Pulmonary Hypertension Association

Why Potassium Channel Dysfunction May Be 'Missing Link' in Pathophysiology of PAH

Evangelos D. Michelakis, MD
Assistant Professor of Medicine
Department of Medicine (Cardiology)
Director, Pulmonary Hypertension Program,
University of Alberta Hospital
University of Alberta
Edmonton, Alberta, Canada

Stephen L. Archer, MD
Heart and Stroke Chair in Cardiovascular Research
Chair, Cardiology Division,
Department of Medicine
University of Alberta
Edmonton, Alberta, Canada

Potassium (K+) channel dysregulation in pulmonary artery smooth muscle cells (PASMCs) is important in understanding the etiology of pulmonary arterial hypertension (PAH), which is characterized by abnormalities in the vascular biology of the pulmonary circulation at all levels from lumen to adventitia (Figure 1).¹ The challenge for any proposed mechanism for PAH is to account for the entire spectrum of the syndrome. The number of abnormalities in PAH suggests a “multiple hit” etiology. A permissive “primary” abnormality, such as a mutation of the gene for the serotonin transporter or the bone morphogenetic protein receptor 2 (BMPR2) gene or a Kv channel deficiency, may be inadequate to provoke PAH unless accompanied by one or more exogenous stimuli (eg, ingestion of dexfenfluramine), a situation analogous to atherosclerosis or cancer. A gene chip-based comparison of gene expression in lungs from patients with PAH versus controls found upregulation of genes that favor proliferation (ie, oncogenes like v-myc, jun D proto-oncogene, etc) and suppress apoptosis (eg, apoptosis inhibitory genes).² In support of the theory that Kv channels are important to PAH,^3,4 Kv channel gene expression was also suppressed in PAH.² It is encouraging that the K+ channel hypothesis (Figure 2), derived by conventional means in humans and rats, is supported by a nonbiased genomic screening approach. Since PAH is rarely detected in an early or asymptomatic phase, there are no natural history data to indicate the relationship between the onset of pulmonary hypertension and the decrease in Kv channel expression and function.

Overview of K+ Channels in Vascular
K+ channels are transmembrane spanning proteins that contain a pore with great selectivity for K+.⁵ There are three major classes K+ channels: Kv channels (including Ca2+-sensitive channels, KCa), the inward rectifier channels (Kir), and a family with a tandem, 2-pore motif (TASK). Kv channels are tonically active in vascular smooth muscle cells, allowing a basal efflux of K+ down the ion’s concentration gradient. It is this efflux of K+ that, in balance with the intra- and
extracellular K+ concentrations (145 and 5 mM, respectively), largely establishes the resting membrane potential of the PASMC (Figure 3). Healthy PASMCs from resistance arteries have a membrane potential of ~ -60 mV. Inhibition of certain K+ channels (only those that are open at resting membrane potential) results in accumulation of positively charged K+ ions within the cell, depolarizing the membrane potential to more positive levels. This activates the voltagegated, L-type calcium channel. Calcium then enters the cell through this selective conduit, flowing down a 10,000/1 concentration gradient. The increase in Ca2+ activates the PASMC’s contractile apparatus, leading to vasoconstriction. If depolarization is long-term, the cell enters a proliferative phase and, interestingly, expression of the specific Kv channels occurs (Figure 4). The loss of channels, particularly
Kv1.5, is not restricted to PASMCs and was first described in pituitary cells.⁶

The Kv1.x channel family is particularly relevant for PAH. This family of channels is selectively inhibited by correolide. ⁷ It is noteworthy that correolide, like hypoxia, selectively constricts “resistance” pulmonary arteries, having little effect on large, proximal pulmonary arteries.⁷ The Kv1.x family of channels includes several O2 sensitive channels found in PASMCs, most notably Kv1.5 and Kv1.2, and it appears the constrictor effects of correolide mostly relate to inhibition of Kv1.5.

Lessons from the Mechanism of Hypoxic Pulmonary Vasoconstriction
Hypoxia initiates hypoxic pulmonary vasoconstriction (HPV), at least in part, by inhibition of Kv channels in the PASMC.⁸ O2-sensitive K+ channels are involved in O2 sensing in virtually all mammalian O2-sensitive tissues, including the mechanism of HPV in the pulmonary circulation. The resulting depolarization activates the voltage-gated, L-type Ca2+ channel. The importance of these two voltage gated channels to the pulmonary circulation is clear. K+ channel blockers
cause vasoconstriction; K+ channel openers cause pulmonary vasodilatation. Moreover, inhibiting the L-type Ca2+ channel using drugs such as nifedipine, which are also useful therapy for PAH,³ largely inhibits HPV.⁹ The Kv channels and the L-type Ca2+ channel are functionally coupled through membrane potential, which is established by the K+ channels.¹⁰ As will be discussed subsequently, Kv1.5 is particularly important in the PASMC in both HPV and PAH.

Homo- or heterotetramers composed of Kv1.2, Kv1.5, Kv2.1, Kv3.1b, and Kv2.1/Kv9.3 channels have been proposed as potential “O2-sensitive” channels mediating HPV.^11,12 It appears that some of these same channels are downregulated in pulmonary hypertension. In humans with PAH, PASMCs are depolarized and calcium overloaded, as a consequence of decreased expression of certain Kv channels, most notably Kv1.5.¹³ In rats, chronic hypoxia-induced pulmonary hypertension recapitulates many of the hallmarks of human pulmonary hypertension, including increased pulmonary vascular resistance (PVR), medial hypertrophy of small pulmonary arteries, and right ventricular hypertrophy. ¹⁴ Interestingly, acute HPV is selectively suppressed in rodents with chronic hypoxia-induced pulmonary hypertension, even though constriction to other stimuli is enhanced.¹⁵ This is associated with a functional deficiency of a select group of PASMC K+ channels (Kv1.5 and Kv2.1).¹⁶ This Kv expression is downregulated early, probably before pulmonary hypertension occurs.¹⁶ Indeed, chronic hypoxia reduces Kv1.5 expression in cell culture models, suggesting that it is the channel deficiency that drives the pulmonary hypertension, rather than the reverse.^{13, 17} In chronic hypoxia-induced pulmonary hypertension PASMCs are depolarized, much as has been described in studies of human PAH.^{13, 17}

As would be predicted if the K+ channel hypothesis were correct, increasing the expression of Kv channels ameliorates established chronic hypoxia-induced pulmonary hypertension. Increasing K+ current can be accomplished by increasing expression of Kv channels by stimulating endogenous channel production (Figure 4), activating existing channels (Figures 5 and 6), or increasing channel expression via gene transfer (Figure 7). Sildenafil elevates cyclic guanosine monophosphate (c-GMP) in PASMCs, at least in part, by opening PASMC BKCa channels via a protein kinase G-dependent mechanism.^18,19 This hyperpolarizes and relaxes the pulmonary artery (Figure 5). In humans with PAH, acutely sildenafil is as effective and selective a pulmonary vasodilator as inhaled nitric oxide,²⁰ likely because of the relative enrichment of the pulmonary arteries in phosphodiesterase 5. Long-term administration of sildenafil (50 mg tid) improves the 6-minute walk by more than 100 m in functional class II-II patients (Figure 6).²¹

Dichloroacetate (an inhibitor of the mitochondrial enzyme, pyruvate dehydrogenase kinase, reverses established chronic hypoxia-induced pulmonary hypertension by both increasing the opening and upregulating the expression of Kv2.1 and Kv1.5 channels (Figure 4).¹⁴ Dichloroacetate’s mechanism of action remains unclear, but might include metabolic actions that occur at higher doses (dichloroacetate is an inhibitor of pyruvate dehydrogenase kinase that increases the pyruvate/lactate ratio) and more rapid kinase-dependent mechanisms that occur at lower doses. Dichloroacetate causes rapid activation of Kv channels by a tyrosine kinase-dependent mechanism. Dichloroacetate is a very attractive drug to be tested in the treatment of primary pulmonary hypertension since it has little effect on K+ channels in nonhypertensive PASMCs. Moreover, it has been tried in humans with coronary disease and heart failure, with a very good safety profile. Dichloroacetate is also beneficial in monocrotaline models (our unpublished data).

If Kv channels are crucial to the pathogenesis of PAH, restoration of channels directly, via gene therapy, should also be beneficial. This has been tested in the chronic hypoxiainduced pulmonary hypertension model using serotype 5 adenovirus (Ad5) as the vector to deliver human pulmonary artery-Kv1.5 to the pulmonary circulation.¹⁵ Rodents exposed to chronic hypoxia selectively lose HPV, which appears to relate to the loss of Kv1.5 and Kv2.1 expression in PASMCs. Kv1.5 expression and function were restored by adenoviral gene therapy (Figure 7). Lightly anesthetized rats with established chronic hypoxia-induced pulmonary hypertension were nebulized with 100 µL of sterile saline, Ad5- GFP, or Ad5-GFP-Kv1.5 using an intratracheal microspray device. Transgene expression was evident 2 days after nebulization, and persisted for more than 14 days, gradually disappearing by 1 month. PVR was lower in the gene therapy group, largely because of increased CO.

This group also displayed partial regression of right ventricular hypertrophy and medial hypertrophy of small pulmonary arteries, but had no significant fall in pulmonary artery pressure. The decreased PASMC K+ current density in chronic hypoxia-induced pulmonary hypertension was reversed by Kv1.5 gene therapy and there was a corresponding restoration of HPV in rats that received Kv1.5 gene therapy (Figure 7). Thus, restoration of a single Kv channel to resistance pulmonary arteries restored HPV and reduced PVR, despite ongoing exposure to hypoxia.

Kv1.5 and Pulmonary Arterial Hypertension
Kv1.5 is an appealing candidate for involvement in PAH not only because to is central to the control of membrane potential in the PASMC⁷ and involved in HPV,^{11, 22} but also because loss of Kv1.5 expression appears to be important to the pathogenesis of various forms of pulmonary hypertension, including chronic hypoxia-induced pulmonary hypertension. In addition, anorexigens (eg, dexfenfluramine) that cause pulmonary vasoconstriction²³ and have led to outbreaks of PAH, inhibit K+ current in PASMCs by directly blocking Kv1.5 and Kv2.1. Finally, targeted deletion of Kv1.5 in mice reduces HPV and eliminates the hypoxia and 4-AP-sensitive component of the K+ current.²² Kv1.5 is an interesting therapeutic target because its expression is dynamically regulated, both at the level of transcription and post-translationally. The rapid pathophysiological changes in Kv1.5 expression in chronic hypoxia-induced pulmonary hypertension may reflect decreasing Kv1.5 gene transcription, destabilization of Kv1.5 mRNA or accelerating turnover of Kv1.5 protein. The fact that the CMV-promoted Kv1.5 transgene product was not suppressed by chronic hypoxia suggests that downregulation of the endogenous rat Kv1.5 occurs at the transcriptional level, as this would not be expected to alter episomal Kv1.5 expression. Chronic hypoxia- induced pulmonary hypertension may activate KRE (Kv1.5 repressor element), a dinucleotide repetitive DNA sequence forming a cell-specific silencer that inhibits transcription of the Kv1.5 gene.^24,25 Other regulators of gene expression may also be important. The fact that Kv1.5 expression can be rapidly suppressed offers hope that it might also be rapidly restored to a therapeutic end.

Channel Diversity in the Pulmonary Circulation
K+ channels manifest a diversity in expression and function that could explain the pulmonary circulation-specificity of the vascular disease in PAH. Use of patch-clamp techniques to directly record K+ currents from freshly dispersed PASMCs, isolated from various segments of the pulmonary circulation, reveals a differential distribution of ionic currents along the length of the pulmonary artery tree. Relevant to PAH, there is enrichment of the Kv channels involved in PAH, most notably Kv1.5,⁷ in the resistance arteries that largely determine PVR. In addition to longitudinal differences in whole-cell K+ current are electrophysiologically distinct cell populations, coexisting side by side, within an arterial segment.²⁶ The smooth muscle cells of conduit pulmonary arteries are large and predominantly express BKCa current; conversely, the resistance arteries are enriched in smaller smooth muscle cells with a high density of 4-APsensitive Kv current. Consistent with this finding, small pulmonary arteries vigorously contract in response to inhibition of Kv1.x channels by correolide but do not respond to pharmacological block of BKCa channels, indicating that Kv1.x channels are the primary determinants of resting membrane potential. K+ channel diversity can result from one or more of the following mechanisms:

Heterogeneous distribution of a-subunits. The concept that a particular segment of the pulmonary vascular bed uniquely expresses HPV or develops pulmonary hypertension because of heterogeneous expression or loss of a single a-subunit is appealing, but has not been demonstrated.

Heterotetramer formation. The “mix and match” of various a- or ?-subunits in a tissue-specific manner could yield functionally discrete channels that are unique to the pulmonary circulation. This concept awaits formal testing.

Alternate splicing variants of genes for a- and ß-subunits. ²⁷ This is an appealing mechanism by which PAH could be focused on the pulmonary circulation, but testing this requires sophisticated sequencing of the relevant channel in the tissue of interest, since results from immunoblotting are likely not to distinguish among splice variants unless a unique epitope is identified and targeted.

Association with ß-subunits. Kvß-subunits (Kvß 1-4, each with splice variants) are small cytosolic proteins (~30 KDa) that associate intimately with the channels. The ß-subunits create heterogeneity by modifying the gating and cellsurface expression of Kv channels.²⁸

Transcriptional regulation of K+ gene expression. Some K+ channels, such as Kv1.5, have very rapid rates of turnover, which can account for diversity in expression in pathological situations, such as in response to hypoxia. The Kv1.5 gene has cyclic adenosine monophosphate and glucocorticoid response elements. Local variation in promoters and silencers, such as KRE, CREB, and KCRF may be important, particularly in light of the short half-life of Kv channel mRNA and protein (~8 hours for Kv1.5).⁶

Environmental regulation. The regional fine-tuning of ionic expression is also regulated by the phosphorylation of these channels by various protein kinases, notably protein kinase A, G, and C. Phosphorylation can activate (as when nitric oxide activates protein kinase G, which phosphorylates and activates BKCa channels) or inhibit channels (as occurs with endothelin and serotonin, resulting in protein kinase Cmediated K+ channel inhibition). The local kinase environment may create tissue heterogeneity independent of variation in the a, ß, or ?-subunits expression.

Thus, Kv channels have a remarkable propensity for heterogeneity, and the PASMCs appear to take full advantage of this to generate diverse populations of Kv channels.

Genetics of Primary Pulmonary Hypertension
The established biological consequences of activating the BMPR2 pathway include cell differentiation, growth inhibition, and stimulation of collagen synthesis. BMP 2, 4, and 7 suppress PASMC proliferation in cells from normal subjects and patients with secondary PAH, but not in PASMCs from PAH patients.²⁹ In normal PASMCs, BMP 2 or 7 markedly increase the percentage of cells undergoing apoptosis and cause phosphorylation of Smad1.³⁰ The 5’ sites of the Kv1.5 gene may be regulated by Smad activation. Since PAH is associated with reduced expression of the same channel that is crucial to HPV, Kv1.5, we speculate that there is a link between the pathways that is relevant to both.

Environmental Stimuli: Diet Drugs, Toxic Oil, HIV
Both with aminorex and dexfenfluramine outbreaks only a small proportion of the patients exposed to anorexigen developed PAH, suggesting a requirement for one or more predisposing conditions. What are predisposing conditions for anorexigen-induced PAH?

First, many of the anorexigens are in fact also serotonin transporter substrates³¹ and thus get translocated into pulmonary vascular cells where, depending on the degree of retention, their intrinsic toxicity is amplified. Second, endothelial dysfunction, acquired or genetic, is almost certainly a predisposing factor. There is also a potential link between the anorexigens, PASMC Kv channels, and the endothelium. Kv channel inhibition and membrane depolarization partially account for the pulmonary and systemic vasoconstriction that dexfenfluramine and aminorex induce.^{23, 32} Recent studies show that Kv1.5 and Kv2.1 are directly inhibited by the anorexigens, even when the channels are studied in expression systems. In addition to their effects on smooth muscle cells, anorexigens also promote vasoconstriction by enhancing Ca++ release from the sarcoplasmic reticulum.³³ The anorexigens also block Kv channels in platelet progenitor cells, megakaryocytes.⁴ Kv channel inhibition in platelets leads to serotonin release. Furthermore, fenfluramine reduces Kv1.5 mRNA levels by 50% in PASMCs from normotensive patients,³⁴ suggesting inhibited gene transcription and expression of Kv channels play an important role in anorexigen-associated PAH.

Thus anorexigens are in reality Kv channel blockers that cause their intended effects (appetite suppression and alteration of serotonin levels) and their unintended effects (altered platelet function and increased vascular tone) through their control of membrane potential and cytosolic Ca++ levels. Are K+ channel abnormalities also found in HIVassociated PPH? Interestingly, expression of Kv1.3, an important molecular target for immunosuppressive agents in T lymphocytes, is inhibited, via a PKC-dependent mechanism, in HIV-PPH,³⁵ suggesting a link between cellular electrophysiology, immunity, and PAH.³⁶

The Link
There is a potential unifying link between the K+ channel hypothesis and the many other abnormalities observed in the platelets, endothelium, serotonin handling, and vascular tone in PAH (Figure 1). That link may be the common role of K+ channels in controlling membrane potential and thus intracellular concentrations of Ca2+ and K+ in platelets, smooth muscle cells, and possibly endothelial cells (Figure 3). By regulating cytosolic Ca2+, K+ channels can control vascular tone and cell proliferation (Figure 2). By regulating cytosolic K+, K+ channels modulate apoptosis and thus influences vascular remodeling. The loss or inhibition of Kv channels that occurs in PPH may also account for the observed decrease in platelet serotonin stores and the rise in plasma serotonin levels. The elevated serotonin, in the presence of endothelial dysfunction, would act as a vasoconstrictor and reinforce pulmonary hypertension, particularly in the setting of PASMC membrane depolarization and elevated cytosolic Ca2+. Recently it has been recognized that inhibition of K+ channels reduces and activation of K+ channels increases programmed cell death. By blocking the tonic egress of K+, Kv channel inhibition elevates intracellular K+. Because intracellular K+ tonically inhibits caspases, the inhibition or lack of Kv channels, as occurs in PAH and experimental pulmonary hypertension, contributes to a resistance to apoptosis.^37,38 Whether primary or secondary, the loss of K+ channels, the increased endothelin levels, and the increased serotonin levels all eventually result in increased intracellular Ca++ levels in PASMCs. In turn, this results in vasoconstriction and activation of genes that lead to cell proliferation. Normally, the BMPR2 , via the Smad pathway, exhibits a negative tonic control of gene transcription, promoting apoptosis rather than proliferation. The “loss of function” mutations in the BMPR2, in addition to one or more other abnormalities, create an imbalance that favors vasoconstriction and proliferation. It is uncertain how this K+ channel hypothesis relates to the matrix metalloproteinase and prothrombotic theories. Once initiated, PPH may be sustained and exacerbated by elastase and matrix metalloproteinase- induced matrix remodeling and a prothrombotic diathesis. Taking a lesson from recent advances in dilated cardiomyopathy, one is mindful of the possibility that PPH may result from a variety of molecular mechanisms.³⁹

Conclusion
PAH is a model disease in vascular biology. Its pathogenesis involves complex interactions among a predisposing genome, a permissive phenotype, and environmental stimuli, and it involves all the elements of the vasculature. An understanding of the pathways that are disordered leading to PPH suggests a number of attractive candidate targets for gene therapy, including Kv1.5, Kv2.1, and BMPR2.

Acknowledgment
Drs Michelakis and Archer are supported by the Canada Foundation for Innovation, the Alberta Heart and Stroke Foundation, the Alberta Heritage Foundation for Medical Research, and the Canadian Institutes for Health Research. Dr. Archer is supported by NIH-RO1-HL071115. Drs Archer and Michelakis hold a patent (2001057 US Prov) for K+ channel replacement therapy for vascular
diseases, including pulmonary arterial hypertension.

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