Skip to main content
  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
  • Impact Factor 13.965
  • Facebook
  • Twitter

  • My alerts
  • Sign In
  • Join

  • Advanced search

Header Publisher Menu

  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

Circulation Research

  • My alerts
  • Sign In
  • Join

  • Impact Factor 13.965
  • Facebook
  • Twitter
  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
Reviews

Regulation of Ion Channel Expression

Barbara Rosati, David McKinnon
Download PDF
https://doi.org/10.1161/01.RES.0000124921.81025.1F
Circulation Research. 2004;94:874-883
Originally published April 15, 2004
Barbara Rosati
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David McKinnon
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Tables
  • Info & Metrics

Jump to

  • Article
    • Abstract
    • An Ideal Model of Homeostatic Regulation
    • Regulated Parameters and Biological Feedback
    • Limitations of Cardiac Ion Channel Regulation
    • Is There Any Feedback System Capable of Sensing Changes in Electrophysiological Phenotype?
    • Compensatory Pathways
    • Cognate Pathways
    • Is Transcriptional Regulation a Suitable Mechanism for the Control of Ion Channel Expression?
    • Future Directions
    • Conclusions
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Tables
  • Info & Metrics
  • eLetters
Loading

Abstract

A potentially important mechanism controlling ion channel expression is homeostatic regulation, which can act to maintain a stable electrophysiological phenotype in cardiac myocytes as well as to provide plasticity in response to genetic, pathological, or pharmacological insults. The capabilities and limitations of the homeostatic regulatory mechanisms that contribute to the control of cardiac ion channel expression are the primary topic of this review.

  • heart
  • ion channel
  • gene regulation

This Review is part of a thematic series on the Biology of Cardiac Arrhythmias, which includes the following articles:



Antiarrhythmic Drug Target Choices and Screening
Inherited Arrhythmogenic Diseases: The Complexity Beyond Monogenic Disorders
Genomics in Sudden Cardiac Death
Regulation of Ion Channel Expression
Ion Channel Protein Processing Computational Insights: Chaos and Wave Theory
Gene Therapy and Cell Therapy of Cardiac Arrhythmias


This series is in honor of Harry A. Fozzard, 8th Editor of Circulation Research.
Gordon Tomaselli Editors

Much of the initial work on the molecular biology of cardiac ion channel genes focused on a single issue: identifying the molecular components of the ion channels that were identified so painstakingly during decades of prior electrophysiology studies.1 Because of the efforts of a large number of scientists, this research program has been largely successful, and we now have most of the tools necessary to study how virtually any cardiac ion channel is regulated at the molecular level. It is appropriate at this juncture, therefore, to ask what issues must be addressed to understand how ion channel genes are regulated and what steps can be taken to gain a more detailed understanding of this regulation.

Three processes determine the number and kinds of ion channels expressed in any given cardiac myocyte. These are developmental regulation, homeostatic regulation, and evolution.

Developmental regulation typically involves activation of complex programs of gene regulation affecting large numbers of genes to produce predetermined outcomes.2 During the course of cardiac development, a program of gene expression plays out in a stereotypical fashion, resulting in the production of several phenotypically distinct subsets of cardiac myocytes in the adult organism.3,4 As one aspect of a larger pattern of tissue-specific gene regulation, these differentiated cell types display marked differences in ion channel expression. For example, myocytes in the SA node have multiple unique phenotypic properties and represent a differentiated state distinct from that of the surrounding atrial myocytes.5 As part of this program of differentiation, the expression pattern of ion channel genes important for pacemaker function is differentiated in the sinoatrial (SA) node. In particular, there is a unique and very sharp upregulation of HCN1, HCN4, and Cav3.1 gene expression in SA nodal myocytes.6–8 Similar tissue-specific patterns of ion channel expression are seen in other cardiac tissues.9,10 The study of how cardiac tissues differentiate through developmentally regulated programs of gene expression is a very active research area that is largely outside the scope of this review (recent reviews include those by Cripps and Olson4 and Solloway and Harvey11). It is likely, however, that advances in virtually any aspect of the understanding of the molecular aspects of cardiac development and differentiation will have implications for our understanding of the regulation of cardiac ion channel expression, because these channels are integral to the function of the heart.

Homeostatic regulation refers to two related processes in the adult heart. It is a mechanism by which individual cells can monitor and maintain a stable phenotype under constant physiological conditions. Homeostatic regulation also provides a degree of plasticity for the adult myocyte, allowing it to respond to changing physiological demands. In general, homeostatic regulation acts with high specificity, regulating the expression of a relatively small number of genes and their products in response to physiological signals and generating small changes in phenotype rather than large-scale differentiation events. In this respect, it is directly analogous to the regulation of metabolic enzyme genes in bacteria and yeast,12 where physiological stimuli such as changes in the level of various metabolites are monitored and the response is the upregulation or downregulation of the appropriate metabolic enzymes. The capabilities and limitations of the homeostatic regulatory mechanisms that control cardiac ion channel expression are only now coming into focus and comprise the primary topic of this review.

Homeostatic regulation also acts during the course of development to help adjust the physiological properties of the heart to the changing demands placed on fetal, neonatal, and juvenile cardiovascular systems. The robustness of cardiac development13,14 is likely to reflect, at least in part, the feedback action of homeostatic regulation. Presently there is limited experimental work directly addressing the interplay of homeostatic regulation and development in heart. More detailed studies have been performed on developing neurons and skeletal muscle,15–17 and it is to be anticipated that there will be similarities with the heart, particularly in the roles of electrical activity and calcium signaling.

Striking differences in the patterns of ion channel expression in the hearts of different species have developed during the course of mammalian evolution.9,18 These species-specific differences are more dramatic in cardiac myocytes than in other comparable, electrically excitable cell types, eg, sympathetic neurons. In particular, there is a systematic decrease in heart rate with increasing body size, and this is accompanied by a corresponding increase in action potential duration as well as changes in action potential morphology. An example of evolutionary effects on channel expression patterns is the absence of Ito expression in guinea pigs.19,20 Both humans and rodents, which bracket the guinea pig both phylogenetically21 and in terms of body size, express Ito currents with similar biophysical properties. Misexpression of Ito in guinea pig myocytes results in premature action potential repolarization,22 suggesting that guinea pig electrophysiology has evolved in such a way that significant Ito expression would be deleterious to normal function. Why this has occurred is unknown.

In addition to quantitative changes in channel expression, evolution can produce qualitative changes in ion channel function. For example, in rodents, Ito is a primary repolarizing current and strongly affects action potential duration.23 In contrast, in large mammals, Ito has little or no effect on action potential duration. In large mammals, the regulatory apparatus ensures that the Ito does not become so large relative to depolarizing currents that it produces premature repolarization,24 whereas in rats it ensures that the Ito is large enough to produce rapid repolarization. How these different physiological roles are reflected in the homeostatic regulatory apparatus controlling expression of this current is not known.

An Ideal Model of Homeostatic Regulation

Before discussing homeostatic regulation, it is useful to first consider a simple model of possible homeostatic mechanisms to help organize the presently fragmented experimental data.

Voltage-gated ion channels are multimeric membrane proteins with a complicated biosynthetic pathway,25 and, as such, the expression of functional channels in the plasma membrane potentially can be regulated at multiple levels. An ideal physiological regulatory system for the control of ion channel expression in heart would look something like that shown in Figure 1. In such a system, there are multiple levels of control and multiple fail-safe systems. Channel expression can be modified at several levels along the biosynthetic pathway. As a consequence, if a partial failure occurs at an early step in the pathway, there can be changes in biosynthesis rates at downstream levels that help to maintain the final phenotype. Information about the final product, in this case the electrophysiological phenotype, would feed back into the biosynthesis pathway via both cognate and compensatory feedback pathways to maintain the phenotype within limits appropriate to the function of a particular cell type and the physiological stresses on that cell.

Figure1
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 1. Idealized model of homeostatic regulation of ion channel expression. Black arrows represent the biosynthetic or efferent pathway of the regulatory circuit. Colored arrows represent the feedback or afferent arm of the circuit. The feedback pathway has two components. The cognate feedback pathway (crimson) regulates expression within the biosynthetic pathway of a single gene. The compensatory feedback pathway (purple) regulates the expression of complementary or antagonistic genes. The modulatory pathway (teal) represents all the ways in which neurohumoral signal molecules can produce covalent or allosteric modification of channel function. These changes are generally reversible, resulting in transient changes in the level of functional channel expression.

Regulated Parameters and Biological Feedback

The first issue to address in trying to understand the homeostatic regulation of ion channel expression is to determine exactly what is regulated. In the idealized model, the primary regulated parameter is the electrophysiological phenotype, or the number of each type of functional ion channel expressed in the cell membrane. Myocytes do not seem to have any mechanism that keeps track of the absolute numbers of a particular ion channel in the cell membrane. This implies that any feedback signal is likely to be a complicated reflection of the actions of many different ion channels and auxiliary proteins. Of the electrophysiological properties listed in Table 1, none are thought to be directly sensed, in large part because there is presently no established mechanism by which the myocyte can directly sense the trajectory of its membrane potential. Although voltage-gated ion channels are membrane potential sensors par excellence, to date there is little evidence that the voltage sensor function of ion channels has any feedback function beyond the immediate regulation of ion flows.

View this table:
  • View inline
  • View popup
Table 1.

Physiological Properties That Are Dependent on the Number and Type of Ion Channels Expressed

A compounding problem is that the syncytial properties of the myocardium will limit the value of the membrane potential as a source of information for the regulation of ion channel expression at the cellular level. An individual myocyte could express no ion channels and still have a membrane voltage profile similar to that of its neighbors, although there may be some blunting of the fastest components of the membrane voltage signal because of cable properties.

This seems to leave changes in intracellular ion concentrations as the only source of feedback regarding the electrophysiological behavior of individual myocytes. However, ion concentrations are an even more indirect measure of ion channel expression than membrane voltage because they are also strongly dependent on the concentrations and function of buffers, pumps, and transporters.

Steady-state calcium concentrations will represent weighted averages with surrounding cells, but dynamic changes in cell calcium levels will be cell specific. The relatively large size of the myocytes combined with the slow rate of intracellular calcium ion diffusion means that dynamic changes in calcium levels will be transmitted poorly from cell to cell. Because of this, transient calcium concentration changes are likely to reflect, albeit indirectly, the electrophysiological behavior of individual cells. If this is the only physiological property that can provide feedback to the regulatory apparatus, it is likely that there is a considerable loss of information.

It is widely accepted, for many different electrically excitable cells, that voltage-gated calcium channels provide an indirect measure of membrane voltage changes and that the subsequent influx of calcium ions can trigger changes in gene expression, including changes in ion channel expression.16,26,27 Calcium ions influence gene expression through multiple transcription factors; some well-studied examples include members of the CREB, NFAT, and MEF2 families,28–31 although many more transcription factors may be sensitive to changing calcium levels.32 A broadly held assumption is that calcium channels represent the most important feedback link between electrical activity and the regulation of ion channel gene expression, in large part because there is no other well-established pathway. It has been proposed that the flux of other ions, including Na+ and Zn2+, can also affect either gene expression or ion channel expression in excitable cells.33–35

Further removed from the electrical activity of the cell is the contractile activity induced by electrical excitation. Mechanical stress can affect gene expression in myocytes via intrinsic mechanoreceptors or via autocrine/paracrine pathways.36,37 Whether it has a direct role in the regulation of ion channel expression in vivo is not clearly established.

Analogous to mechanical stress, ion channel expression can be influenced by both intrinsic pathways (Ca2+ ions) and extrinsic pathways (neurohumoral). The extrinsic pathways are potentially numerous. A range of signaling molecules has been implicated in regulating long-term changes in ion channel expression (a partial list is shown in Table 2), with delivery to myocytes mediated through either neuronal release or autocrine/paracrine/endocrine systems. Many of these molecules have pleiotropic effects, however, and the changes in ion channel expression seen in vivo may be secondary or tertiary responses. The broader the action of a particular molecule, the less likely it is to form part of a feedback pathway specifically regulating ion channel expression.

View this table:
  • View inline
  • View popup
Table 2.

Signaling Molecules That Produce Changes in Ion Channel Gene Expression

Some of these neurohumoral pathways are active in normal adult hearts and could therefore act as part of homeostatic feedback loops. For example, the sympathetic nervous system through the actions of the signaling molecules norepinephrine and neuropeptide Y (NPY) regulates ion channel expression in normal hearts.38,39 Whether it simply provides a chronic, unregulated input or can be activated in such a way as to provide meaningful feedback in response to changes in the state of the electrophysiological phenotype of the cardiac myocytes that it innervates is not established. What the source of sensory information would be and whether there is sufficient specificity in the connectivity of the sympathetic system to produce targeted delivery of signaling molecules to different regions in the heart is also unknown.

Several of the molecules listed in Table 2 are thought to be released via autocrine/paracrine pathways,40,41 and these are the molecules most likely to produce specific actions on cardiac ion channel expression. Whether these molecules exert a tonic influence on channel expression or participate in physiological feedback loops in normal adult hearts has not been definitively established.

The changes in ion channel expression that are induced in several pathophysiological conditions, including cardiac hypertrophy,42 myocardial infarction,43 thyroid dysfunction,44 and diabetes,45 reflect in part the activation of neurohumoral pathways. In every case, however, these are complex responses that can also involve the reactivation of developmental pathways46 and tissue remodeling pathways.47 Although the changes in ion channel expression seen in these pathologies may reflect appropriate responses to changing demands on the electrical function of the heart, at least in some cases these changes seem to be epiphenomena that may actually contribute to the pathology.42

Limitations of Cardiac Ion Channel Regulation

The myocardium could still approach the performance of an idealized system if it could make unexpectedly effective use of the feedback information that is available. Alternatively, we may not understand the full nature of the feedback information available. To date, neither of these possibilities seems to be the case, and, if anything, the system seems more insensitive than might be expected.

The most basic fail-safe system in both the regulatory model (Figure 2A) and in biological systems is the two alleles for each ion channel gene. One potential response to a loss-of-function mutation in one allele would be to increase the expression of the other allele at the level of transcription. Alternatively, the efficiency of posttranscriptional biosynthesis could be increased through mechanisms such as increased channel subunit synthesis, increased transport of assembled channels to the cell surface, or reduced degradation rates. Any of these mechanisms could, in principle, be activated through a cognate feedback pathway (Figure 1) so that the final phenotype would be affected imperceptibly.

Figure2
  • Download figure
  • Open in new tab
  • Download powerpoint

Figure 2. In recessive loss-of-function mutations, the loss of one allele can be compensated by increased gene transcription or increased efficiency of the biosynthetic pathway. In haploinsufficiency, there is either no effective feedback mechanism or no flexibility to increase channel production. For the wild-type (A), both alleles of the gene encoding a given ion channel contribute in equal amounts (×1) to the production of mRNA, which is in turn translated into functional channels that play a role in the determination of the wild-type electrophysiological phenotype. In the case of a loss-of-function recessive mutation in one of the alleles (B and C), compensation can occur at two different levels. Either there is an increase in the transcription rate of the normal allele that reconstitutes the normal (×2) levels of mRNA for that channel (B) or there is a posttranscriptional compensatory mechanism that produces the same amount of functional channels (2 years) from half the normal amount of mRNA (C). In haploinsufficiency (D), there is no effective feedback pathway to initiate a compensatory response, and as a consequence there is a reduced level of channel expression.

In practice, loss-of-function mutations in a single allele often produce no detectable phenotype.48 Even a significant fraction, possibly a majority, of homozygous gene knockouts have limited or no phenotypic expression under laboratory conditions, indicating the activation of compensatory pathways or the presence of functional redundancies in the system.49–51 One potential contributing factor to this genetic robustness, which is seen in both heterozygous and homozygous knockouts, is likely to be homeostatic regulation.52,53 In the case of heterozygous knockouts, homeostatic feedback from an expressed phenotype could result in either an increase in transcription from the second allele or increased production of functional proteins from a reduced level of mRNA (Figures 2B and 2C). Activation of either mechanism would result in the maintenance of a constant level of channel production and a maintained electrophysiological phenotype.

The phenomenon of haploinsufficiency (Figure 2D) demonstrates that the fail-safe systems that might be expected in an ideal regulatory system are lacking for at least some cardiac ion channels. Loss-of-function mutations in either the KCNQ154–56 or KCNH2 (HERG)57,58 potassium channel genes can produce long-QT syndrome, even when the gene product encoded by the mutant allele is functionally silent and has little or no effect on the subunits produced from the normal allele. Despite the continued presence of one wild-type allele and at least eight potential levels of regulation downstream from transcriptional regulation (Figure 1), there is not enough flexibility within any one of these levels to compensate adequately for the loss.

The likelihood that heterozygosity for a homozygous-null mutation will produce a haploinsufficiency defect depends strongly on the nature of the gene that is mutated.48 Null mutations in transcription factor genes seem to have a high probability of producing haploinsufficiency defects,48,59 unless they contribute a feedback signal to their own regulatory pathway.60 This sensitivity is not entirely surprising, because transcription factors act at the top of a hierarchy of genes and a reduction in transcription factor production will produce pleiotropic effects that will be difficult to sense in a way that could provide intelligible feedback to the regulatory apparatus. In contrast, ion channels are final effector proteins whose expression controls, in a relatively simple way, the final phenotype of electrically excitable cells. In this case, there is a direct relationship between the product, the electrophysiological phenotype, and its primary constituent components, the ion channels. It would seem to be a much simpler problem to devise quality controls for the final product. Yet the loss of one allele of one potassium channel gene can result in a significantly increased mortality rate.61 One might not have expected a priori that the cardiac system would be quite so vulnerable to the loss of a single copy of a single potassium channel gene.

Even in the case of mutations in the KCNQ1 and KCNH2 genes that produce nonfunctional, dominant-negative subunits, expression of wild-type channels is not entirely eliminated,54,57 and increased transcriptional activity or posttranscriptional processing would reduce the physiological effect of the mutation. In addition, compensatory mechanisms, such as the upregulation of expression of other potassium channels, could be activated to largely eliminate the functional impact of the mutation.

These results suggest that there are constraints on the system over and above those of good engineering design principles. Although genetic robustness can compensate for a certain level of genetic variability within a species and the resultant variability may actually be selectively favored,52 there may be limits to the adaptive advantage that this process confers. If mechanisms for the compensation of genetic defects become too effective, the load of defective genes within a population may become too high, with negative long-term consequences for individual reproductive fitness. The need to prune out the more extreme genetic defects may be a competing process, resulting in limited evolutionary pressure to create a perfectly robust system.

Is There Any Feedback System Capable of Sensing Changes in Electrophysiological Phenotype?

The schema shown in Figure 1 assumes that there is a feedback system that is sensitive to electrophysiological phenotype. The observation that a 50% reduction in KCNQ1 or KCNH2 gene dosage can disrupt normal cardiac electrical function calls this assumption into question. There are, however, examples of homeostatic regulation of ion channel expression in the heart that support the idea that feedback mechanisms exist.

In mice that have a dominant-negative knockout of the Kv4.2 and Kv4.3 channels, which underlie Ito,f, expression of the Kv1.4 channel is significantly and selectively upregulated.62,63 Presumably, the cardiac system senses that there is something wrong with the electrical function of the heart, possibly because of an abnormal prolongation of the ventricular action potential duration in the knockout animals,64 and acts to compensate for this defect, in this case via a compensatory feedback pathway. This result suggests that there is a mechanism that can sense and initiate a response to prolongation of the ventricular action potential duration in mouse heart.

Similarly, treatment with the sodium channel blocker mexiletine results in the upregulation of sodium channel expression65 attributable to increased sodium channel gene transcription.66 Treatment with the calcium channel blocker verapamil produces a similar effect to that seen with sodium channel blockade, suggesting that the response to sodium channel blockade is mediated by changes in calcium ion fluxes.66 In this case, homeostatic regulation seems to act in response to a reduction in the number of functional sodium channels, suggesting the presence of a feedback pathway in rat heart.

An indirect argument favoring the possibility that a feedback system also exists in humans is the relatively tight control of ventricular action potential duration in the normal adult population. The hallmark clinical manifestation of LQT syndrome (lengthening of the QT interval) is only detectable because the normal range of variation is maintained within relatively narrow limits.67 It is hard to imagine that the trajectory of development could be so reliably predetermined as to arrive at such a consistent final end point in the absence of at least some feedback.

Compensatory Pathways

Compensation for the loss of an allele is not limited to modulation of the output of the other allele via a cognate feedback system (Figure 1). Changes in the expression of genes that encode channels with similar functions could be initiated by compensatory feedback pathways, and this could partially or completely compensate for the loss of one or even both alleles.

One example of compensatory homeostatic regulation is seen in calcium channel Cav1.2 knockout mice. Expression of the Cav1.3 gene is significantly upregulated during embryonic development in response to knockout of the Cav1.2 gene.68 Activation of this compensatory pathway seems to extend the embryonic life of the knockout mice, although the Cav1.2 knockout mutation remains embryonic lethal, indicating the limits of this compensation pathway. In a complementary experiment using Cav1.3 knockout mice, there seems to be compensatory upregulation of Cav1.2 channel expression.69

Expression of a Kv1 dominant-negative construct in transgenic mice largely eliminates IK,slow1.70 In these mice, there is a specific compensatory response involving increased expression of the IK,slow2 component of the delayed rectifier, which seems to be mediated by increased Kv2.1 mRNA expression.70

In Kv4 dominant-negative knockout mice, Kv1.4 channel expression is selectively upregulated to compensate for the loss of Ito,f.63 The limits of this compensation pathway are shown in mice in which both the Kv4 and Kv1.4 channels are knocked out.63 In this case there are only minor subsequent changes in ion channel expression, suggesting that even though the system is aware of the deficit, as shown by the response to the loss of the Kv4 channels alone, it cannot mount an appropriate response.

This last example highlights some of the limitations of the compensatory pathway. In the case of the Kv4 channel knockouts, channels that might better compensate for the loss of Kv4 channel expression are not significantly upregulated. Similarly, when both the Kv4 and Kv1.4 channels are eliminated, expression of other transient potassium channels, not normally expressed in heart, is not activated. Both of these failures reflect the fact that the developmental process creates a network of transcription factors in the heart that significantly limits the universe of possible homeostatic responses. In other words, the cells are differentiated and no longer pluripotent. This limitation applies whether the response is transcriptional or posttranscriptional. If the compensatory response involves transcriptional mechanisms, it is dependent on the preexisting network of transcription factors expressed by the differentiation program active in the cell, which places significant limits on which genes can be expressed. If the response involves posttranscriptional mechanisms, these mechanisms can only act on those channels whose genes have been transcriptionally activated during development, such as the Kv1.4 channel, which is expressed at low but finite levels in the adult.

Cognate Pathways

There is a more limited set of data on cognate homeostatic regulatory pathways. As noted above, if the cognate pathway was always effectively activated after loss of a single allele, simple null mutations in the KCNQ1 and KCNH2 genes would not produce arrhythmias in long-QT syndrome.

One example where the cognate pathway seems to be activated is in SA nodal cells of heterozygous Cav1.3 knockout mice. In these cells, calcium channel function is unaffected by the loss of one Cav1.3 allele, suggesting that there is upregulation at some level of the output from the remaining functional allele.69 A similar finding has been described for the heterozygous knockout of the clcn1 chloride channel in skeletal muscle.71 However, in both cases the data are incomplete, and it is difficult to be absolutely certain that there has been compensation via a cognate pathway. As a consequence, whether a transcriptional or posttranscriptional response can be activated by the heterozygous loss of an ion channel gene remains an open question. Transcriptional compensation activated by a cognate feedback pathway has been described for genes other than ion channels,60,72 although these may be special cases.

Heterozygous knockout of the Scn5a or KChIP2 genes produces a 50% reduction in INa or Ito expression, respectively,73,74 suggesting that no cognate homeostatic regulatory pathway can compensate for the loss of these genes in mice. The results from the Scn5a mice are at variance with the results obtained using pharmacological blockade of sodium channels65,66 described above. There is one marked complication with the Scn5a knockout mice. Tonic sympathetic drive to the heart is apparently much greater in the heterozygotes than in wild-type mice to compensate for the much slower intrinsic heart rate of the heterozygote hearts. As a consequence, the increased sympathetic activity by itself may have modified channel expression. Intriguingly, this seems to be an example where physiological mechanisms, as opposed to regulatory mechanisms, are activated to maintain homeostasis. This example highlights the importance of obtaining temporally restricted knockouts for the study of homeostatic regulation.

Is Transcriptional Regulation a Suitable Mechanism for the Control of Ion Channel Expression?

The classic model systems used for the study of physiologically controlled transcriptional regulation have been monomeric soluble proteins such as metabolic enzymes.12 Ion channels, which are typically both heteromeric and multimeric membrane proteins, seem, at least at first glance, to be much poorer candidates for transcriptional regulation. Yet there are multiple examples where either developmentally determined patterns of ion channel expression or subsequent changes in expression induced by various cardiac disorders seem to be mediated primarily at the level of transcription.6,38,66,68,70,73–84

Transcriptional regulation can provide both specificity and precision to the control of ion channel expression. Specificity is necessary because multiple, functionally distinct ion channels with nonoverlapping or only partially overlapping functions are expressed in the heart.85 Recruitment of varying combinations of multiple different transcription factors can result, in principle, in the independent regulation of each ion channel gene.86 Precise regulation of channel expression levels will be important for some channels, such as those active during the plateau phase of the action potential, where small changes in current levels can result either in premature repolarization or significant prolongation of action potential duration.22,24,87–89 Although less well studied, transcriptional regulation can produce precisely graded levels of gene expression.90

It is important to note that even when transcriptional regulation of channel gene expression is the primary regulated process, it will not be the sole influence on channel expression levels. For example, posttranscriptional steps may control the final level of channel expression by contributing a rate-limiting step to the biosynthesis pathway. A possible example of this is KCNH2 channel expression in rat heart. KCNH2 mRNA and protein expression levels are high in rat ventricular myocytes, whereas the IKr current is very small.91,92 One explanation for these results is that a step in the posttranslational biosynthetic pathway is rate limiting. Alternatively, however, a component of the IKr channel complex might be missing or expressed at low levels in ventricular myocytes.93,94

One major complication in determining the relative contributions of transcriptional versus posttranscriptional regulatory mechanisms is the heteromeric nature of most ion channels. An example of this is Ito expression in the ventricular myocytes of canine and human heart. Although there is a large gradient of Ito expression across the ventricular walls, Kv4.3 mRNA is expressed at constant levels throughout the ventricle.79,80 For many years this seemed to be an example of posttranscriptional regulation, because of the marked disjuncture between current levels and mRNA expression. It was only with the cloning and elucidation of the function of the KChIP2 ß subunit95 that an alternative hypothesis became available, that regulation of ß subunit expression controls expression of the Ito channel.74,79,80,82,96 To definitively conclude that posttranscriptional mechanisms are predominant in the control of a particular ion channel requires a complete knowledge of all of the auxiliary subunits that are either necessary for expression or can affect the expression of a particular channel. This is a very high standard to reach at the moment, and candidate genes will not always be intuitively obvious. The difficulty of this problem is well illustrated by the unanticipated results obtained from the study of mutations of the cytoskeletal protein ankyrin B gene, which alters the expression or function of several ion channels and pumps.97,98 The development of high-throughput proteomic assays that can be used to detect protein-protein interactions for membrane proteins may address this problem in the near future.99

Most specific long-term changes in ion channel expression are likely to be mediated at the level of gene transcription, although there will undoubtedly be exceptions to this general principle. For example, an activated signal transduction pathway could, in principle, modify any step in the biosynthetic pathway (Figure 1). To be useful for physiological regulation, the pathway should selectively modify the biosynthesis of a small number of ion channels without producing global changes in channel biosynthesis. This criterion can be achieved, at least in certain circumstances. Signal transduction pathways triggered by the influx of calcium ions through NMDA receptors regulate the trafficking of AMPA receptors to the postsynaptic membrane at some excitatory synapses without significantly affecting NMDA receptor expression at those same synapses.100 Whether there are similar examples of highly specific regulated trafficking of ion channel complexes in cardiac myocytes has yet to be established. The specificity seen at excitatory synapses is attributable in part to compartmentalization, and in particular to the unique subcellular anatomy of the dendritic spine. Microdomains for calcium signaling pathways do not necessarily require such an elaborate anatomical basis101–103 and may exist in cardiac myocytes.

Future Directions

Cardiac organogenesis is mediated by the interactions of complex networks of transcription factors, and many of these networks remain active in the adult heart. As a consequence, virtually every aspect of the study of ion channel regulation in the heart will benefit greatly if it can be more closely linked to the rapidly increasing knowledge of transcription factor function during cardiac myocyte development and differentiation.104–106 This important task has only just begun and will need some time to yield useful results.

The networks of transcription factors that are created during development both limit and enable the capabilities of the homeostatic regulatory processes in the adult heart. For developmental regulation of ion channel expression, the problem is well defined. We need to know how ion channel genes are turned on or off during cardiac development. In contrast, for homeostatic regulation, the issues are not as clearly formulated. There have been very few systematic studies on homeostatic regulation, and as a consequence we only have a broad outline of the capabilities and limitations of this regulatory system. To date, most results come from genetics, both traditional and reverse. Traditional human genetic studies have provided considerable information about the limitations of these regulatory systems.107 The use of reverse genetics in mouse to modify gene dosage is an important paradigm for future studies on the capabilities of the homeostatic regulatory system.63,68,69,74 One issue that could benefit from more detailed analysis is channel expression in heterozygous knockouts, which are particularly useful for studying cognate feedback pathways. Overexpression and misexpression of ion channel genes are other ways to test the capabilities of the homeostatic regulatory mechanisms,108 although overexpression can produce nonspecific effects.109

One limitation of all the gene dosage experiments conducted to date is that modified gene function has been active during development, which could lead to the disruption of normal developmental processes and the misinterpretation of results. The development of reliable systems to produce changes in ion channel gene dosage that are restricted to adult heart would be an extremely important addition to the field.

The nature of the feedback mechanisms involved in homeostatic regulation remains puzzling. It is difficult to imagine that calcium transients are by themselves the sole source of information that a cardiac myocyte receives about the state of its electrophysiological phenotype, because these transients could be regulated almost solely by increases or decreases in the level of calcium channel expression and other proteins affecting calcium metabolism. Obviously the temporal pattern of the calcium transients is important, including both duration and frequency. But again, changes in calcium handling proteins will have a disproportionately large effect. It seems almost necessary to assume that other pathways are important, but to date only calcium signaling pathways have much experimental validation.

Our understanding of intracellular sensing may not be complete at the moment, and it is possible that important mechanisms have been overlooked. This is a difficult area, but it will be important to establish whether sensory systems exist in addition to calcium signaling. One simple approach will be to establish whether there are examples of homeostatic regulation that cannot be accounted for by calcium signaling pathways. In this context, neurohumoral signaling pathways may prove to be important effectors in homeostatic regulatory pathways.

With regard to intracellular calcium sensing, there is a large and rapidly increasing literature on transcription factors activated by calcium influxes29–32 but relatively few studies that have attempted to link this knowledge to the regulation of cardiac ion channel expression.110 More efforts in this area are likely to yield important results.

In this review we have largely ignored the problem of coordinate regulation. In many cases there is a yin-yang nature to the regulation of ion channel expression, or a necessity to balance inward currents with outward currents. This is particularly evident in the plateau phase of the action potential, during which very small and closely balanced currents flow. One way to create and maintain this balance would be to coregulate the expression of antagonistic inward and outward currents by coordinate regulation of the relevant channel genes. Alternatively, functional expression of antagonist channels could be regulated by expression of a common auxiliary subunit93 or by common recruitment to the cell membrane by a scaffolding protein. Whatever the mechanism, coordinate regulation may be an important contributor to the stability of the electrophysiological phenotype and should receive more direct experimental analysis than has been attempted to date.

Conclusions

Waddington111 coined the term “canalization” to describe developmental robustness, implying that development moves down certain well-trodden pathways or canals. These canalized pathways were imagined to have relatively steep walls, explaining why the developmental process and the establishment of the final adult phenotype are relatively impervious to environmental or genetic variation. The topology that seems to best characterize the robustness of the cardiac electrophysiological phenotype is a broad valley with several shallow local minima. This is perhaps best illustrated by the phenomena of pacing-induced atrial fibrillation.112 Repetitive pacing readily shifts the electrophysiological phenotype of atrial myocytes to a new semi-stable state, which, unfortunately, actually promotes the maintenance of atrial fibrillation.77,112 Obviously a balance must be established between phenotypic stability and phenotypic plasticity. The sensitivity to genetic, pharmacological, or pathological insults that the cardiac electrophysiological phenotype displays seems surprisingly high, however, and creates a genuine challenge for the design of effective therapies. Understanding those homeostatic regulatory mechanisms that promote phenotypic robustness is an important first step toward understanding how to shift an inappropriate electrophysiological phenotype toward a normal, healthy phenotype.

Acknowledgments

This work was supported by NIH grants HL-28958, NS-29755, and American Heart Association 0235467T. All opinions, errors, and omissions are the sole responsibility of the authors. We would like to thank Drs P. Boyden, I. Cohen, M. Kernan, J. Nerbonne, R. Robinson, M. Rosen, and M. Sanguinetti for comments and suggestions.

Footnotes

  • Original received December 15, 2003; revision received February 18, 2004; accepted February 20, 2004.

References

  1. ↵
    Fozzard HA. Cardiac sodium and calcium channels: a history of excitatory currents. Cardiovasc Res. 2002; 55: 1–8.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Davidson EH. Genomic Regulatory Systems: Development and Evolution. New York, NY: Academic Press; 2001.
  3. ↵
    Harvey RP, Rosenthal N. Heart Development. New York, NY: Academic Press; 1998.
  4. ↵
    Cripps RM, Olson EN. Control of cardiac development by an evolutionarily conserved transcriptional network. Dev Biol. 2002; 246: 14–28.
    OpenUrlCrossRefPubMed
  5. ↵
    Boyett MR, Honjo H, Kodama I. The sinoatrial node: a heterogeneous pacemaker structure. Cardiovasc Res. 2000; 47: 658–687.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, Robinson RB, Dixon JE, McKinnon D, Cohen IS. Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ Res. 1999; 85: e1–e6.
    OpenUrlPubMed
  7. ↵
    Bohn G, Moosmang S, Conrad H, Ludwig A, Hofmann F, Klugbauer N. Expression of T- and L-type calcium channel mRNA in murine sinoatrial node. FEBS Lett. 2000; 481: 73–76.
    OpenUrlCrossRefPubMed
  8. ↵
    Moosmang S, Stieber J, Zong X, Biel M, Hofmann F, Ludwig A. Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem. 2001; 268: 1646–1652.
    OpenUrlPubMed
  9. ↵
    Nerbonne JM. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol. 2000; 525: 285–298.
    OpenUrlCrossRefPubMed
  10. ↵
    Schram G, Pourrier M, Melnyk P, Nattel S. Differential distribution of cardiac ion channel expression as a basis for regional specialization in electrical function. Circ Res. 2002; 90: 939–950.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Solloway MJ, Harvey RP. Molecular pathways in myocardial development: a stem cell perspective. Cardiovasc Res. 2003; 58: 264–277.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Ptashne M. A Genetic Switch Gene Control and Phage Lambda. Cambridge, Mass: Cell Press & Blackwell Scientific Publications; 1986.
  13. ↵
    Freeman M. Feedback control of intercellular signalling in development. Nature. 2000; 408: 313–319.
    OpenUrlCrossRefPubMed
  14. ↵
    Keller EF. Developmental robustness. Ann N Y Acad Sci. 2002; 981: 189–201.
    OpenUrlPubMed
  15. ↵
    Lomo T, Westgaard RH. Control of ACh sensitivity in rat muscle fibers. Cold Spring Harbor Symp Quant Biol. 1976; 40: 263–274.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Buonanno A, Fields RD. Gene regulation by patterned electrical activity during neural and skeletal muscle development. Curr Opin Neurobiol. 1999; 9: 110–120.
    OpenUrlCrossRefPubMed
  17. ↵
    Wong RO, Ghosh A. Activity-dependent regulation of dendritic growth and patterning. Nat Rev Neurosci. 2002; 3: 803–812.
    OpenUrlCrossRefPubMed
  18. ↵
    Zicha S, Moss I, Allen B, Varro A, Papp J, Dumaine R, Antzelevich C, Nattel S. Molecular basis of species-specific expression of repolarizing K+ currents in the heart. Am J Physiol Heart Circ Physiol. 2003; 285: H1641–H1649.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Hume JR, Uehara A. Ionic basis of the different action potential configurations of single guinea-pig atrial and ventricular myocytes. J Physiol. 1985; 368: 525–544.
    OpenUrlCrossRefPubMed
  20. ↵
    Findlay I. Is there an A-type K+ current in guinea pig ventricular myocytes? Am J Physiol Heart Circ Physiol. 2003; 284: H598–H604.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Murphy WJ, Eizirik E, O’Brien SJ, Madsen O, Scally M, Douady CJ, Teeling E, Ryder OA, Stanhope MJ, de Jong WW, Springer MS. Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science. 2001; 294: 2348–2351.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Hoppe UC, Marbán E, Johns DC. Molecular dissection of cardiac repolarization by in vivo Kv4.3 gene transfer. J Clin Invest. 2000; 105: 1077–1084.
    OpenUrlPubMed
  23. ↵
    Clark RB, Bouchard RA, Salinas-Stefanon E, Sanchez-Chapula J, Giles WR. Heterogeneity of action potential waveforms and potassium currents in rat ventricle. Cardiovasc Res. 1993; 27: 1795–1799.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Antzelevitch C. The Brugada syndrome: ionic basis and arrhythmia mechanisms. J Cardiovasc Electrophysiol. 2001; 12: 268–272.
    OpenUrlCrossRefPubMed
  25. ↵
    Deutsch C. The birth of a channel. Neuron. 2003; 40: 265–276.
    OpenUrlCrossRefPubMed
  26. ↵
    Fields RD. Effects of ion channel activity on development of dorsal root ganglion neurons. J Neurobiol. 1998; 37: 158–170.
    OpenUrlCrossRefPubMed
  27. ↵
    Spitzer NC, Lautermilch NJ, Smith RD, Gomez TM. Coding of neuronal differentiation by calcium transients. Bioessays. 2000; 22: 811–817.
    OpenUrlCrossRefPubMed
  28. ↵
    Xia Y, McMillin JB, Lewis A, Moore M, Zhu WG, Williams RS, Kellems RE. Electrical stimulation of neonatal cardiac myocytes activates the NFAT3 and GATA4 pathways and up-regulates the adenylosuccinate synthetase 1 gene. J Biol Chem. 2000; 275: 1855–1863.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    West AE, Griffith EC, Greenberg ME. Regulation of transcription factors by neuronal activity. Nat Rev Neurosci. 2002; 3: 921–931.
    OpenUrlCrossRefPubMed
  30. ↵
    McKinsey TA, Zhang CL, Olson EN. MEF2: a calcium-dependent regulator of cell division, differentiation and death. Trends Biochem Sci. 2002; 27: 40–47.
    OpenUrlCrossRefPubMed
  31. ↵
    Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2003; 17: 2205–2232.
    OpenUrlFREE Full Text
  32. ↵
    Dolmetsch R. Excitation-transcription coupling: signaling by ion channels to the nucleus. Sci STKE. 2003; 2003:P E4.
    OpenUrl
  33. ↵
    Linden DJ, Smeyne M, Connor JA. Induction of cerebellar long-term depression in culture requires postsynaptic action of sodium ions. Neuron. 1993; 11: 1093–1100.
    OpenUrlCrossRefPubMed
  34. ↵
    Rose CR. Na+ signals at central synapses. Neuroscientist. 2002; 8: 532–539.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Atar D, Backx PH, Appel MM, Gao WD, Marbán E. Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J Biol Chem. 1995; 270: 2473–2477.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Sadoshima J, Izumo S. The cellular and molecular response of cardiac myocytes to mechanical stress. Annu Rev Physiol. 1997; 59: 551–571.
    OpenUrlCrossRefPubMed
  37. ↵
    Tarone G, Lembo G. Molecular interplay between mechanical and humoral signalling in cardiac hypertrophy. Trends Mol Med. 2003; 9: 376–382.
    OpenUrlCrossRefPubMed
  38. ↵
    Bru-Mercier G, Deroubaix E, Capuano V, Ruchon Y, Rucker-Martin C, Coulombe A, Renaud JF. Expression of heart K+ channels in adrenalectomized and catecholamine-depleted reserpine-treated rats. J Mol Cell Cardiol. 2003; 35: 153–163.
    OpenUrlCrossRefPubMed
  39. ↵
    Protas L, Barbuti A, Qu J, Rybin VO, Palmiter RD, Steinberg SF, Robinson RB. Neuropeptide Y is an essential in vivo developmental regulator of cardiac ICa,L. Circ Res. 2003; 93: 972–979.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Russell FD, Molenaar P. The human heart endothelin system: ET-1 synthesis, storage, release and effect. Trends Pharmacol Sci. 2000; 21: 353–359.
    OpenUrlCrossRefPubMed
  41. ↵
    Carey RM, Siragy HM. Newly recognized components of the renin-angiotensin system: potential roles in cardiovascular and renal regulation. Endocr Rev. 2003; 24: 261–271.
    OpenUrlCrossRefPubMed
  42. ↵
    Tomaselli GF, Marbán E. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res. 1999; 42: 270–283.
    OpenUrlFREE Full Text
  43. ↵
    Pinto JM, Boyden PA. Electrical remodeling in ischemia and infarction. Cardiovasc Res. 1999; 42: 284–297.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Dillmann WH. Cellular action of thyroid hormone on the heart. Thyroid. 2002; 12: 447–552.
    OpenUrlCrossRefPubMed
  45. ↵
    Pandit SV, Giles WR, Demir SS. A mathematical model of the electrophysiological alterations in rat ventricular myocytes in type-I diabetes. Biophys J. 2003; 84: 832–841.
    OpenUrlPubMed
  46. ↵
    Akazawa H, Komuro I. Roles of cardiac transcription factors in cardiac hypertrophy. Circ Res. 2003; 92: 1079–1088.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Chen PS, Chen LS, Cao JM, Sharifi B, Karagueuzian HS, Fishbein MC. Sympathetic nerve sprouting, electrical remodeling and the mechanisms of sudden cardiac death. Cardiovasc Res. 2001; 50: 409–416.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Veitia RA. Exploring the etiology of haploinsufficiency. Bioessays. 2002; 24: 175–184.
    OpenUrlCrossRefPubMed
  49. ↵
    Miklos GL, Rubin GM. The role of the genome project in determining gene function: insights from model organisms. Cell. 1996; 86: 521–529.
    OpenUrlCrossRefPubMed
  50. ↵
    Gu Z, Steinmetz LM, Gu X, Scharfe C, Davis RW, Li WH. Role of duplicate genes in genetic robustness against null mutations. Nature. 2003; 421: 63–66.
    OpenUrlCrossRefPubMed
  51. ↵
    Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 2003; 421: 231–237.
    OpenUrlCrossRefPubMed
  52. ↵
    Kirschner M, Gerhart J. Evolvability. Proc Natl Acad Sci U S A. 1998; 95: 8420–8427.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Hartman JL, Garvik B, Hartwell L. Principles for the buffering of genetic variation. Science. 2001; 291: 1001–1004.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Wang Z, Tristani-Firouzi M, Xu Q, Lin M, Keating MT, Sanguinetti MC. Functional effects of mutations in KvLQT1 that cause long QT syndrome. J Cardiovasc Electrophysiol. 1999; 10: 817–826.
    OpenUrlCrossRefPubMed
  55. ↵
    Franqueza L, Lin M, Shen J, Splawski I, Keating MT, Sanguinetti MC. Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits. J Biol Chem. 1999; 274: 21063–21070.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Yamashita F, Horie M, Kubota T, Yoshida H, Yumoto Y, Kobori A, Ninomiya T, Kono Y, Haruna T, Tsuji K, Washizuka T, Takano M, Otani H, Sasayama S, Aizawa Y. Characterization and subcellular localization of KCNQ1 with a heterozygous mutation in the C terminus. J Mol Cell Cardiol. 2001; 33: 197–207.
    OpenUrlCrossRefPubMed
  57. ↵
    Sanguinetti MC, Curran ME, Spector PS, Keating MT. Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia. Proc Natl Acad Sci U S A. 1996; 93: 2208–2212.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Paulussen A, Raes A, Matthijs G, Snyders DJ, Cohen N, Aerssens J. A novel mutation (T65P) in the PAS domain of the human potassium channel HERG results in the long QT syndrome by trafficking deficiency. J Biol Chem. 2002; 277: 48610–48616.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Seidman JG, Seidman C. Transcription factor haploinsufficiency: when half a loaf is not enough. J Clin Invest. 2002; 109: 451–455.
    OpenUrlCrossRefPubMed
  60. ↵
    Trieu M, Ma A, Eng SR, Fedtsova N, Turner EE. Direct autoregulation and gene dosage compensation by POU-domain transcription factor Brn3a. Development. 2003; 130: 111–121.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    Priori SG, Schwartz PJ, Napolitano C, Bloise R, Ronchetti E, Grillo M, Vicentini A, Spazzolini C, Nastoli J, Bottelli G, Folli R, Cappelletti D. Risk stratification in the long-QT syndrome. N Engl J Med. 2003; 348: 1866–1874.
    OpenUrlCrossRefPubMed
  62. ↵
    Guo W, Xu H, London B, Nerbonne JM. Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes. J Physiol. 1999; 521: 587–599.
    OpenUrlCrossRefPubMed
  63. ↵
    Guo W, Li H, London B, Nerbonne JM. Functional consequences of elimination of Ito,f and Ito,s: early afterdepolarizations, atrioventricular block, and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 α subunit. Circ Res. 2000; 87: 73–79.
    OpenUrlAbstract/FREE Full Text
  64. ↵
    Barry DM, Xu H, Schuessler RB, Nerbonne JM. Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 α subunit. Circ Res. 1998; 83: 560–567.
    OpenUrlAbstract/FREE Full Text
  65. ↵
    Taouis M, Sheldon RS, Duff HJ. Upregulation of the rat cardiac sodium channel by in vivo treatment with a class I antiarrhythmic drug. J Clin Invest. 1991; 88: 375–378.
    OpenUrlPubMed
  66. ↵
    Duff HJ, Offord J, West J, Catterall WA. Class I and IV antiarrhythmic drugs and cytosolic calcium regulate mRNA encoding the sodium channel α subunit in rat cardiac muscle. Mol Pharmacol. 1992; 42: 570–574.
    OpenUrlAbstract
  67. ↵
    Priori SG, Napolitano C, Schwartz PJ. Low penetrance in the long-QT syndrome: clinical impact. Circulation. 1999; 99: 529–533.
    OpenUrlAbstract/FREE Full Text
  68. ↵
    Xu M, Welling A, Paparisto S, Hofmann F, Klugbauer N. Enhanced expression of L-type Cav1.3 calcium channels in murine embryonic hearts from Cav1.2-deficient mice. J Biol Chem. 2003; 278: 40837–40841.
    OpenUrlAbstract/FREE Full Text
  69. ↵
    Zhang Z, Xu Y, Song H, Rodriguez J, Tuteja D, Namkung Y, Shin HS, Chiamvimonvat N. Functional roles of Cav1.3 (α1D) calcium channel in sinoatrial nodes: insight gained using gene-targeted null mutant mice. Circ Res. 2002; 90: 981–987.
    OpenUrlAbstract/FREE Full Text
  70. ↵
    Zhou J, Kodirov S, Murata M, Buckett PD, Nerbonne JM, Koren G. Regional upregulation of Kv2.1-encoded current, IK,slow2, in Kv1DN mice is abolished by crossbreeding with Kv2DN mice. Am J Physiol Heart Circ Physiol. 2003; 284: H491–H500.
    OpenUrlAbstract/FREE Full Text
  71. ↵
    Chen MF, Niggeweg R, Iaizzo PA, Lehmann-Horn F, Jockusch H. Chloride conductance in mouse muscle is subject to post-transcriptional compensation of the functional Cl− channel 1 gene dosage. J Physiol. 1997; 504: 75–81.
    OpenUrlCrossRefPubMed
  72. ↵
    Guidi CJ, Veal TM, Jones SN, Imbalzano AN. Transcriptional compensation for loss of an allele of the Ini1 tumor suppressor. J Biol Chem. 2004; 279: 4180–4185.
    OpenUrlAbstract/FREE Full Text
  73. ↵
    Papadatos GA, Wallerstein PM, Head CE, Ratcliff R, Brady PA, Benndorf K, Saumarez RC, Trezise AE, Huang CL, Vandenberg JI, Colledge WH, Grace AA. Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a. Proc Natl Acad Sci USA. 2002; 99: 6210–6215.
    OpenUrlAbstract/FREE Full Text
  74. ↵
    Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH, Ross J, Giles WR, Chien KR. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of Ito and confers susceptibility to ventricular tachycardia. Cell. 2001; 107: 801–813.
    OpenUrlCrossRefPubMed
  75. ↵
    Kubo Y, Reuveny E, Slesinger PA, Jan YN, Jan LY. Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature. 1993; 364: 802–806.
    OpenUrlCrossRefPubMed
  76. ↵
    Dixon JE, McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res. 1994; 75: 252–260.
    OpenUrlAbstract/FREE Full Text
  77. ↵
    Yue L, Melnyk P, Gaspo R, Wang Z, Nattel S. Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation. Circ Res. 1999; 84: 776–784.
    OpenUrlAbstract/FREE Full Text
  78. ↵
    Huang B, Qin D, Deng L, Boutjdir M, E1-Sherif N. Reexpression of T-type Ca2+ channel gene and current in post-infarction remodeled rat left ventricle. Cardiovasc Res. 2000; 46: 442–449.
    OpenUrlAbstract/FREE Full Text
  79. ↵
    Rosati B, Grau F, Rodriguez S, Li H, Nerbonne JM, McKinnon D. Concordant expression of KChIP2 mRNA, protein and transient outward current throughout the canine ventricle. J Physiol. 2003; 548: 815–822.
    OpenUrlCrossRefPubMed
  80. ↵
    Rosati B, Pan Z, Lypen S, Wang HS, Cohen I, Dixon JE, McKinnon D. Regulation of KChIP2 potassium channel β subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J Physiol. 2001; 533: 119–125.
    OpenUrlCrossRefPubMed
  81. ↵
    Dobrev D, Graf E, Wettwer E, Himmel HM, Hala O, Doerfel C, Christ T, Schuler S, Ravens U. Molecular basis of downregulation of G-protein-coupled inward rectifying K+ current (IK,ACh) in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced IK,ACh and muscarinic receptor-mediated shortening of action potentials. Circulation. 2001; 104: 2551–2557.
    OpenUrlAbstract/FREE Full Text
  82. ↵
    Kobayashi T, Yamada Y, Nagashima M, Seki S, Tsutsuura M, Ito Y, Sakuma I, Hamada H, Abe T, Tohse N. Contribution of KChIP2 to the developmental increase in transient outward current of rat cardiomyocytes. J Mol Cell Cardiol. 2003; 35: 1073–1082.
    OpenUrlCrossRefPubMed
  83. ↵
    Le Bouter S, Demolombe S, Chambellan A, Bellocq C, Aimond F, Toumaniantz G, Lande G, Siavoshian S, Baro I, Pond AL, Nerbonne JM, Leger JJ, Escande D, Charpentier F. Microarray analysis reveals complex remodeling of cardiac ion channel expression with altered thyroid status: relation to cellular and integrated electrophysiology. Circ Res. 2003; 92: 234–242.
    OpenUrlAbstract/FREE Full Text
  84. ↵
    Fernandez-Velasco M, Goren N, Benito G, Blanco-Rivero J, Bosca L, Delgado C. Regional distribution of hyperpolarization-activated current (If) and hyperpolarization-activated cyclic nucleotide-gated channel mRNA expression in ventricular cells from control and hypertrophied rat hearts. J Physiol. 2003; 553: 395–405.
    OpenUrlCrossRefPubMed
  85. ↵
    Roden DM, Balser JR, George AL Jr, Anderson ME. Cardiac ion channels. Annu Rev Physiol. 2002; 64: 431–475.
    OpenUrlCrossRefPubMed
  86. ↵
    Ptashne M, Gann A. Genes and Signals. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2002.
  87. ↵
    Liu DW, Antzelevitch C. Characteristics of the delayed rectifier current (IKr and IKs) in canine ventricular epicardial, midmyocardial, and endocardial myocytes: a weaker IKs contributes to the longer action potential of the M cell. Circ Res. 1995; 76: 351–365.
    OpenUrlAbstract/FREE Full Text
  88. ↵
    Viswanathan PC, Shaw RM, Rudy Y. Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study. Circulation. 1999; 99: 2466–2474.
    OpenUrlAbstract/FREE Full Text
  89. ↵
    Pandit SV, Clark RB, Giles WR, Demir SS. A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes. Biophys J. 2001; 81: 3029–3051.
    OpenUrlCrossRefPubMed
  90. ↵
    Biggar SR, Crabtree GR. Cell signaling can direct either binary or graded transcriptional responses. EMBO J. 2001; 20: 3167–3176.
    OpenUrlAbstract
  91. ↵
    Wymore R, Gintant GA, Wymore RT, Dixon JE, McKinnon D, Cohen IS. Tissue and species distribution of mRNA for the IKr-like K+ channel, erg. Circ Res. 1997; 80: 261–268.
    OpenUrlAbstract/FREE Full Text
  92. ↵
    Pond AL, Scheve BK, Benedict AT, Petrecca K, Van Wagoner DR, Shrier A, Nerbonne JM. Expression of distinct ERG proteins in rat, mouse, and human heart: relation to functional IKr channels. J Biol Chem. 2000; 275: 5997–6006.
    OpenUrlAbstract/FREE Full Text
  93. ↵
    Yu H, Wu J, Potapova I, Wymore RT, Holmes B, Zuckerman J, Pan Z, Wang H, Shi W, Robinson RB, El-Maghrabi MR, Benjamin W, Dixon J, McKinnon D, Cohen IS, Wymore R. MinK-related peptide 1: a β subunit for the HCN ion channel subunit family enhances expression and speeds activation. Circ Res. 2001; 88: e84–e87.
    OpenUrlCrossRef
  94. ↵
    Pourrier M, Zicha S, Ehrlich J, Han W, Nattel S. Canine ventricular KCNE2 expression resides predominantly in Purkinje fibers. Circ Res. 2003; 93: 189–191.
    OpenUrlAbstract/FREE Full Text
  95. ↵
    An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature. 2000; 403: 553–556.
    OpenUrlCrossRefPubMed
  96. ↵
    Patel SP, Campbell DL, Morales MJ, Strauss HC. Heterogeneous expression of KChIP2 isoforms in the ferret heart. J Physiol. 2002; 539: 649–656.
    OpenUrlCrossRefPubMed
  97. ↵
    Chauhan VS, Tuvia S, Buhusi M, Bennett V, Grant AO. Abnormal cardiac Na+ channel properties and QT heart rate adaptation in neonatal ankyrinB knockout mice. Circ Res. 2000; 86: 441–447.
    OpenUrlAbstract/FREE Full Text
  98. ↵
    Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogne K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H, Bennett V. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature. 2003; 421: 634–639.
    OpenUrlCrossRefPubMed
  99. ↵
    Stagljar I, Fields S. Analysis of membrane protein interactions using yeast-based technologies. Trends Biochem Sci. 2002; 27: 559–563.
    OpenUrlCrossRefPubMed
  100. ↵
    Malinow R. AMPA receptor trafficking and long-term potentiation. Philos Trans R Soc Lond B Biol Sci. 2003; 358: 707–714.
    OpenUrlAbstract/FREE Full Text
  101. ↵
    Naraghi M, Neher E. Linearized buffered Ca2+ diffusion in microdomains and its implications for calculation of. J Neurosci. 1997; 17: 6961–6973.
    OpenUrlAbstract/FREE Full Text
  102. ↵
    Rios E, Stern MD. Calcium in close quarters: microdomain feedback in excitation-contraction coupling and other cell biological phenomena. Annu Rev Biophys Biomol Struct. 1997; 26: 47–82.
    OpenUrlCrossRefPubMed
  103. ↵
    Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415: 198–205.
    OpenUrlCrossRefPubMed
  104. ↵
    Harvey RP, Lai D, Elliott D, Biben C, Solloway M, Prall O, Stennard F, Schindeler A, Groves N, Lavulo L, Hyun C, Yeoh T, Costa M, Furtado M, Kirk E. Homeodomain factor Nkx2-5 in heart development and disease. Cold Spring Harb Symp Quant Biol. 2002; 67: 107–114.
    OpenUrlCrossRefPubMed
  105. ↵
    Srivastava D, Gottlieb PD, Olson EN. Molecular mechanisms of ventricular hypoplasia. Cold Spring Harb Symp Quant Biol. 2002; 67: 121–125.
    OpenUrlCrossRefPubMed
  106. ↵
    Wang D, Passier R, Liu ZP, Shin CH, Wang Z, Li S, Sutherland LB, Small E, Krieg PA, Olson EN. Regulation of cardiac growth and development by SRF and its cofactors. Cold Spring Harb Symp Quant Biol. 2002; 67: 97–105.
    OpenUrlCrossRefPubMed
  107. ↵
    Keating MT, Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell. 2001; 104: 569–580.
    OpenUrlCrossRefPubMed
  108. ↵
    Bodi I, Muth JN, Hahn HS, Petrashevskaya NN, Rubio M, Koch SE, Varadi G, Schwartz A. Electrical remodeling in hearts from a calcium-dependent mouse model of hypertrophy and failure: complex nature of K+ current changes and action potential duration. J Am Coll Cardiol. 2003; 41: 1611–1622.
    OpenUrlCrossRefPubMed
  109. ↵
    Nerbonne JM, Nichols CG, Schwarz TL, Escande D. Genetic manipulation of cardiac K+ channel function in mice: what have we learned, and where do we go from here? Circ Res. 2001; 89: 944–956.
    OpenUrlAbstract/FREE Full Text
  110. ↵
    Patberg KW, Plotnikov AN, Quamina A, Gainullin RZ, Rybin A, Danilo P Jr, Sun LS, Rosen MR. Cardiac memory is associated with decreased levels of the transcriptional factor CREB modulated by angiotensin II and calcium. Circ Res. 2003; 93: 472–478.
    OpenUrlAbstract/FREE Full Text
  111. ↵
    Waddington CH. The Strategy of the Genes. London, UK: George Allen and Unwin; 1957.
  112. ↵
    Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation: a study in awake chronically instrumented goats. Circulation. 1995; 92: 1954–1968.
    OpenUrlAbstract/FREE Full Text
  113. ↵
    Shimoni Y, Liu XF. Role of PKC in autocrine regulation of rat ventricular K+ currents by angiotensin and endothelin. Am J Physiol Heart Circ Physiol. 2003; 284: H1168–H1181.
    OpenUrlPubMed
  114. ↵
    Ferron L, Capuano V, Ruchon Y, Deroubaix E, Coulombe A, Renaud JF. Angiotensin II signaling pathways mediate expression of cardiac T-type calcium channels. Circ Res. 2003; 93: 1241–1248.
    OpenUrlAbstract/FREE Full Text
  115. ↵
    Takimoto K, Levitan ES. Glucocorticoid induction of Kv1.5 K+ channel gene expression in ventricle of rat heart. Circ Res. 1994; 75: 1006–1013.
    OpenUrlAbstract/FREE Full Text
  116. ↵
    Takimoto K, Li D, Nerbonne JM, Levitan ES. Distribution, splicing and glucocorticoid-induced expression of cardiac α1C and α1D voltage-gated Ca2+ channel mRNAs. J Mol Cell Cardiol. 1997; 29: 3035–3042.
    OpenUrlCrossRefPubMed
  117. ↵
    Wickenden AD, Kaprielian R, Parker TG, Jones OT, Backx PH. Effects of development and thyroid hormone on K+ currents and K+ channel gene expression in rat ventricle. J Physiol. 1997; 504: 271–286.
    OpenUrlCrossRefPubMed
  118. ↵
    Gloss B, Trost S, Bluhm W, Swanson E, Clark R, Winkfein R, Janzen K, Giles W, Chassande O, Samarut J, Dillmann W. Cardiac ion channel expression and contractile function in mice with deletion of thyroid hormone receptor α or β. Endocrinology. 2001; 142: 544–550.
    OpenUrlCrossRefPubMed
View Abstract
Back to top
Previous ArticleNext Article

This Issue

Circulation Research
April 16, 2004, Volume 94, Issue 7
  • Table of Contents
Previous ArticleNext Article

Jump to

  • Article
    • Abstract
    • An Ideal Model of Homeostatic Regulation
    • Regulated Parameters and Biological Feedback
    • Limitations of Cardiac Ion Channel Regulation
    • Is There Any Feedback System Capable of Sensing Changes in Electrophysiological Phenotype?
    • Compensatory Pathways
    • Cognate Pathways
    • Is Transcriptional Regulation a Suitable Mechanism for the Control of Ion Channel Expression?
    • Future Directions
    • Conclusions
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Tables
  • Info & Metrics

Article Tools

  • Print
  • Citation Tools
    Regulation of Ion Channel Expression
    Barbara Rosati and David McKinnon
    Circulation Research. 2004;94:874-883, originally published April 15, 2004
    https://doi.org/10.1161/01.RES.0000124921.81025.1F

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
  •  Download Powerpoint
  • Article Alerts
    Log in to Email Alerts with your email address.
  • Save to my folders

Share this Article

  • Email

    Thank you for your interest in spreading the word on Circulation Research.

    NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

    Enter multiple addresses on separate lines or separate them with commas.
    Regulation of Ion Channel Expression
    (Your Name) has sent you a message from Circulation Research
    (Your Name) thought you would like to see the Circulation Research web site.
  • Share on Social Media
    Regulation of Ion Channel Expression
    Barbara Rosati and David McKinnon
    Circulation Research. 2004;94:874-883, originally published April 15, 2004
    https://doi.org/10.1161/01.RES.0000124921.81025.1F
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects

  • Basic, Translational, and Clinical Research
    • Ion Channels/Membrane Transport
  • Genetics
    • Gene Expression & Regulation

Circulation Research

  • About Circulation Research
  • Editorial Board
  • Instructions for Authors
  • Abstract Supplements
  • AHA Statements and Guidelines
  • Permissions
  • Reprints
  • Email Alerts
  • Open Access Information
  • AHA Journals RSS
  • AHA Newsroom

Editorial Office Address:
3355 Keswick Rd
Main Bldg 103
Baltimore, MD 21211
CircRes@circresearch.org

Information for:
  • Advertisers
  • Subscribers
  • Subscriber Help
  • Institutions / Librarians
  • Institutional Subscriptions FAQ
  • International Users
American Heart Association Learn and Live
National Center
7272 Greenville Ave.
Dallas, TX 75231

Customer Service

  • 1-800-AHA-USA-1
  • 1-800-242-8721
  • Local Info
  • Contact Us

About Us

Our mission is to build healthier lives, free of cardiovascular diseases and stroke. That single purpose drives all we do. The need for our work is beyond question. Find Out More about the American Heart Association

  • Careers
  • SHOP
  • Latest Heart and Stroke News
  • AHA/ASA Media Newsroom

Our Sites

  • American Heart Association
  • American Stroke Association
  • For Professionals
  • More Sites

Take Action

  • Advocate
  • Donate
  • Planned Giving
  • Volunteer

Online Communities

  • AFib Support
  • Garden Community
  • Patient Support Network
  • Professional Online Network

Follow Us:

  • Follow Circulation on Twitter
  • Visit Circulation on Facebook
  • Follow Circulation on Google Plus
  • Follow Circulation on Instagram
  • Follow Circulation on Pinterest
  • Follow Circulation on YouTube
  • Rss Feeds
  • Privacy Policy
  • Copyright
  • Ethics Policy
  • Conflict of Interest Policy
  • Linking Policy
  • Diversity
  • Careers

©2018 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. The American Heart Association is a qualified 501(c)(3) tax-exempt organization.
*Red Dress™ DHHS, Go Red™ AHA; National Wear Red Day ® is a registered trademark.

  • PUTTING PATIENTS FIRST National Health Council Standards of Excellence Certification Program
  • BBB Accredited Charity
  • Comodo Secured