Faculty Bibliography

Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca2+ entry via Na-Ca exchange current (INaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1-5 day), juvenile (JV, 10-14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD90: 108-378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (QExout) and inward (QExin) Ni2+-sensitive charge movement in response to AP prolongation. QExout and QExin measured during age-appropriate APs declined postnatally [QEXout: NB (2 day) 0.19 +/- 0.02, JV (10 day) 0.10 +/- 0.01, AD 0.04 +/- 0.002; QEXin: NB -0. 2 +/- 0.01, JV -0.11 +/- 0.02; AD -0.04 +/- 0.003 pC/pF] despite the significantly shorter APD90 of newborn myocytes (NB 122 +/- 10; AD 268 +/- 22 ms). When Ca2+ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 +/- 0.5, JV 1.2 +/- 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth

The cardiac inward rectifying K+ current (IK1) is important in maintaining the maximum diastolic potential. We used antisense oligonucleotides to determine the role of Kir2.1 channel proteins in the genesis of native rat ventricular IK1. A combination of two antisense phosphorothioate oligonucleotides inhibited heterologously expressed Kir2.1 currents in Xenopus oocytes, either when coinjected with Kir2.1 cRNA or when applied in the incubation medium. Specificity was demonstrated by the lack of inhibition of Kir2.2 and Kir2.3 currents in oocytes. In rat ventricular myocytes (4-5 days culture), these oligonucleotides caused a significant reduction of whole cell IK1 (without reducing the transient outward K+ current or the L-type Ca2+ current). Cell-attached patches demonstrated the occurrence of multiple channel events in control myocytes (8, 14, 21, 35, 43, and 80 pS). The 21-pS channel was specifically knocked down in antisense-treated myocytes (fewer patches contained this channel, and its open frequency was reduced). These results demonstrate that the Kir2.1 gene encodes a specific native 21-pS K(+)-channel protein and that this channel has an essential role in the genesis of cardiac IK1

Previous indirect studies of newborn hearts have suggested a diminished functional role of the SR and a greater dependency upon trans-sarcolemmal Ca2+ fluxes to directly elicit contraction and promote relaxation. We tested the hypothesis that the SR in newborn rabbit hearts is functionally incompetent by measuring contraction and relaxation in ventricular myocytes isolated from the hearts of 1-2-day-old (newborn), 10-12-day-old (juvenile) and >150-day-old (adult) rabbits. Electrically stimulated twitch characteristics were compared to those elicited by the rapid application of 10 mm caffeine in the presence and absence of functional sarcolemmal Na-Ca exchange (disabled using a Na+- and Ca2+-free extracellular solution). During steady state, electrically-induced contractions were lower in amplitude in newborn and juvenile compared to adult myocytes (2.9+/-0.5 and 3.4+/-0.3 v 8.5+/-0.9% of resting cell length, respectively; n=24-29) and relaxation was slower in immature myocytes (t0.75 values: newborn 250+/-20; juvenile 240+/-10; adult 130+/-20 ms, n=14-21). Contrary to our hypothesis, caffeine triggered sufficient SR Ca2+ release from immature myocytes to elicit contractions of similar magnitude to adults (newborn 12.8+/-1. 1; juvenile 14.0+/-0.9; adult 15.0+/-1.6% of resting cell length, n=25-29). The amplitude of indo-1 Ca2+ transients during steady-state twitch was 36+/-12% of the maximal caffeine-induced Ca2+ transient in newborns (n=6) and 59+/-4% in adults (n=6). Caffeine slightly prolonged relaxation in adult myocytes (t0. 75=200+/-30 ms), but accelerated relaxation in newborn and juvenile myocytes (t0.75=180+/-20 and 150+/-30 ms, respectively). When both the SR and Na-Ca exchanger were disabled, the rate of relaxation (attributable to the sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter) of newborn and juvenile myocytes was significantly faster than in the adults (1660+/-210 and 3030+/-180 v 4530+/-310 ms, respectively; n=14-21). We conclude that neonatal and adult rabbit ventricular myocytes have comparable SR Ca2+ load, but neonatal cells exhibit smaller fractional SR Ca2+ release during steady-state contractions and greater Ca2+ removal by sarcolemmal Na-Ca exchange during relaxation.

Current evidence suggests that members of the Kv4 subfamily may encode native cardiac transient outward current (I(to)). Antisense hybrid-arrest with oligonucleotides targeted to Kv4 mRNAs specifically inhibited rat ventricular I(to), supporting this hypothesis. To determine whether protein kinase C (PKC) affects I(to) by an action on these molecular components, we compared the effects of PKC activation on Kv4.2 and Kv4.3 currents expressed in Xenopus oocytes and rat ventricular I(to). Phorbol 12-myristate 13-acetate (PMA) suppressed both Kv4.2 and Kv4.3 currents as well as native I(to), but not after preincubation with PKC inhibitors (e.g., chelerythrine). An inactive stereoisomer of PMA had no effect. Phenylephrine or carbachol inhibited Kv4 currents only when coexpressed, respectively, with alpha1C-adrenergic or M1 muscarinic receptors (this inhibition was also prevented by chelerythrine). The voltage dependence and inactivation kinetics of Kv4.2 were unchanged by PKC, but small effects on the rates of inactivation and recovery from inactivation of native I(to) were observed. Thus Kv4.2 and Kv4.3 proteins are important subunits of native rat ventricular I(to), and PKC appears to reduce this current by affecting the molecular components of the channels mediating I(to).