Mechano-arrhythmogenicity is enhanced during late repolarisation in ischemia and driven by a TRPA1-, calcium-, and reactive oxygen species-dependent mechanism

Background: Cardiac dyskinesis in regional ischemia results in arrhythmias through mechanically-induced changes in electrophysiology ('mechano-arrhythmogenicity') that involve ischemic alterations in voltage-calcium (Ca2+) dynamics, creating a vulnerable period (VP) in late repolarisation. Objective: To determine cellular mechanisms of mechano-arrhythmogenicity in ischemia and define the importance of the VP. Methods and Results: Voltage-Ca2+ dynamics were simultaneously monitored in rabbit ventricular myocytes by dual-fluorescence imaging to assess the VP in control and simulated ischemia (SI). The VP was longer in SI than in control (146±7 vs 54±8 ms; p<0.0001) and was reduced by blocking KATP channels with glibenclamide (109±6 ms; p<0.0001). Cells were rapidly stretched (10-18% increase in sarcomere length over 110-170 ms) with carbon fibres during diastole or the VP. Mechano-arrhythmogenicity, associated with stretch and release in the VP, was greater in SI than control (7 vs 1% of stretches induced arrhythmias; p<0.005) but was similar in diastole. Arrhythmias during the VP were more complex than in diastole (100 vs 69% had sustained activity; p<0.05). In the VP, incidence was reduced with glibenclamide (2%; p<0.05), by chelating intracellular Ca2+ (BAPTA; 2%; p<0.05), blocking mechano-sensitive TRPA1 (HC-030031; 1%; p<0.005), or by scavenging (NAC; 1%; p<0.005) or blocking reactive oxygen species (ROS) production (DPI; 2%; p<0.05). Ratiometric Ca2+ imaging revealed that SI increased diastolic Ca2+ (+9±1%, p<0.0001), which was not prevented by HC-030031 or NAC. Conclusion: In ischemia, mechano-arrhythmogenicity is enhanced specifically during the VP and is mediated by ROS, TRPA1, and Ca2+.

Beat-By-Beat Cardiomyocyte T-Tubule Deformation Drives Tubular Content Exchange

Rationale: The sarcolemma of cardiomyocytes contains many proteins that are essential for electro-mechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is non-uniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electro-mechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. Objective: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. Methods and Results: Using electron tomography, we explore the 3D nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. Conclusions: Our data confirm the existence of an advective component to TT content exchange. This points towards a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodelling (e.g. cardiac hypertrophy or failure) forms an exciting target for further research.

Abstract 14409: TRPA1 Channels Are a Source of Calcium-driven Cellular Mechano-arrhythmogenicity

Introduction: Pathologic changes in myocardial mechanics and hemodynamic load result in arrhythmias via mechanically-induced changes in electrophysiology or intracellular Ca 2+ (‘mechano-arrhythmogenicity’). While molecular mechanisms driving mechano-arrhythmogenicity are poorly defined, they are associated with disease-related alterations in voltage-Ca 2+ dynamics. Objective: Define mechanisms of mechano-arrhythmogenicity during alterations in voltage-Ca 2+ dynamics in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100ms) during diastole or late repolarisation in control or during K ATP channel activation (pinacidil). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), non-selectively block stretch-activated channels (streptomycin), or specifically block (HC-030031) or activate (AITC) mechano-sensitive TRPA1 channels. Voltage-Ca 2+ dynamics were simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system and diastolic Ca 2+ was measured using Fura Red. Results: Pinacidil caused greater shortening of the AP than Ca 2+ transient (-144±17 vs -74±11ms; n =24 cells, N =7 rabbits; p <0.001) with no change in cell stiffness or contractility. Stretch during pinacidil application caused arrhythmias in both diastole and late repolarisation (8 and 10% of stretches; n =46, N =5), which voltage-Ca 2+ imaging revealed were Ca 2+ -driven. Arrhythmias were reduced with BAPTA (3 and 0% of stretches; p <0.05), streptomycin (4 and 2%; p <0.05), and HC-030031(2 and 1%; p <0.01), while dantrolene had no effect ( n =40, N =5 for each). Stretch in diastole during AITC application also caused arrhythmias (15%; p <0.001), which were blocked by HC-030031 (4%; p <0.001) or BAPTA (3%; p <0.001; n =40, N =5 each). Both AITC and pinacidil caused an increase in diastolic Ca 2+ (112±29 and 78±29% of control; p<0.05), which was reduced by HC-030031 with AITC (26±24%; p <0.05), but not with pinacidil ( n =25, N =5 each). Conclusions: TRPA1 activation increases mechano-arrhythmogenicity via a Ca 2+ -driven mechanism and may represent a novel anti-arrhythmic target in pathologies involving altered cardiac mechanics.

Abstract 14416: Mechano-arrhythmogenicity is Enhanced in Late Repolarisation During Acute Ischemia by a Calcium- and TRPA1-dependent Mechanism

Introduction: Altered tissue mechanics in acute regional ischemia contribute to arrhythmias by a mechano-sensitive, Ca 2+ -dependent mechanism. This is facilitated by uncoupling of voltage-Ca 2+ dynamics, creating a vulnerable period (VP) in late repolarisation for stretch-induced arrhythmias (‘mechano-arrhythmogenicity’). However, cellular mechanisms driving mechano-arrhythmogenicity in acute ischemia are unknown. Objective: Define cellular mechanisms of mechano-arrhythmogenicity in the VP during acute ischemia in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100 ms) during diastole or the VP in normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia, acidosis, metabolic inhibition). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), block mechano-sensitive TRPA1 channels (HC-030031), or block (DPI) or increase (bi-product of di-4-ANBDQPQ excitation) ROS production. Voltage-Ca 2+ was simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system. Results: SI shortened AP duration (APD NT =384 vs APD SI =219ms; p <0.0001) more than Ca 2+ transient duration (CaTD NT =424 vs CaTD SI =357 ms; p <0.0001) and increased the length of the VP (=CaTD-APD; VP NT =54 vs VP SI =146ms; n =50 cells for N NT =6 and N SI =14 rabbits ; p <0.0001). Mechano-arrhythmogenicity (single ectopy and complex sustained activity) was increased in SI compared to NT, but only for stretch in the VP (7 vs 1% of stretches; n =50, N =6 each ; p <0.005), and arrhythmias in the VP were proportionally more complex than those that occurred with stretch in diastole (100 vs 69%; n =50, N =6; p <0.05). Arrhythmia incidence in the VP during SI was reduced by BAPTA (2% of stretches; p <0.05), HC-030031 (1%; p <0.005), and DPI (2%; p <0.05), while dantrolene had no effect ( n =50, N =6 each). Fluorescence imaging during SI further increased mechano-arrhythmogenicity in both the VP and diastole (29 and 14%; n =42, N =4; p <0.05). Conclusions: Acute ischemia enhances cellular mechano-arrhythmogenicity specifically in the VP through a mechanism involving Ca 2+ , ROS, and TRPA1, suggesting potential targets for anti-arrhythmic therapy.

Altered K+ current profiles underlie cardiac action potential shortening in hyperkalemia and β-adrenergic stimulation

Hyperkalemia is known to develop in various conditions including vigorous physical exercise. In the heart, hyperkalemia is associated with action potential (AP) shortening that was attributed to altered gating of K+ channels. However, it remains unknown how hyperkalemia changes the profiles of each K+ current under a cardiac AP. Therefore, we recorded the major K+ currents (inward rectifier K+ current, IK1; rapid and slow delayed rectifier K+ currents, IKr and IKs, respectively) using AP-clamp in rabbit ventricular myocytes. As K+ may accumulate at rapid heart rates during sympathetic stimulation, we also examined the effect of isoproterenol on these K+ currents. We found that IK1 was significantly increased in hyperkalemia, whereas the reduction of driving force for K+ efflux dominated over the altered channel gating in case of IKr and IKs. Overall, the markedly increased IK1 in hyperkalemia overcame the relative decreases of IKr and IKs during AP, resulting in an increased net repolarizing current during AP phase 3. β-Adrenergic stimulation of IKs also provided further repolarizing power during sympathetic activation, although hyperkalemia limited IKs upregulation. These results indicate that facilitation of IK1 in hyperkalemia and β-adrenergic stimulation of IKs represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility.

Sodium Houttuyfonate Inhibits Voltage-Gated Peak Sodium Current and Anemonia Sulcata Toxin II-Increased Late Sodium Current in Rabbit Ventricular Myocytes

Aim: Sodium houttuyfonate (SH), a chemical compound originating from Houttuynia cordata, has been reported to have anti-inflammatory, antibacterial, and antifungal effects, as well as cardioprotective effects. In this study, we investigated the effects of SH on cardiac electrophysiology, because to the best of our knowledge, this issue has not been previously investigated. Methods: We used the whole-cell patch-clamp technique to explore the effects of SH on peak sodium current (INa.P) and late sodium current (INa.L) in isolated rabbit ventricular myocytes. To test the drug safety of SH, we also investigated the effect of SH on rapidly activated delayed rectifier potassium current (IKr). Results: SH (1, 10, 50, and 100 μmol/L) inhibited INa.P in a concentration-dependent manner with an IC50 of 78.89 μmol/L. In addition, SH (100 μmol/L) accelerated the steady state inactivation of INa.P. Moreover, 50 and 100 μmol/L SH inhibited Anemonia sulcata toxin II (ATX II)-increased INa.L by 30.1 and 57.1%, respectively. However, SH (50 and 100 μmol/L) only slightly affected IKr. Conclusions: The inhibitory effects of SH on ATX II-increased INa.L may underlie the electrophysiological mechanisms of the cardioprotective effects of SH; SH has the potential to be an effective and safe antiarrhythmic drug.