Original traces demonstrating the effect of AITC (100 μmol/L) on steady-state sarcomere length (µm; panel A) and [Ca²⁺]_i (340/380 ratio; panel B) in an individual mouse ventricular myocyte. AITC was added where indicated on the figure. Exploded views depicting the dose-dependent changes in sarcomere length and [Ca²⁺]_i (panels C and D, respectively) before (control) and after addition of AITC (1–300 µmol/L) to single ventricular myocyte. AITC was added to the bath in a cumulative fashion. n = 20 cells from 6 hearts.

Table 1. Comparison of AITC- and isoproterenol (ISO)-induced changes in CM [Ca^2±]_i and contractile function. Data are expressed as mean ± SEM. * P < 0.05 compared with untreated control (ctrl).

AITC (µmol/L) n = 20	0 (Ctrl)	1	10	100	300
Fractional Shortening (% sarcomere length)	4.5 ± 0.1	5.0 ± 0.2	6.3 ± 0.2*	8.7 ± 0.3*	10.8 ±0.3*
Maximum Velocity of Cell Shortening (μm/sec)	4.7 ± 0.1	5.4 ± 0.2	7.1± 0.3*	9.9 ± 0.3*	11.2 ± 0.3*
Maximum Velocity of Cell Relaxation (μm/sec)	3.6 ± 0.1	4.1 ± 0.1	5.1 ± 0.3*	7.1 ± 0.3*	8.3 ± 0.3*
Peak [Ca^2+]i (change in 340/380 ratio)	0.13 ± 0.02	0.15 ± 0.04	0.19 ± 0.04*	0.22 ± 0.06*	0.26 ± 0.04*
Tp [Ca^2+]I (msec)	158 ± 9.4	144 ± 8.4	112 ± 9.0*	58 ± 6.1*	46 ± 3.3
[Ca^2+]i Decay (msec)	610 ± 19	563 ± 18	458 ± 15*	326 ± 12*	235 ± 9*
ISO (nmol/L) n = 12	0 (Ctrl)	1	5	10	100
Fractional Shortening (% sarcomere length)	4.6 ± 0.2	5.5 ± 0.3	7.1 ± 0.3*	10.7 ± 0.6*	11.5 ±0.5*
Maximum Velocity of Cell Shortening (μm/sec)	4.7 ± 0.1	5.1 ± 0.1	7.4 ± 0.2*	11.1 ± 0.4*	12.7 ± 0.4*
Maximum Velocity of Cell Relaxation (μm/sec)	3.6 ± 0.1	4.0 ± 0.1	5.6 ± 0.2*	8.6 ± 0.3*	10.0 ± 0.2*
Peak [Ca^2+]i (change in 340/380 ratio)	0.11 ± 0.02	0.12 ± 0.03	0.17 ± 0.05*	0.22 ± 0.05*	0.23 ± 0.05*
Tp [Ca^2+]I (msec)	140 ± 17	124 ± 16	104 ± 9	56 ± 7*	52 ± 6*
[Ca^2+]i Decay (msec)	615 ± 16	586 ± 15	381 ± 16*	231 ± 21*	174 ± 16*

Figure 2. AITC increases fractional shortening, maximum velocity of shortening and maximum velocity of relengthening in CMs. Overlays of individual cell shortening and relengthening events illustrating the dose-dependent effects of AITC (1–300 μmol/L) on fractional shortening are depicted in panel A. Representative overlays assessing changes in sarcomere length normalized to peak height (set at 100%) and aligned at initiation of stimulus or at peak shortening to illustrate dose-dependent changes in timing of shortening and relengthening are depicted in panels C and E, respectively. Summarized data for panels A (fractional shortening), C (maximal velocity of shortening) and E (maximal velocity of relengthening) are depicted in panels B, D and F, respectively. Results are expressed as percent of steady-state baseline control (Ctrl) value set at 100%. Changes in fractional shortening were measured as percent of sarcomere length. Changes in velocity were measured in micrometers/sec. * P < 0.05 compared with Ctrl. n = 20 cells from 6 hearts.

Figure 3. AITC increases peak [Ca^2±]_I and accelerates time to peak [Ca^2±]_I and the rate of [Ca^2±]_I decay in CMs. Overlays illustrating the dose-dependent effects of AITC (1–300 μmol/L) on peak [Ca²⁺]_i are depicted in panel A. Representative overlays assessing dose-dependent changes in [Ca²⁺]_i peak amplitude normalized to peak height (set at 100%) to illustrate changes in time to peak [Ca²⁺]_i and the time of [Ca²⁺]_i decay are depicted in panels C and E, respectively. Summarized data for panels A ([Ca²⁺]_i peak amplitude), C (time to peak [Ca²⁺]_i) and E ([Ca²⁺]_i decay) are depicted in panels B, D and F, respectively. Results are expressed as percent of steady-state baseline control (Ctrl) value set at 100%. Changes in peak [Ca²⁺]_i are measured as the change in the 340/380 ratio. Changes in timing are measured in milliseconds. Individual traces were smoothed using the Savitzky-Golay filter to increase the signal-to-noise ratio. * P < 0.05 compared with Ctrl. n = 20 cells from 6 hearts.

Figure 4. AITC has no effect on [Ca^2±]_I and shortening in CMs obtained from TRPA1 null mice. Original traces demonstrating the lack of effect of AITC (100 μmol/L) on steady-state sarcomere length (µm; panel A) and [Ca²⁺]_i (340/380 ratio; panel B) in an individual mouse ventricular myocyte obtained from TRPA1 null mice (TRPA1^−/−). AITC was added where indicated on the figure. Exploded views of changes in sarcomere length and [Ca²⁺]_i before and after addition of AITC are depicted in panels C-F. n = 8 cells from 4 hearts.

Figure 5. TRPA1 stimulation does not signal through the cAMP/Protein Kinase A (PKA) pathway in CMs. Summarized data demonstrating the effects of ISO (1–100 nmol/L) and AITC (1–300 µmol/L) on cAMP production in CMs obtained from WT mice are depicted in panels A and B, respectively. Results are expressed as total cAMP production (pmol/mL; n = CMs isolated from 5 separate hearts repeated in triplicate). Summarized data demonstrating the effects of AITC (100 µmol/L) or ISO (10 nmol/L) in the presence or absence of PKA inhibitor, PKI 14–22 (1–100 nmol/L), on PKA activity is shown in panel C (n = CMs isolated from 3 separate hearts repeated in triplicate). The dose-dependent effects of PKI 14–22 treatment following isoproterenol-induced increases in contractile function are shown in the subfigure on the right side of panel C (n = 6 CMs from 3 hearts). * P < 0.05 compared with untreated control value. # P < 0.05 compared with isoproterenol-treated CMs.

Figure 6. AITC-induced increases in contractile function and [Ca^2±]_i occur independently of PKA in CMs. Overlays of fractional shortening and [Ca²⁺]_i peak amplitudes in electrically-paced CMs illustrating the effects of AITC (100 µmol/L) in the presence and absence of PKI 14–22 (100 nmol/L) are depicted in panels A and B, respectively. Summarized data for parameters measuring contractile function are depicted in panels C (fractional shortening), E (maximum velocity of shortening) and G (maximum velocity of relengthening). Summarized data analyzing [Ca²⁺]_i dynamics are depicted in panels D ([Ca²⁺]_i peak amplitude), F (time to peak [Ca²⁺]_i) and H ([Ca²⁺]_i decay). Results are expressed as percent of steady-state baseline control (Ctrl) value set at 100%. Individual [Ca²⁺]_i traces were smoothed using the Savitzky-Golay filter to increase the signal-to-noise ratio. N.S. = not statistically significant. * P < 0.05 compared with Ctrl. n = 18 cells from 6 hearts.

Figure 7. AITC-induced phosphorylation of CaMKII at threonine 286 occurs in a time- and TRPA1-dependent manner in CMs. Representative immunoblots demonstrating the effect of AITC (100 µmol/L) on CaMKII phosphorylation at threonine 286 (pCaMKII-t²⁸⁶) at 0–5 minutes in CMs obtained from WT mice are depicted in panel A. All samples were lysed at the indicated time points (mins). Summarized data are shown as the % of the untreated mean control value (pCaMKII-t²⁸⁶/total protein) in panel B (n = CMs obtained from 5 separate hearts). Representative immunoblots demonstrating the effects of AITC (100 µmol/L) on CaMKII phosphorylation in the presence (+) and absence (−) of KN93 (10 µmol/L) is shown in panel C. Samples from WT and TRPA1^−/− were either treated with AITC or pretreated with KN93 for 10 minutes before AITC administration. All samples were lysed 5 minutes following treatment of AITC. Summarized data are expressed as the percent of the untreated WT mean control value in panel D (n = CMs isolated from 5 separate hearts). GAPDH was probed as the loading control. N.S. = not statistically significant. P.L. = protein ladder. * P < 0.05 compared with the WT mean control value. # P < 0.05 compared with the AITC-treated WT CMs.

Figure 8. TRPA1 stimulation modulates contractile function and [Ca^2±]_i through a CaMKII-dependent mechanism in CMs. Overlays of fractional shortening and [Ca²⁺]_i peak amplitudes in electrically-paced CMs illustrating the effects of AITC (100 µmol/L) in the presence and absence of KN93 (1–10 µmol/L) are depicted in panels A and B, respectively. Summarized data for parameters measuring contractile function are depicted in panels C (fractional shortening), E (maximum velocity of shortening) and G (maximum velocity of relengthening). Summarized data analyzing [Ca²⁺]_i dynamics are depicted in panels D ([Ca²⁺]_i peak amplitude), F (time to peak [Ca²⁺]_i) and H ([Ca²⁺]_i decay). Results are expressed as percent of steady-state baseline control (Ctrl) value set at 100%. Individual [Ca²⁺]_i traces were smoothed using the Savitzky-Golay filter to increase the signal-to-noise ratio. * P < 0.05 compared with Ctrl. # P < 0.05 compared with AITC-treated cells. n = 15 cells from 5 hearts.