Transient receptor potential melastatin 4 inhibitor 9-phenanthrol abolishes arrhythmias induced by hypoxia and re-oxygenation in mouse ventricle

Abstract

Background and purpose: Hypoxia and subsequent re-oxygenation are associated with cardiac arrhythmias such as early afterdepolarizations (EADs), which may be partly explained by perturbations in cytosolic calcium concentration. Transient receptor potential melastatin 4 (TRPM4), a calcium-activated non-selective cation channel, is functionally expressed in the heart. Based on its biophysical properties, it is likely to participate in EADs. Hence, modulators of TRPM4 activity may influence arrhythmias. The aim of this study was to investigate the possible anti-arrhythmic effect of 9-phenanthrol, a TRPM4 inhibitor in a murine heart model of hypoxia and re-oxygenation-induced EADs.

Experimental approach: Mouse heart was removed, and the right ventricle was pinned in a superfusion chamber. After a period of normoxia, the preparation was superfused for 2 h with a hypoxic solution and then re-oxygenated. Spontaneous electrical activity was investigated by intracellular microelectrode recordings.

Key results: In normoxic conditions, the ventricle exhibited spontaneous action potentials. Application of the hypoxia and re-oxygenation protocol unmasked hypoxia-induced EADs, the occurrence of which increased under re-oxygenation. The frequency of these EADs was reduced by superfusion with either flufenamic acid, a blocker of Ca(2+) -dependent cation channels or with 9-phenanthrol. Superfusion with 9-phenanthrol (10(-5) or 10(-4) mol·L(-1) ) caused a dramatic dose-dependent abolition of EADs.

Conclusions and implications: Hypoxia and re-oxygenation-induced EADs can be generated in the mouse heart model. 9-Phenanthrol abolished EADs, which strongly suggests the involvement of TRPM4 in the generation of EAD. This identifies non-selective cation channels inhibitors as new pharmacological candidates in the treatment of arrhythmias.

Figure 1

Hypoxia–re-oxygenation-induced arrhythmias. (A) Action potentials were recorded in a mouse ventricle superfused with a standard, oxygenated solution (a), followed by a hypoxic solution (b), followed by the oxygenated solution again (c). EADs are indicated by stars. Bars represent time interval (ms) or voltage (mV) as indicated. (B) The number of EADs recorded as above were normalized to that of action potentials (EAD/AP) for 10 s periods and plotted against time. Same experiment as in (A). Arrows indicate the time corresponding to a, b and c recordings. Bar represents a 30 min interval. (C) pO₂ in the superfused solution, normalized to the value of a saturated solution, recorded during the above hypoxia–re-oxygenation protocol (mean ± SEM; n= 4). (D) Number of EAD/AP is compared for the three phases: start (O₂), hypoxia and re-oxygenation. Open circles linked by dotted lines indicate single experiments, while black circles linked by black line correspond to the mean of experiments (mean ± SEM; n= 6).

Figure 2

Anti-arrhythmic effect of 9-phenanthrol. (A) Action potentials recorded in a mouse ventricle during re-oxygenation in the standard solution (a), or in the presence of 9-phenanthrol, 10⁻⁴ mol·L⁻¹ (b), or after washout of 9-phenanthrol (c). Bars represent time interval (ms) or voltage (mV) as indicated. (B) Number of EAD/AP for 10 s periods during re-oxygenation for the above a, b and c phases, as indicated. The phase of superfusion with 9-phenanthrol, 10⁻⁴ mol·L⁻¹, is highlighted in grey. Bar represents a 10 min interval. (C) Number of EAD/AP after re-oxygenation before and during superfusion of 10⁻⁵ or 10⁻⁴ mol·L⁻¹ 9-phenanthrol. Open circles linked by dotted lines indicate single experiments while black circles linked by black line correspond to the mean of experiments (mean ± SEM; n = 6). (D) Reduction in EAD/AP (mean ± SEM) during superfusion with 10⁻⁵ or 10⁻⁴ mol·L⁻¹ 9-phenanthrol, followed by washing with standard solution, given as a percentage of the value during re-oxygenation before application of the drug and calculated as follows: % reduction = 100 −[(EAD/AP with inhibitor)/(EAD/AP in control) x 100]. (E) Resting membrane potential variation induced by 9-phenanthrol 10⁻⁴ mol·L⁻¹. Open circles linked by dotted lines indicate single experiments while black circles linked by black line correspond to the mean of the 6 experiments. Control corresponds to RMP in re-oxygenation solution before application of the drug.

Figure 3

9-Phenanthrol effect on I_Ca,L and I_K. (A) Representative example of 9-phenanthrol effect (10⁻⁵ mol·L⁻¹, left, and 10⁻⁴ mol·L⁻¹, right) on L-type calcium current elicited by a 300 ms step from −80 to 0 mV in mouse ventricular myocytes. (B) Means ± SEM of 9-phenanthrol effect on L-type calcium current (in pA/pF). Number of cells is indicated in each column. (NS: not significant, * indicates P < 0.05). (C) Representative example of 9-phenanthrol effect (10⁻⁵ mol L⁻¹, left, and 10⁻⁴ mol L⁻¹, right) on K current elicited by voltage step (bottom) in mouse ventricular myocytes. (D) Means ± SEM of 9-phenanthrol effect (10⁻⁵ mol L⁻¹ left and 10⁻⁴ mol L⁻¹ right) on global charge carrying by potassium (in pC/pF).

Figure 4

Anti-arrhythmic effect of 9-phenanthrol is independent of PKA. (A) Action potential recordings in a mouse ventricle under re-oxygenation in four successive superfusion solutions: (i) initial re-oxygenation without drug, (ii) re-oxygenation with solution containing H-89 10⁻⁶ mol·L⁻¹, (iii) re-oxygenation with solution containing H-89, 10⁻⁶ mol·L⁻¹+ 9-phenanthrol, 10⁻⁴ mol·L⁻¹ or (iv) re-oxygenation with solution containing H-89, 10⁻⁶ mol·L⁻¹ with washout of 9-phenanthrol. Bars represent time interval (ms) or voltage (mV) as indicated. Note that superfusion with H-89 does not suppress EADs, while that with 9-phenanthrol does. (B) Percentage reduction of EAD/AP (mean ± SEM) due to superfusion with re-oxygenation solution containing H-89 (10⁻⁶ mol·L⁻¹) alone or H-89 (10⁻⁶ mol·L⁻¹) + 9-phenanthrol (10⁻⁴ mol·L⁻¹) (n= 3) compared with initial re-oxygenation solution without the drugs.

Figure 5

Anti-arrhythmic effect of flufenamic acid. (A) Action potentials recorded in a mouse ventricle superfused with standard re-oxygenation solution (a), followed by that containing flufenamic acid, 10⁻⁵ mol·L⁻¹ (b), followed by wash with re-oxygenation solution without drug again (c). Bars represent the time interval (ms) and voltage (mV) as indicated. (B) Number of EAD/AP for 10 s periods during the above phases, as indicated by arrows. Phase (b) is highlighted in grey. Bar represents a 5 min interval. (C) EAD/AP (mean ± SEM) recorded during re-oxygenation with standard solution or with that containing flufenamic acid, 10⁻⁵ mol·L⁻¹ are compared (n= 5).