Modulator - Issue No. 15

Regional expression of cardiac ion channels and cardiac electrical activity

Gernot Schram, M.D., Marc Pourrier, B.Sc., Peter Melnyk, B.Sc. and Stanley Nattel, M.D.


Important differences in electrophysiological properties have been noted between different regions of the heart. Electrophysiological heterogeneity has also been detected within different parts of a given tissue, such as the ventricular subendocardium, midmyocardium and subepicardium. Although many molecular candidates for native ionic currents have been identified, the molecular basis of most currents is not completely understood. Heterogeneity of channel protein composition might well underlie the differences observed among the properties of ionic currents or the shape of the action potential in different regions of the heart. Techniques such as immunocytochemistry, immunohistochemistry and Western blotting have played an important role in identifying tissue expression of channel proteins as well as their cellular localization. This review summarizes the electrophysiological differences observed in different regions of the heart and correlates them with information regarding expression patterns of ion channel subunits that might account for regional functional variation.

The inward rectifier current, IK1

IK1 is responsible for the maintenance of the cardiac resting potential and the final phase of cardiac repolarization. It is believed that differences between atrial and ventricular IK1 are responsible for the differences observed between the atrial and ventricular action potential. The atrial action potential has a short plateau phase and relatively slow repolarization, whereas the ventricular action potential has a long plateau and a much faster repolarization phase1. Patch clamp studies have revealed significant differences between atrial and ventricular IK1. Atrial IK1 has a 4-10 fold lower current density and conducts less outward current than ventricular IK11-3. It is believed that a smaller IK1 contributes to the less negative resting potential and slower terminal repolarization in atrial cells2.

Furukawa et al. reported a larger outward component of IK1 in endocardial cells than in epicardial cells in the cat. No differences in single channel conductance and open probability were observed, suggesting that observed differences are due to differences in current density and not to differences in molecular composition4. However, no differences in IK1 current density or kinetics have been found across the left ventricular wall in myocytes isolated from guinea pig5 or dog6. The observed discrepancies might be due to species-specific variations.

Clones of the Kir2 family are believed to underlie IK1. Four different α subunits have been cloned and shown to be present in the human heart2, 7-9. Kir2.1 mRNA is the most abundant subunit, with similar concentrations in atrium and ventricle2. Kir2.2 mRNA expression was relatively weak in both atrium and ventricle. Kir2.3 concentration is approximately 12-fold higher in atrium than in ventricle, but about 10-fold lower than Kir2.12. The abundance of Kir2.4 in human heart has not been determined. It has been speculated that the comparatively high abundance of Kir2.3, a lower-conductance subunit, in atrium might contribute to the smaller current density of atrial IK12. Recent data suggests that Kir5.1 can act like an endogenous dominant negative subunit, co-assemble with Kir2.1 and lead to the formation of nonconducting inward rectifier channels in the brain10. It is unclear if this mechanism operates in the heart. Nothing is known at present about the cellular localization of Kir2 subunits.

The delayed rectifier current, IK

IK consists of two distinct components, the rapidly activating IKr and the slowly activating IKs11. IK is the main phase 3 repolarizing current in the heart. Comparison of IK between canine right and left atrium showed a consistently higher current density of IKr in the left atrium12. No differences in current kinetics were observed. Western blot experiments showed a significantly greater expression of human ethera-go-go-related gene (Herg) protein in the left atrium, consistent with the electrophysiological data. No significant differences were found in IKs current densities12. These results are significant, given the prominent role of the left atrium in AF maintenance13, which may in part be due to abbreviated left atrial refractoriness due to larger IKr.

Comparison of Herg protein expression with functional IKr expression in rat found Herg protein and IKr to be more prominent in atria than in ventricles, whereas Herg protein expression in mice and humans was higher in the ventricle14. RNase protection assays found similar levels of Herg mRNA in rabbit left atrium, left ventricle, SA node and in canine Purkinje fibres, left and right ventricle suggesting that either interspecies differences or post-translational mechanisms may lead to differences in the level of expressed protein. Herg mRNA levels are ~1.5-fold more abundant than those of Kv4.3 in canine right ventricle15. Voltage clamp studies using left ventricular guinea pig myocytes revealed a higher IK current density in sub-epicardial and mid-myocardial than in endocardial myocytes.

Both components, IKr and IKs, were significantly smaller in sub-endocardial than in midmyocardial or sub-epicardial cells16. Furukawa et al. reported a higher current density of IK, with faster activation and delayed deactivation, in left ventricular epicardial compared to endocardial cells of the cat. No changes were found in open probability and single channel conductance leading to the suggestion that the observed differences in current density are due to a higher expression of channel protein and not to differences in molecular composition4.

Liu and Antzelevitch described a smaller IK in mid-myocardial cells compared to cells isolated from the endocardium or epicardium in dogs. Further evaluation showed that the observed difference was due to a significantly smaller IKs in mid-myocardium. No changes were found in the current density of IKr nor the kinetics of IKr or IKs in the three cell types17. In ferret, Herg mRNA transcripts and protein levels were found to be higher in epicardial cell than endocardial cell layers18.

To further investigate the lower IKs density in mid-myocardial cells Péréon and colleagues used RNase protection assays to determine KvLQT1 message expression across the human right and left ventricular wall. Overall KvLQT1 expression was similar throughout the ventricular wall. Midmyocardial cells, however, were found to contain a higher percentage of an endogenous N-terminal truncated KvLQT1 splice variant, referred to as KvLQT1 isoform 2, which acts as a dominant negative.

Co-expression of isoform 1 with isoform 2 in COS 7 cells in a stoichiometry mimicking that in midmyocardium resulted in a 75% reduction in current amplitude, consistent with the data provided by Liu & Antzelevitch. Current kinetics were not changed by co-expression with KvLQT1 isoform 2. KvLQT1 concentrations in atrium were found to be similar to those in endocardial and epicardial left and right ventricular cells. The regulatory subunit minK was found to be similarly expressed in midmyocardium, endocardial and epicardial left and right ventricular cells.

The transient outward K+ current, Ito

Regional differences in Ito are well described and contribute to the observed differences in action potential configuration in various species19-23. Ito density has been found to be greater in epicardial cells than in cells isolated from the endocardium in both dog20 and rat21,22. In human ventricle, Ito is larger in sub-epicardial than in subendocardial myocytes24. Differences in inactivation kinetics and 4-AP sensitivity have been found between atrial and ventricular human myocytes suggesting potential differences in their molecular basis23. Action potentials recorded from regions with a high Ito current density show a typical “spike and dome” configuration20.

Based upon differences in activation and inactivation kinetics as well as recovery from inactivation, Ito1 has been divided into two types, fast (Ito,f) and slow (Ito,s)25-31. Ito,s density is high in left ventricular epicardial myocytes in human28 and ferret27. It is believed that Kv4.2 and Kv4.3 contribute to the rapid component, Ito,f , and kv1.4 appears to underlie the slow component, Ito,s22. Important differences among various species as well as differences in the distribution of these subunits across the ventricular wall are well documented. The distribution of various subunits contributing to Ito has been most extensively studied in rat. In the atrium, only Kv4.2 has been found32, whereas both Kv4.2 and Kv4.3 have been identified in ventricle33. Kv4.3 and Kv1.4 mRNA expression across the ventricular wall of rat heart is almost uniform, whereas Kv4.2 shows a marked gradient with higher expression levels in epicardial than endocardial cells34, 35. Kv4.2 expression has been shown to parallel Ito current density across the left ventricular wall34 suggesting that Kv4.2 is the main subunit underlying Ito in rat.

Kv4.2 expression predominates in the right ventricular wall22. Confocal and immunohistochemical studies show that Kv4.2 is localized in the transverse-axial tubular system of the rat myocytes36. Kv4.3 and Kv1.4 may be proportionately more important in the septum22. Important differences have been found among various species. In the mouse, Kv1.4 has been found in high concentration in the septum and is believed to underlie Ito,s, whereas α subunits of the Kv4 subfamily underlie Ito,f in the ventricular apex and septum37. The properties of Ito in rabbit myocytes are similar to those of Kv1.4, suggesting an important role of Kv1.4 in rabbit Ito38-40In situ hybridization and immunohistochemistry has shown regional differences in the expression of Kv1.4 and Kv4.2/4.3 in ferret heart, suggesting that Kv1.4 and Kv4.2/4.3 underlie Ito in ferret left ventricular endocardial and epic ardial myocytes, respectively27. The molecular basis for canine and human Ito has been attributed to Kv4.335. RNase protection assays showed that neither Kv4.1 nor Kv4.2 mRNA is expressed at detectable levels in canine ventricular muscle, whereas Kv4.3 is abundant. Kv4.3 mRNA is expressed in human ventricle at similar levels to those found in canine ventricle35. Han et al. reported the presence of TEA sensitive Ito in canine Purkinje fibers. Their data suggest that the molecular basis of canine Purkinje Ito is different from Kv4.2/4.3 and Kv1.4, since these channels are TEA-insensitive. It has been proposed that Kv3.3 and Kv3.4 subunits might play an important role in Purkinje Ito, but the exact molecular basis still remains to be determined41.

The Na+-Ca2+ exchanger (NCX)

The Na+-Ca2+ exchanger (NCX) catalyzes the exchange of three Na+ for one Ca2+ across the plasma membrane in many mammalian cells42. The transport is reversible and can facilitate Ca2+ entry, leading to Ca2+ release from the sarcoplasmic reticulum43. The exchange activity is especially high in cardiomyocytes and plays a key role in the maintenance of Ca2+ homeostasis and relaxation of Ca2+ muscle42. The exchange of Ca2+ for sodium was first observed in guinea pig atria44. Western blotting and Northern blot experiments have shown increased expression levels of the NCX1 isoform in heart failure45-48.

NCX proteins in mammals are encoded by three genes: NCX1, NCX2 and NCX349-51. NCX2 and NCX3 are found only in the skeletal muscle and in the brain52. NCX1 transcripts undergo alternative splicing of 6 internal exons (A,B,C,D,E and F) to produce tissue-specific isoforms53. This splicing confers distinct functional characteristics to tissue-specific isoforms of the Na+-Ca2+ exchanger53-56. For example, the cardiac isoform (NCX1.1) is less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform. In addition, NCX1.1 is more sensitive to PKA activation than the renal form57. Komuro et al. cloned and characterized the human cardiac Na+-Ca2+ exchanger in 199258.

Confocal microscopy of adult guinea pig and rat heart cells has shown that the NCX is present in all membranes of the myocytes that face the extracellular space59. Dilly et al. showed NCX to be present on the surface and in T-tubule membranes on all cardiac myocytes60. Immunohistochemistry has revealed a specific localization in some types of cells59. Confocal microscopy has shown that in guinea pig myocytes, the NCX is located at the intercalated disks, the transverse tubules and exterior surface of the membrane61. In rabbit myocytes, the NCX appears to be more prominent in T-tubule membranes than in peripheral sarcolemma62.

The calcium current, ICa

Two principal Ca2+ currents have been described. The voltage-gated L-type (”long lasting” ) ICa is responsible for triggering sarcoplasmic reticulum Ca2+ release and consequently the initiation of excitation contraction coupling in heart63-65 and for the plateau phase of the action potential66. L-type Ca2+ channels cluster in the surface plasma membrane overlying junctional sarcoplasmic reticulum in guinea pig myocytes67. N, P, Q and R-type channels have also been identified66,68,69 as high voltage activated calcium channels70 but appear not to be expressed in cardiomyocytes.

T-type (”transient”) ICa inactivates very rapidly and at relatively negative potentials64. It is found mainly in sinoatrial node, Purkinje or atrial cells, but its expression is also species dependent64. The fact that ICa.T is at high density in nodal cells71 and embryonic cardiomyocytes67 ,72 suggests that it is important in pacemaker function65,67. T-type current is present in the guinea pig heart but has not been found in rat or in human adult myocytes67,73.

L-type channels were initially purified from skeletal muscle74. They are heteromultimers of at least 3 different subunits: α1C, β and α2δ. The α1C subunit encodes the basic pore-forming protein of the channel, whereas the auxiliary β subunit modulates the expression, the open probability, activation and inactivation67,75-77. The α2δ subunit is a disulfide-linked dimer78-80 and is ubiquitously expressed in all types of high voltage-dependent Ca2+ channels81. The α1C subunit is encoded by three different genes (CaCh1-3). Only the product of CaCh2a is present in the heart. Four β subunit genes have been identified (β1 to β4). Genes encoding ICa.T were identified in human heart in 199870,82. The molecular basis for T-type calcium channels has been associated with α1G, α1H and α1I genes70,82,83. α1G and α1H are expressed in heart and brain at different levels70. The cell or tissue specificity hasn’t yet been established. Immunoconfocal and immunogold electron microscopy labelling were used81 to show the distribution of L-type Ca2+ channels in cardiac myocytes isolated from rabbit and rat ventricle. The channels were localized on the surface of the plasma membrane and transverse T-tubules in rabbit myocytes, whereas the labelling was more intense in T-tubules than in the surface sarcolemma in rat myocytes.

Connexins/Gap Junctions

Gap junctions are more than simple conduits allowing for enhanced current flow between adjacent cardiac myocytes: they allow for the passage of various cations, anions and small, non-charged molecules from one cell to another. Though gap junctions are not directly affected by membrane potential, they can respond to differences in transcellular voltage by altering their conductance84.

As determined by immunocytochemistry, among the channel proteins expressed in cardiac gap junctional plaques are three principal connexins: connexin (Cx)40, Cx43 and Cx4585. Two other connexins – Cx37 and Cx46 – have also been identified in the heart, albeit in trace amounts86. Different ratios of these connexins give rise to multiple gap junction phenotypes. These different phenotypes appear to have a deliberate and functionally important distribution pattern throughout the heart. Electron microscopy has shown SA nodal myocytes to have small, scattered gap junctions87. This finding has implications regarding conduction velocity in this region, which is very slow88. The number and distribution of gap junctions, as well as their composition, are important factors in modulating conduction propagation velocity. In a study of mice heterozygous for a null mutation in the Cx43 gene (Cx43 +/- mice), it was determined that this mutation was responsible for a reduction in the total number of gap junctions while the remaining gap junctions were unaffected in terms of size89. These mice have a ~40% slower ventricular epicardial conduction velocity90.

Cx43 is the most abundant gap junctional channel in human91 and rat89 ventricles and atria. The expression level of Cx43 in the four chambers of the human heart is more or less uniform91. Studies have shown that the human SA node has little92 or no Cx4386. Western blotting of the AV node reveals a weak signal found primarily in the middle of the region86, consistent with the important slowly-conducting properties of the AV node. When Purkinje fibres are probed with an antibody for Cx43, the signal is more intense at longitudinal ends than transversely86. This distribution pattern is likely important in the known functionally-important anisotropy of conduction.

Cx40 is expressed in human atrium91 and to a much lesser degree in ventricular subendocardium86 A study of Cx40 expression in dog also showed that there was a much higher level of this channel in the crista terminalis compared with left ventricular subepicardium93. In both man86 and dog85, Purkinje fibres show a far more robust signal for Cx40 than ventricular myocardium. Canine92 and human86 SA nodal cells clearly express Cx40. Human AV nodal cells also express Cx4086.

Low levels of Cx45 can be detected in human ventricles, with more being seen in the atria91. Both rat94 and human86 conduction systems (including the SA node, AV node and Purkinje fibres) have an abundant expression of Cx45. In a recent study of Cx45 knockout mice, it was found that these mice die of heart failure at embryonic day 10. Though the hearts of these mice do contract, conduction block through the AV node region is observed95.

The pacemaker current If

A slow membrane depolarization phase between action potentials is responsible for the rhythmic activity of the heart96, 97. The principal current underlying this phenomenon is referred to as If or Ih. Both Na+ and K+ ions carry this current with the selectivity being fourfold higher for K+.

If is activated on membrane hyperpolarization. The hyperpolarization-activated cyclic nucleotide gated cation (HCN) family of channels have many of the characteristics of If channels. Four members (HCN1-4) have been isolated from mouse, rabbit and human tissues98-101, and share a homology of ~60% on the amino acid level101. Northern blot showed that only HCN2 and HCN4 are expressed in the human heart. HCN2 currents activate faster than HCN4. Two different native currents with distinct kinetics have been identified in the heart102-104. Semi quantitative RT-PCR showed that human HCN2 and HCN4 mRNA is found to be approximately equally abundant throughout the atria and ventricles105. Northern blotting revealed that in rabbit, HCN4 mRNA is more abundant in SA nodal cells than in ventricle or atrium100. SA nodal total HCN message in general is 140 times the HCN message for the ventricle and 25 times that of Purkinje fibres106, corresponding to the primary pacemaking role of the SA node and the greater pacemaking activity in Purkinje fibres compared to working muscle.

The sodium current, INa

INa is responsible for the rising phase (phase 0 upstroke) of action potentials in electrically excitable cells78, 107, 108 and for rapid impulse conduction through cardiac tissue107.

The functional channel consists of a principal α pore-forming subunit composed of four homologous domains ( I- IV) of six transmembrane segments S1-S6107 and 2 distinct β-subunits involved in the modulation of channel gating and cell surface expression. Ten genes encoding Na+ channel α-subunits have been described (NaV1.1 to NaV1.9, and NaX)108 and 3 β-subunits (β1 to β3) have been identified to date108-110. Most of the subunits in the NaV1.x family have been studied in heterologous expression systems and, therefore, the most detailed observations are for this family. At least 2 α- subunit mRNAs are expressed in human heart: NaV1.5 and NaV2.1108,111,112. β1- Subunit mRNA is expressed in rat and human heart113,114 but not found in mouse heart115. β1 and β2 subunits have been localized in rat and mouse heart by immunofluorescence111. Both proteins are expressed in cardiac muscle along the Z lines111. The differences in the functional properties of Na+ channel isoforms result in unique conductances in specific cell types108. Immunocytochemical studies have shown that β1, β2, NaV1.1 and NaV1.5 localize along Z lines in adult rat and mouse cardiac myocytes111,116. NaV1.5 has been localized to the surface and T-tubular system of rat hearts and does not display a large variation other than a somewhat enhanced labelling at the intercalated disks of ventricular myocytes116. It is possible that this enhanced localization is related to fast conduction in ventricular myocardium. In the compact rabbit AV node, a gradient of Na+ channel expression exists, with peripherally-located cells having stronger signals compared with centrally located ones117. This gradient is likely important in the slow conduction through this region.

Functional importance

The cardiac action potential consists of a fine interplay between a large number of inward and outward currents of which the main ones are carried by K+, Na+ or Ca2+ ions. Characteristic action potential shapes have been found in different regions of the heart as well as in different layers of the ventricular wall26,118. Clones encoding a large number of channel proteins have been identified, some of which clearly reconstitute native channel properties35, 34, 94, 119, 120. However, many discrepancies between native currents and putative underlying clones remain to be resolved.

Important regional differences in IK11 are not readily explained by the mRNA distribution of corresponding Kir2 family subunits2.

Future studies using techniques like Western blotting, immunocytochemistry and immunohistochemistry may help to determine Kir2 protein expression levels, tissue expression pattern and cellular localization of Kir2 subunits giving further insight into the molecular basis of IK1. Transmural gradients in Ito are likely important in governing action potential shape and explaining many important ECG phenomena6, 118, 120-123. M-cells have longer action potentials than in other ventricular regions, playing an important role in arrhythmias due to excessive prolongation of repolarization (long QT syndrome). Transmural gradients in IKs17, likely related to differential distribution of KvLQT1 isoforms124, is probably responsible. Ito downregulation may be important in ventricular arrhythmogenesis in heart failure and appears to parallel a decrease in Kv4.3 mRNA125, although the nature of any regional or transmural differences is not well-known. Changes in connexin expression may also contribute to arrhythmogenesis. For example, Peters et al. observed a reduction in ventricular Cx43 signal from hypertrophied and infarcted human hearts126.

The above are only limited examples of the importance of regional variations in ion channel expression in physiological and pathological states. In addition to differential expression of pore- forming subunits, the formation of functionally distinct heterotetramers and the presence of regulatory subunits likely contribute importantly to regional diversity in channel function, and relatively little is known about them. Much more work clearly remains to be done in this important area.

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