Stretch-Induced Increase in Ca2+-Spark Rate in Rabbit Atrial Cardiomyocytes Requires TRPA1 and Intact Microtubule Network

Abstract

Background: Mechanical stretch of the myocardium is proarrhythmic and alters cellular Ca²⁺ handling, potentially involving cation nonselective mechano-sensitive ion channels. This study aimed to assess the presence and mechanisms of stretch-induced increase in Ca²⁺-spark rate (SiS) in isolated atrial cardiomyocytes.

Methods: Freshly isolated rabbit, pig, and human left atrial cardiomyocytes were stretched axially using glass microrods. Free cytosolic Ca²⁺ concentration was monitored using confocal microscopy at resting sarcomere length (≈1.79 μm) and during severe (≈12%) increase in sarcomere length.

Results: Diastolic stretch provoked SiS, which was prevented by disrupting microtubules with colchicine, but unaffected by inhibition of NADPH oxidase 2 or scavenging of reactive oxygen species. SiS was absent in Na⁺- and Ca²⁺-free external solution, suggesting that it requires transsarcolemmal influx of Na⁺ or Ca²⁺. Activation of Piezo1 increased baseline spark rate, which was further increased by stretch. TRPA1 (transient receptor potential ankyrin 1) activation also increased baseline spark rate, with no further change upon stretch. SiS was not detectable in the presence of streptomycin (a blocker of nonselective mechano-sensitive ion channels), and HC-030031 and A-967079 (selective blockers of TRPA1), even when Piezo1 was activated. SiS was also observed in pig and human atrial cardiomyocytes.

Conclusions: In atrial cardiomyocytes, diastolic stretch enhances Ca²⁺-spark rate through a mechanism that requires microtubular integrity and TRPA1 but that is independent of redox signaling. TRPA1 emerges as a key regulator of stretch-induced Ca²⁺ handling in atrial cells, with potential implications for arrhythmogenesis.

Keywords: atrial electrophysiology; mechano‐transduction; stretch‐activated ion channels.

Figure 1. Technical approach to single atrial myocyte stretch.

A, Rabbit atrial cardiomyocyte, attached to glass microrods coated with a biological adhesive. Top: nonstretched; Bottom: during severe stretch. Yellow box: area used for calculation of SL. B, Example of force recordings at different levels of isometric stretch, increasing resting SL. Force traces shown were recorded at the end of each 20‐second stretch step. C, Relation between amount of stretch (calculated as the relative increase in distance between glass rods) and change in SL. The right y axis shows relative changes in mean SL (ΔSL, expressed as percentage difference relative to mean SL at 0% stretch). Friedman test with Dunn's post hoc test was performed to test for statistical significance. n/N=5/3. D, Isometric peak force as a function of SL, linearly fitted, showing 95% CIs. n/N=8/3. All experiments were performed with physiological saline solution 1 containing 1.8 mmol·L⁻¹ CaCl₂ at 37 °C. SL, sarcomere length.

Figure 2. Changes in systolic Ca²⁺ and force parameters in rabbit atrial cardiomyocytes during moderate and severe stretch.

Representative example of (A) force of contraction and (B) CaT, recorded simultaneously at 20 seconds after the onset of stretch in an atrial cardiomyocyte paced at 2 Hz. Superimposed black, blue, and red traces are representing baseline, moderate, and severe stretch, respectively. C, Summary data illustrating change in force (mN·mm⁻²) and maximum velocity of contraction (+dF/dt_max) or relaxation (−dF/dt_max) as a function of diastolic stretch. n/N=7/3. D, Left, CaT amplitude (F/F ₀); Middle, time to 50% peak fluorescence (TF₅₀, in ms); Right, time constant of decay of Ca²⁺ transient (tau, in ms). n/N=9/4. Friedman test with Dunn's post hoc test used for (C) and (D). All experiments shown in this figure were performed with physiological saline solution 1 containing 1.8 mmol·L⁻¹ CaCl₂ at 37 °C. CaT indicates Ca²⁺ transients; +dF/dt_max, maximum velocity of force generation; −dF/dt_max, maximum velocity of relaxation; Tau, time constant of decay of Ca²⁺ transient; and TF₅₀, time from peak force to 50% relaxation.

Figure 3. Stretch‐activated diastolic Ca²⁺‐release events in rabbit atrial cardiomyocytes.

A, Representative example of a fluorescence surface plot of a confocal line scan through the center of a rabbit atrial cardiomyocyte, showing diastolic Ca²⁺‐release events (Ca²⁺ sparks) before and during severe stretch in cells preconditions by 60‐second pacing at 2 Hz. B, Effect of stretch on Ca²⁺‐spark rate during rest and moderate or severe stretch. n/N=15/4 and 65/12, respectively. C, The histogram (1 second bin size) shows the count of Ca²⁺ sparks illustrating a stretch‐induced increase in Ca²⁺‐spark rate upon severe stretch. n/N=65/12. D, Enlarged XT‐plot of fluorescence intensity of a single Ca²⁺ spark, illustrating the analysis of spark properties, including (E) amplitude (ΔF/F ₀), duration (FDHM), and width (FWHM), at baseline and during severe stretch. n/N=11/3. Wilcoxon matched pairs signed rank test was for (B) and (E). All experiments shown in this figure were performed with physiological saline solution 1 containing 3.6 mmol·L⁻¹ CaCl₂ at 37 °C. FDHM indicates full duration at half maximum fluorescence; FWHM, full width at half maximum fluorescence.

Figure 4. Role of microtubules and ROS in stretch‐induced increase in Ca²⁺‐spark rate in rabbit atrial cardiomyocytes.

A, Fluorescence surface plots of atrial cardiomyocytes before and during severe stretch, in control cells and following administration of colchicine (an inhibitor of microtubule polymerization, 10 μmol·L⁻¹), gp91ds (an inhibitor of NOX2, 3 μmol·L⁻¹) and scrambled (nontargeted) peptides (3 μmol·L⁻¹), and N‐acetylcysteine (a ROS scavenger, 10 mmol·L⁻¹). B through D, Ca²⁺‐spark rate at baseline and during stretch in atrial cardiomyocytes exposed to the aforementioned pharmacological agents. n/N=24/5, 14/4, 12/5, 9/4, and 24/5, respectively. E, Ca²⁺‐spark rate at baseline and during stretch in control or Na⁺‐ and Ca²⁺‐free solution, n/N=42/9 and 15/4, respectively. Wilcoxon matched‐pairs signed rank test was used for (B), (C), and (D) to assess statistical significance. Mixed‐effects analysis followed by Šídák's multiple comparisons test was used for control, Na⁺/Ca²⁺‐free (E) and GsMTx‐4 (Figure S5) to assess statistical significance. All experiments were carried out in physiological saline solution 1 containing 3.6 mmol·L⁻¹ CaCl₂ at 37 °C. NAC indicates N‐acetylcysteine; NOX2, NADPH oxidase 2; and ROS, reactive oxygen species.

Figure 5. Role of MSC_NS in the stretch‐induced increase in Ca²⁺‐spark rate in rabbit atrial cardiomyocytes.

A, Fluorescence surface plots of atrial cardiomyocytes before and during application of severe stretch in control conditions, and following treatment with streptomycin (STP, a nonspecific blocker of MSC_NS, 40 μmol·L⁻¹), Yoda1 (a chemical activator of Piezo1, 20 μmol·L⁻¹), allylisothiocyanate (AITC, a selective agonist of TRPA1, 50 μmol·L⁻¹), HC‐030031 (HC, a selective blocker of TRPA1, 10 μmol·L⁻¹), A‐967079 (A96, a selective blocker of TRPA1, 10 μmol·L⁻¹); HC‐030031 plus Yoda1 (HC + Yoda1, 10 μmol·L⁻¹ and 20 μmol·L⁻¹, respectively), and A‐967079 plus Yoda1 (A96 + Yoda1, 10 μmol·L⁻¹ and 20 μmol·L⁻¹, respectively). B, Summary data on Ca²⁺‐spark rate at baseline and during stretch in atrial cardiomyocytes exposed to the aforementioned pharmacological agents. n/N=20/9, 10/4, 10/3, 10/4, 10/4, 11/4, 11/3 and 10/4, respectively. C, Role of MSC_NS and potential candidates in baseline Ca²⁺‐spark rate. D, Role of MSC_NS and potential candidates in Ca²⁺‐spark rate during stretch. Mixed‐effects analysis followed by Šídák's multiple comparisons test for (B), (C), and (D) was used. Pairwise comparisons were performed among the groups in (B). All experiments were carried out in physiological saline solution 2 containing 1.8 mmol·L⁻¹ CaCl₂ at room temperature. MSC_NS, cation nonselective mechanosensitive ion channel(s); and TRPA1, transient receptor potential ankyrin 1.

Figure 6. Expression and function of TRPA1 channel in rabbit atrial cardiomyocytes.

A, TRPA1 and GAPDH protein expression in rabbit left atrial cardiomyocytes using the Western blot technique. N=3. B, Representative recording of control and allylisothiocyanate (50 μmol·L⁻¹) at rest (0 mm Hg) and during stretch (−80 mm Hg) in a cell‐attached patch at holding potential of −80 mV. C and D, Quantification of channel open probability and average currents in control and allylisothiocyanate at rest and during stretch. n/N=12/3 in control and 19/3 in allylisothiocyanate. Wilcoxon matched‐pairs signed rank test and Mann–Whitney U test for (C) and (D) were used for assessment of statistical significance. AITC indicates allylisothiocyanate; and TRPA1, transient receptor potential ankyrin 1.

Figure 7. SiS in atrial cardiomyocytes from pig and human.

A, Fluorescence surface plots of atrial cardiomyocytes from pig (left) and human (right) before and during application of severe stretch. B and C, Summary data on Ca²⁺‐spark rate at baseline and during stretch in atrial cardiomyocytes from pig and human, respectively. D and E, Amplitude (ΔF/F ₀), duration (FDHM), and width (FWHM) at baseline and during stretch in atrial cardiomyocytes from pig (n/N=14/3) and human (11/3), respectively. Wilcoxon matched‐pairs signed rank test was used for (B through E) to assess statistical significance. All experiments were carried out in physiological saline solution 2 containing 1.8 mmol·L⁻¹ CaCl₂ at room temperature. FDHM indicates full duration at half maximum fluorescence; FWHM, full width at half maximum fluorescence; and SiS, stretch‐induced increase in Ca²⁺‐spark rate.