Comparison of Ito in young and adult human atrial myocytes: evidence for developmental changes


Crumb, William J., Jr., John D. Pigott, and Craig W. Clarkson. Comparison of Ito in young and adult human atria myocytes: evidence for developmental changes. Am. J. Physiol. 268 (Heart Circ. physiol. 37): Hl335-H1342, 1995.-In an effort to understand the ionic basis for the developmental changes that have been reported to occur in the configuration of the human atria1 action potential, we characterized the transient outward current (Ito) and the inward rectifier current in atria1 myocytes isolated from 20 young (ages 1 day-2.5yr) and 8 adult (11-68 yr) human hearts using the whole cell patch-clamp technique. We found evidence for statistically significant (P < 0.05) age-related changes in the Ito, including 1) the presence of an Ito, in only 67% of the cells isolated from young hearts vs. 100% of the cells isolated from adult hearts, 2) an almost twofold increase in the current density of Ito, in adult cells vs. young cells, and 3) recovery kinetics that are approximately twofold slower in young myocytes relative to adult myocytes. In contrast, there were no age-related changes found in the current density of the inward rectifier current or the sustained current measured after the decay of Ito. These results suggest important current-dependent changes that occur with age in human atria.
potassium currents; transient outward current; inward rectifier

STUDIES PUBLISHED during the past few years have documented that there are significant developmental changes in the density and characteristics of potassium currents observed in mammalian cardiac tissue. For example, in 1990, Kilborn and Fedida (10) documented that there are significant developmental changes in the transient outward current (Ito) in rat ventricular myocytes, with cells from l-day-old rats displaying a significantly smaller Ito compared with cells from adult rats. Similarly, in 1992, Jeck and Boyden (9) showed that neonatal canine ventricular myocytes completely lack a definable Ito in contrast to adult myocytes. Developmental changes have also been observed for the inward rectifier (Ito, with significant increases in the current density of Ikl occurring during postnatal development in both rat (14) and rabbit (8, 13) ventricular myocytes. Although these studies collectively suggest that there may be a common pattern of developmental changes in potassium channel expression in cardiac tissue in lower mammals, it is still unclear whether a similar pattern also occurs in higher primates or humans. Perhaps the best evidence for developmental changes in potassium currents in humans comes from the work of Escande et al. (4), who in 1985 documented that there are developmental changes in the shape, duration, and rate dependence of the duration of the atria1 action potential, as well as a developmental change in the sensitivity of phase 1 of the action potential to alteration by the potassium channel blocker, 4-aminopyridine. Escande et al. (4) also found that the shape of action potentials recorded from adult tissue could be made to mimic the shape of neonatal action potentials during exposure to concentrations of 4-aminopyridine known to block Ito. These results seem consistent with there being significant developmental changes in Ito in humans, similar to other species. However, to date, the only direct supporting evidence for this hypothesis has been the recent report that Ito is absent in atria1 cells isolated from the hearts of clearly diseased young individuals (ages 2 mo-5 yr) in contrast to cells from adult patients, which contain a robust Ito (11). Unfortunately, cardiac disease ( e. g ., cardiac hypertrophy) is also known to reduce significantly the amplitude of Ito, (2, 11). Therefore, it remains unclear whether the observation of an absence of Ito, in young diseased atria1 cells is due to disease, stage of development, or a combination of these two factors. In light of this controversy, we investigated whether developmental changes could be defined in potassium currents expressed in human tissue free from significant pathology. The primary purpose of this developmental study using human atria1 cells was to answer four basic questions: 1) Are there significant developmental changes in the amplitude dependence of &,? 2) Are there developmental changes in the time dependence of Ito 3) Are there developmental changes in the voltage-dependent behavior of Ito? and 4) Are there developmental changes in the IK1?

Materials And Methods

Isolation of cardiac myocytes. Human myocytes were obtained from specimens of human right atria1 appendage obtained during surgery from hearts of patients undergoing cardiopulmonary bypass. Tissue was obtained in accordance with Tulane University School of Medicine institutional guidelines. All atrial specimens were described as grossly normal at the time of excision. The cell isolation procedure was similar to that described in Fermini et al. (5) based on an earlier method by Escande et al. (3). Briefly, samples were quickly immersed in a cardioplegia solution consisting of (in mM) 50 KH2P04, 8 MgS04, 10 NaHC03,5 adenosine, 25 taurine, 140 glucose, and 100 mannitol, titrated to a pH of 7.4 and bubbled with 100% O2 at 0-4°C. Specimens were minced into 0.5- to l-mm cubes and were transferred to a 50-ml conical tube containing an ultralow calcium wash solution containing (in mM) 137 NaCl, 5 KH2P04, 1 MgS04, 10 taurine, 10 glucose, 5 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), and 100 PM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA); pH = 7.4 (22-24°C). The tissue was gently agitated by continuous bubbling with 100% O2 for 5 min. The tissue was next incubated in 5 ml of solution containing (in mM) 137 NaCl, 5 KHZP04, 1 MgS04, 10 taurine, 10 glucose, 5 HEPES, supplemented with 0.1% bovine albumin, 2.2 mg/ml collagenase (type V), and 1.0 mg/ml protease (type XXIV; Sigma Chemical), pH = 7.4 (37°C) and bubbled continuously with 100% O2.

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