Cellular electrophysiology of canine pulmonary vein cardiomyocytes: action potential and ionic current properties

Abstract

Pulmonary vein (PV) cardiomyocytes play an important role in atrial fibrillation; however, little is known about their specific cellular electrophysiological properties. We applied standard microelectrode recording and whole-cell patch-clamp to evaluate action potentials and ionic currents in canine PVs and left atrium (LA) free wall. Resting membrane potential (RMP) averaged -66 +/- 1 mV in PVs and -74 +/- 1 mV in LA (P < 0.0001) and action potential amplitude averaged 76 +/- 2 mV in PVs vs. 95 +/- 2 mV in LA (P < 0.0001). PVs had smaller maximum phase 0 upstroke velocity (Vmax: 98 +/- 9 vs. 259 +/- 16 V s(-1), P < 0.0001) and action potential duration (APD): e.g. at 2 Hz, APD to 90% repolarization in PVs was 84 % of LA (P < 0.05). Na+ current density under voltage-clamp conditions was similar in PV and LA, suggesting that smaller Vmax in PVs was due to reduced RMP. Inward rectifier current density in the PV cardiomyocytes was approximately 58% that in the LA, potentially accounting for the less negative RMP in PVs. Slow and rapid delayed rectifier currents were greater in the PV (by approximately 60 and approximately 50 %, respectively), whereas transient outward K+ current and L-type Ca2+ current were significantly smaller (by approximately 25 and approximately 30%, respectively). Na(+)-Ca(2+)-exchange (NCX) current and T-type Ca2+ current were not significantly different. In conclusion, PV cardiomyocytes have a discrete distribution of transmembrane ion currents associated with specific action potential properties, with potential implications for understanding PV electrical activity in cardiac arrhythmias.

Figure 1. Canine atrial preparation with adjacent PVs

A, photograph of a typical preparation indicating locations at which LA and PV cells were obtained (dashed lines) and APs were recorded with standard microelectrode technique. B and C, photomicrographs of PV sections (Masson trichrome staining) under low and high magnification, showing the myocardial sleeve that extends from the LA onto PVs. D-F, longitudinal Gomori-stained PV sections at progressively higher magnification (squares highlight areas magnified in subsequent panels), showing connective tissue (green) and myocytes (pink). RSPV: right superior PV; LSPV: left superior PV; RIPV: right inferior PV; LIPV: left inferior PV; S: PV myocardial sleeve; PW: posterior wall.

Figure 2. Action potential properties of LA and PV cardiomyocytes

Recordings of action potentials from the LA (A) and PV (B) and a transmembrane potential in the smooth muscle cell layer (SMC) (C). No spontaneous diastolic depolarizations were observed. D and E, left, typical action potential recordings from LA and PV; right, phase 0 upstrokes of the same APs on an expanded time scale. F, mean (± S.E.M.) action potential properties of LA and PV cardiomyocytes. APA, action potential amplitude; APD₉₀ and APD₅₀, APD at 90 or 50 % repolarization, respectively; RMP, resting membrane potential; OS, overshoot; V_max, maximum phase 0 upstroke velocity. **P < 0.005, ***P < 0.0001 for LA vs. PV.

Figure 3. Sodium current (I_Na)

A and B, representative I_Na recordings (obtained with the protocol shown in inset to A, delivered at 0.1 Hz) from LA and PV cardiomyocytes. C, I_Na density-voltage relations (n = 10 cells per data point). Mean LA and PV current densities were not different (P = n.s., ANOVA). D, voltage dependence of I_Na inactivation, evaluated with 1000 ms prepulses to various voltages followed by a 40 ms test pulse to −40 mV, was not different between cardiomyocytes from LA and PV (V_1/2= −104.3 ± 2.0 vs. −104.1 ± 2.8 mV, respectively, n = 4 and 6 cells, P = n.s.). E, similarly, V_1/2 of activation was not different (−49.9 ± 3.5 mV vs. −48.2 ± 4.3 mV, respectively, n = 5 cells each, P = n.s.). Time constants for current activation and inactivation (F) were similar between the two tissues (e.g. at −35 mV activation τ was 0.20 ± 0.05 for LA and 0.24 ± 0.03 ms for PV, P = n.s. and inactivation τ was 0.81 ± 0.1 vs. 0.90 ± 0.15 ms, respectively, P = n.s.). Open symbols represent PV, filled symbols represent LA. TP, test potential.

Figure 4. Inward rectifier current (I_K1)

A and B, representative 1 mM Ba²⁺-sensitive I_K1 recordings obtained with the voltage protocol shown in the inset (0.1 Hz) in a LA and a PV cardiomyocyte. C, mean ±s.e.m.I_K1 density-voltage relationship (8 cells each for LA and PV), with magnified outward component of the current in the inset. Mean LA currents were significantly greater than PV current as determined by ANOVA analysis (P = 0.002). TP, test potential.

Figure 5. Delayed rectifier potassium current components (I_Ks and I_Kr)

A and B, representative I_Ks recordings obtained with 4 s depolarizations (0.1 Hz) followed by 2 s repolarizations to −30 mV in the presence of 1 μM dofetilide from a LA (A) and a PV (B) cardiomyocyte. C, mean ±s.e.m.I_Ks density-voltage relations from 12 cells. I_Ks denisty was greater in PV than in LA cardiomyocytes (P < 0.0001, ANOVA). D, mean ±s.e.m.I_Ks tail density-voltage relations (n = 12 per group). E, representative recordings of chromanol 293B (50 μM)-resistant I_Kr tail currents before and after application of 1 μM dofetilide obtained with the protocol in A (0.1 Hz). F, mean ±s.e.m.I_Kr tail current-voltage relationships and Boltzmann fits to mean data. I_Kr densities were significantly larger in the PV (P = 0.01, ANOVA). TP, test potential.

Figure 6. Transient outward current (I_to)

A and B, typical I_to recordings obtained with 100 ms depolarizing pulses from −50 mV. C, mean ±s.e.m.I_to density-voltage relations for 43 LA and 37 PV cells. I_to density was greater in LA than in PV cells (P = 0.0001, ANOVA); inset: I_to-V relations of currents normalized to values at +50 mV. D, time to peak I_to and I_to inactivation time constants (τ_inact.; n = 10 cells per group). E, mean ±s.e.m. data for voltage dependence of I_to inactivation (Inact.) and activation (Act.; n = 10 cells per observation). F, mean ±s.e.m. current during P2 normalized to current during P1, as a function of P1-P2 interval (protocol in inset). Curves are mono-exponential fits to mean ±s.e.m. data (n = 9 cells each). All protocols were at 0.1 Hz. TP, test potential.

Figure 7. L-type calcium current (I_Ca,L)

I_Ca,L recordings during 240 ms depolarizing steps from a HP of −50 mV in LA (A) and PV (B) cardiomyocytes (0.1 Hz). C, mean ±s.e.m.I_Ca,L density-voltage relations, (n = 10 cells per group). Mean PV I_Ca,L density was smaller than LA current density (P < 0.001, ANOVA). D, normalized I_Ca,L -voltage relationships for both tissues superimposed. E, voltage dependence of I_Ca,L inactivation and activation. Curves are Boltzmann fits to mean data (n = 10 cells per group). F, inactivation time constants (τ_slow and τ_fast) obtained by bi-exponential curve fitting (n = 10 cells each). G, I_Ca,L recovery kinetics studied with paired 300 ms pulses to +10 mV (0.1 Hz). Mono-exponential fits to mean data are shown (n = 8 cells per group). H, I_Ca,L frequency dependence for 200 ms pulses from −80 mV to +10 mV, with current at steady state normalized to current during first pulse of train (n = 6 cells per group). Open symbols represent PV, closed symbols LA. TP, test potential; act., activation; inact., inactivation.

Figure 8. T-type calcium current (I_Ca,T) and Na⁺,Ca²⁺-exchange current (I_NCX)

A and B, representative recordings of calcium current from LA and PV cardiomyocytes respectively. The top of each panel shows current recorded with holding potentials of −90 mV (open circles) and −50 mV (filled circles). The bottom shows subtracted current, representing I_Ca,T. C, mean I_Ca,T density-voltage relationship (n = 7 and 8 cells for LA and PV, respectively, P = n.s., ANOVA). D and E, representative recordings obtained with the protocol used to evaluate I_NCX from LA and PV cardiomyocytes, respectively at 0.1 Hz. Recordings before (filled squares) and after (open squares) removal of external sodium are shown. F, mean ±s.e.m.I_NCX density-voltage relationship (8 cells each, P = n.s., ANOVA). TP, test potential.