ELECTROPHYSIOLOGY AND ARRHYTHMOGENIC ACTIVITY OF DOG LEFT INFERIOR PULMONARY VEINS

Cheng-I Lin¹, Chien-Chih Ou¹, Yi-Jen Chen^2,3, Yao-Chang Chen¹, Shih-Ann Chen^3
 1Institute of Pharmacology & Department of Biomedical Engineering, National Defense Medical Center, ²Taipei Medical University and ³National Yang-Ming University School of Medicine, Taipei, Taiwan 

Abstract: This study investigated the electrophysiological characteristics of left inferior pulmonary veins (LIPV) and cardiomyocytes isolated from myocardial sleeve of left　PVs. Action potentials (APs) of PV were recorded by means of conventional microelectrode techniques, and whole-cell patch-clamp techniques were used on cardiomyocytes to record APs and ionic currents. Results show that, in the ending of myocardial sleeve of LIPVs obtained from 3 of 14 dogs, spontaneous irregular APs with an average rate of 17±6 Hz and amplitude of 53±3 mV were recorded. Dissociation of inferior PVs, similar to that of superior PV, yielded rod-shaped single cardiomyocytes with or without pacemaker activity. The automaticity was correlated with  smaller inward rectifier K⁺ current (I_K1) and inward current with slow inactivation kinetics on hyperpolarization (I_f). In agreement with the findings of clinical investigations reported in literatures, the arrhythmogenic electrical activities occurred less frequently in LIPV than in superior PV (LSPV). The longer myocardial sleeve existed in LSPV than in LIPV might provide larger cardiac structural and electrical substrates for the genesis of arrhythmias.

INTRODUCTION

Earlier histological study on human embryo and fetuses suggested that all of the thoracic veins containing cardiac muscle have their origin from sinus venosus [1]. In our previous studies in dog left superior pulmonary veins (LSPVs), we found that spontaneous activity (around 3 Hz) and high frequency irregular rhythms (10~24 Hz) often occurred especially during exposure to the β-adrenoceptor agonist isoproterenol or in LSPVs obtained from the rapid atrial pacing dogs (RAP, 780/min for 6-8 weeks) [2]. Whole-cell patch-clamp study revealed that cardiomyocytes isolated from LSPVs contained multiple cardiomyocytes (with vs. without pacemaker activity) with distinct electrophysiological properties [3]. In clinical studies, the incidence of atrial fibrillation originated from ectopic focus located at inferior PVs was low [4] or even absent [5], in contrast to those at superior PVs. The aim of the present experiments on dog PVs was to explore the cellular mechanisms responsible for the different electrophysiological behavior between superior and inferior PVs.

METHODS

Tissue preparations: Fourteen dogs (12-20 kg) were used. The animal was anesthetized with sodium pentobarbital and the heart quickly dissected. In oxygenated Tyrode solution, the LIPV and LSPV were isolated from the left atrium. Proximal segments (diameter around 4 mm) of the PVs were cut longitudinally. The PV strip (3 x 12 mm in size) was superfused with normal Tyrode solution at 37 ^oC. Membrane potentials were recorded through adventitia by mircroelectrode techniques as described previously [2].

PV cardiomyocytes: Inferior PVs (either LIPV or RIPV) from 5 dogs were used for studies on the action potential and ionic current of PV cardiomyocytes. The cardiomyocytes were isolated enzymatically as described recently for LSPV [3]. Membrane potentials were recorded in current-clamp mode and ionic currents in voltage-clamp mode.  All chemicals were obtained from Sigma Chemicals (St. Louis, MO, USA).

Statistical analysis: Statistical analysisValues are expressed as means ± SEM. One-way ANOVA, χ² test and Student t test were used for statistical analysis. A P value less than 0.05 was regarded as statistically significant.

RESULTS

Figure 1A shows traces of a series of high frequency (about 11 Hz) early afterdepolarizations following a large amplitude AP (which failed to repolarize completely) recorded in one LSPV strip. Traces in Fig. 1B and 1C (recorded from two LIPV strips) show slender and high APs intermingled with wide and low amplitude APs, similar to those observed in LSPVs [6]. The firings were rapid (8 and 5 Hz in B and C) and irregular.   

Figure 1. Spontaneous irregular action potentials (APs) recorded in  dog left superior (LSPV) and left inferior pulmonary vein (LIPV). Panel A shows large amplitude APs followed by  small amplitude APs (early afterdepolarizations) in a LSPV strip. Panels B and C show simultaneous recordings of 2 types of cells from single microelectrode impalement in two LIPV strips. Note different rates of discharge and AP configuration.

In 3 of 14 dogs tested, we have recorded electrical activity similar to those shown in Fig. 1 near the ending of myocardial sleeve of LIPV Fig. 1. The average rate and amplitude of spontaneous irregular APs were 17±6 Hz and 53±3 mV, respectively. However, in the remaining 11 dogs, only the driven fast response APs of atrial cells were recorded. The incidence of high frequency irregular rhythms was significantly higher (χ² = 5.591, P<0.05) in LSPV (12/17) as reported previously [2], in agreement with the findings of clinical investigations [4,5]. 

Dissociation of inferior PV with digesting enzymes yielded rod-shaped single cardiomyocytes with or without pacemaker activity. Fig. 2A shows the spontaneously active APs of a cardiomyocyte and Fig. 2B a fast response AP of another cardiomyocyte driven electrically at 1 Hz. From the current tracing, it is obvious that the automatic cell had very small inward rectifier K⁺ current (I_K1) without the hyperpolarization- activated inward current I_f (bottom panel A). In contrast, the myocyte showing fast response AP had an instantaneous I_K1 of 148 pA on hyperpolarizing step to –120 mV and a progressively increased I_f of 64 pA in magnitude at the end of 1 sec pulse.

In a total of 22 cardiomyocytes obtained from IPVs of 5 dogs, 7 cells were spontaneously active and the remaining 15 cells were without pacemaker activity. Automatic cells had  significantly smaller inward rectifier K⁺ current I_K1 (33±7 vs. 220±37 Pa, P<0.05) and I_f current (4±3 vs. 88±19 pA) than cells without pacemaker activity. The results of I_K1 are similar to those observed in cardiac cells of LSPV [3]. 

Figure 2. Action potential configurations (top panels) and membrane currents elicited on hyperpolarization from a holding potential of –40 mV to test potentials (for 1 s) range from –20 to –120 mV in 10 mV steps (bottom panels) in pacemaking (panel A) and non-pacemaking cell (panel B) isolated from dog LIPVs. Inset on top of current traces shows the clamp protocols.

DISCUSSION

In agreement with the findings of clinical investigations reported in literatures [4,5], the arrhythmogenic electrical activities of dog PV occurred less frequently in inferior PVs than in superior PVs. It is known that one important determinant of cardiac arrhythmias is the nature of myocardial substrates [7]. PV myocardial sleeve is a highly discontinuous tissue structure composed of inhomogeneous excitable cells. Since the electrophysiological properties of cardiomyocytes obtained from IPV and LSPV were similar, the longer myocardial sleeve existed in LSPV than in IPV [8] might provide larger cardiac structural and electrical substrates for arrhythmogenesis.

Acknowledgements: The present work was supported by grants NSC-90-2315-B-016-004 and NSC89-2314-B010-017 from the N.S.C. and grant DOD 91-06 from N.D.M.C., Taipei, Taiwan.

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