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Dichloroacetate, a Metabolic Modulator, Prevents and Reverses Chronic Hypoxic Pulmonary Hypertension in Rats

Voltage-gated potassium channel expression and activity have increased.

  1. 1.     Evangelos D. Michelakis, MD;
  2. 2.     M. Sean McMurtry, MD;
  3. 3.     Xi-Chen Wu, PhD;
  4. 4.     Jason R.B. Dyck, PhD;
  5. 5.     Rohit Moudgil, MSc;
  6. 6.     Teresa A. Hopkins, BSc;
  7. 7.     Gary D. Lopaschuk, PhD;
  8. 8.     Lakshmi Puttagunta, MD;
  9. 9.     Ross Waite, BSc;
  10. 10.   Stephen L. Archer, MD

+ Author Affiliations

  1. The authors are from the Department of Medicine (Cardiology) and Vascular Biology Group (E.D.M., M.S.M., X.-C.W., J.R.B.D., R.M., L.P., R.W., S.L.A.), and the Department of Pediatrics and Cardiovascular Research Group (J.R.B.D., T.A.H., G.D.L.), University of Alberta,
  1. Written correspondence should be addressed to Evangelos D. Michelakis, MD, Assistant Professor of Cardiology at the University of Alberta, 2C2.36 Walter Mackenzie Health Sciences Centre, Edmonton, Alberta, Canada. T6G 2B7. E-mail emichela@cha.ab.ca

Comment: We should all be taking NaDCA as a supplement to prevent CH-PHT because there is literally nothing else that will repair the harm caused by heart failure.

 

Abstract

Background— The expression and function of voltage-gated K+ channels (Kv) in pulmonary artery (PA) smooth muscle cells (SMCs) are suppressed in chronic hypoxic pulmonary hypertension (CH-PHT), and the cellular redox balance shifts toward a reduced state. state. We predicted that dichloroacetate (DCA), a metabolic regulator that can raise Kv current in cardiac cells and change the redox balance to an oxidized state, would reverse CH-PHT.

Methods and Results— Normoxic, normoxic+DCA (DCA 70 mg kg1 d1 PO), chronically hypoxic (CH), and CH+DCA were the 4 rat groups that we evaluated. For two to three weeks, CH and CH+DCA mice were housed in a hypoxic room (10% Fio2). DCA was administered either on day 1 to stop CH-PHT or on day 10 to stop its effects. On days 14 through 18, we monitored the hemodynamics of rats with closed chests using catheters with micromanometer tips. When compared to CH rats, CH+DCA animals showed considerably lower levels of right ventricular hypertrophy, PA remodeling, and pulmonary vascular resistance. IK was suppressed by CH, acute hypoxia-sensitive IK was abolished, and Kv2.1 channel expression was reduced. Low-dose DCA (1 mol/L) raised IK in CH-PASMCs temporarily. A tyrosine kinase-dependent pathway allowed DCA to activate Kv2.1 in a mammalian expression system. Long-term administration of DCA resulted in a partial recovery of IK and Kv2.1 expression in PASMCs without affecting the activity of right ventricular pyruvate dehydrogenase, indicating that DCA’s positive effects are caused by nonmetabolic mechanisms.

Conclusions— Through a process involving the restoration of Kv channel expression and function, DCA both prevents and reverses CH-PHT. DCA has been used in people before and could be utilized as a treatment for pulmonary hypertension.

Keywords:

Received August 3, 2001; revision received October 22, 2001; accepted October 29, 2001.

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Introduction

Chronic hypoxia (CH) causes the development of chronic hypoxic pulmonary hypertension (CH-PHT) in humans or animals through an unidentified mechanism. Pulmonary arterial (PA) vasoconstriction and remodeling are features of CH-PHT.1 Although endothelium plays a significant role in the pathogenesis of CH-PHT, vascular smooth muscle cells (SMCs) are becoming more and more understood to play a similar role.2 Both the contractile status and proliferative status of SMCs are controlled by the intracellular Ca2+ concentrations ([Ca2+]i). Ca2+ influx through voltage-gated, L-type Ca2+ channels, whose gating is regulated by the SMC membrane potential, contributes to the regulation of [Ca2+]i levels. The membrane potential in PASMCs is controlled by voltage-gated K+ channels (Kv), such as Kv1.5 and Kv2.1.3 These channels are O2-sensitive and can be inhibited by hypoxia in expression systems, along with Kv1.2, Kv3.1b, and Kv9.1. Acute hypoxia inhibits one or more of these channels, which leads to the start of hypoxic pulmonary vasoconstriction.3

In PASMCs, CH lowers the K+ current density, causing a depolarized state.,4,5 which increases [Ca2+]i, which encourages contraction and proliferation.6 Although the expression of many other channels is unaffected, CH is linked to impaired expression of some Kv channels (such as Kv1.5 and Kv2.1).4,7 Although the exact cause of Kv channel downregulation is unknown, recent research indicates that it may be related to the altered redox state brought on by CH.4 Rats with CH-PHT have more reduced redox state in their lungs than normoxic controls, as shown by a sustained decrease in the production of activated oxygen species and an increase in the levels of reduced glutathione.4 A reduced redox state has potential for both short-term hemodynamic effects (through modulation of K+ channel function8) and Long-term effects (due to the redox regulation of a number of oxygen-responsive genes, including hypoxia-inducible factor) 9).

We proposed that the expression and function of Kv channels are causally linked to the emergence and maintenance of CH-PHT. Dichloroacetate (DCA), a metabolic modulator that has been shown to increase whole-cell K+ current (IK) in cardiac myocytes from a rat myocardial infarction model, was used to improve Kv channel expression and function.10 DCA inhibits mitochondrial pyruvate dehydrogenase kinase (PDK)11 additionally, by raising the pyruvate/lactate ratio, might encourage PASMCs to be in an oxidized state.12 We therefore hypothesized that DCA would ameliorate the activity and expression of Kv channels and reverse CH-PHT, simulating the advantages of a return to normoxia, by reversing the reduced redox state in the PASMCs of CH-PHT rats.

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Methods

Male Sprague-Dawley rats were divided into the following groups according to age and weight: normoxia (n=10), normoxia+DCA (n=5), CH (n=10), or CH+DCA (CH-DCA, n=10). They were kept in a normobaric hypoxic room, the CH and CH-DCA groups (Reming Bioinstruments). Over the course of five days, the inspired oxygen fraction was lowered from 20% to 10% and maintained there. DCA was present in the water for the CH-DCA group at a concentration of 0.75 g/L, pH 7.0 (Aldrich), for a daily dose of 70 mg/kg/d1. This dosage is based on earlier research done on humans. 13–15 Either on day 1 (n = 5) to test for CH-PHT prevention or on day 10 (n = 5) to test for CH-PHT reversal, exposure to DCA began.

Hemodynamic Measurements

The rats were sedated (50 mg/kg IP sodium pentobarbital) and ventilated with room air beginning on day 14, and systemic blood pressure (SBP) was measured as previously mentioned.16 The rats were sedated (50 mg/kg IP sodium pentobarbital) and ventilated with room air beginning on day 14, and systemic blood pressure (SBP) was measured as previously mentioned. A 1.4F catheter (Millar Instruments) was inserted through the right jugular vein to measure PA pressures. Electronic averaging was used to calculate the mean PA pressure (PAP) over a period of 1 minute. Retrograde cannulation through the carotid artery was used to measure the left ventricular end-diastolic pressure (LVEDP). The Fick method was used to measure the cardiac output (CO).17 PAPLVEDP/CO was used to calculate pulmonary vascular resistance (PVR). Right ventricular hypertrophy (RVH) was assessed using the RV/(LV+septum) ratio, which has previously been described..18

Histology

For histology research, a lung segment was formalin-fixed. Researchers who were blinded to the treatment groups examined medial thickness in small and medium-sized PAs (n=88 from 4 rats per group) as previously described..18

Electrophysiology

Third to sixth division PAs were used to isolate and study fresh PASMCs using the amphotericin-perforated patch-clamp method..19,20 Before administration, the pH of 4-aminopyridine (4-AP) and DCA were both brought to 7.4. Currents were elicited by steps from 70 to +50 mV with test pulses of 200 ms while cells were voltage-clamped at a holding potential of 70 mV. Current density was calculated by dividing whole-cell currents by cell capacitance.

Additionally, using the traditional whole-cell method, Chinese hamster ovary (CHO) cells expressing Kv2.1 and green fluorescent protein (GFP) were investigated. Using Adeasy-1, an adenoviral backbone vector containing adenovirus (serotype 5) genomic DNA with E1 and E3 removed, a recombinant, replication-deficient adenovirus encoding GFP and Kv2.1 was created. With the aid of the restriction endonucleases NotI and SalI, a 2609-bp cDNA fragment encoding the open reading frame of the rat Kv2.1 channel was removed from its original pBK-CMV plasmid (provided by Dr. K. Takimoto, University of Pittsburgh, Pittsburgh, Pennsylvania) and ligated into pAdTrack-CMV, which also contains a kanamycin resistance gene and two cytomegalo The Kv2.1 gene undergoes homologous recombination with the adenoviral backbone when pAdTrack-CMV and Adeasy-1 are cotransformed into BJ5183 cells, producing a plasmid that contains the Kv2.1 and GFP genes. After being linearized with a PmeI restriction endonuclease digest, the resulting pAdTrack-CMV Kv2.1 construct was transformed into BJ5183 cells together with the supercoiled adenoviral vector Adeasy-1, and then was plated on LB plates containing kanamycin. Later colonies were isolated, and plasmid purifying columns were used to purify the plasmid DNA (Qiagen). Using LipofectAmine reagent, the Kv2.1 cDNA-containing plasmid from the adenoviral DNA was chosen, amplified, purified, linearized, and transfected into HEK 293 cells. Polymerase chain reaction testing for Kv2.1 was performed on plates that showed complete cell lysis. In HEK 293 cells, Ad5Kv2.1 replication was carried out several times. By using a discontinuous CsCl gradient, the virus that resulted was separated, precipitated, and concentrated. The virus titer was 1.5 109 pfu/mL in the end. In CHO cells cultivated in hypoxia, infection rates with Ad5-GFP-Kv2.1 were 90% after a 24-hour incubation. Using the LSM-510 confocal microscope by Zeiss, infected cells were chosen based on their green fluorescence (excitation 488 nm, detection 505 to 530 nm), and the effects of DCA on the Kv2.1 current were investigated.

With antibodies to K+ channels (Alomone) and isoform 1 of the PDK (Stressgen Biotechnologies), immunoblotting was carried out on pooled PA samples (n=3 rats per group; 25 g protein per group), as previously described.19

Using a radiometric assay, five rats from each group’s RVs had their PDH activity measured.21

Statistics

Mean and SEM are used to express values. An appropriate repeated-measures ANOVA or factorial ANOVA was used to evaluate intergroup differences. Fisher’s protected least significant differences test was utilized in the post hoc analysis (Statview 5.0, SAS Institute). Statistics was considered significant at a value of P 0.05.

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Results

Hemodynamics and Histology

PAP and PVR increased due to CH, which also produced RVH and PA medial hypertrophy. (Figures 1 to 3). Figures 1 to 3 Display the results of the first set of tests, where DCA was administered to the CH-DCA rats on day 10 of the protocol to check for CH-PHT regression. PAP and PVR decreased in CH-DCA rats, reaching levels that were only marginally higher than those in normoxic rats. (Figures 1 and 2). The CO (Figure 2), SBP [mm Hg]: normoxic, 102–6, CH, 110–5, CH-DCA, 100–7, and LVEDP, 9–10 mm Hg for all groups. SBP [mm Hg]: CH, 110–5, CH-DCA, 100–7. Additionally, RVH was dramatically reduced in the CH-DCA rats as compared to the CH rats.(Figure 2).

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Figure 1. PA high-fidelity trace in 3 groups of rats under study. Drinking water containing DCA reduces PA pressure increases brought on by CH.

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Figure 2.Three rat groups’ average hemodynamic data were examined using a regression protocol. While having no discernible impact on CO, DCA reverses the CH-induced increase in PAP, PVR, and RVH.

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Figure 3.A, Medium-sized PAs from 3 rat groups were investigated using histology (H-E stain). Each group’s representative PA is displayed (arrows). B, DCA reduces CH-induced PA medial hypertrophy.

In other studies to stop CH-PHT, DCA was administered on the first day within the hypoxic chamber. An additional 3 groups of rats (normoxic, CH, and CH+DCA, each with 5 animals) were used in this protocol. PVR was significantly decreased in CH-DCA rats from days 14 to 18 (normoxia, 0.090.01, CH, 0.200.02, and CH-DCA, 0.120.02 mm Hg min 1 mL 1, P 0.05). The measures of CO, SBP, and RVH were comparable to those in the regression protocol (not shown). In a different study, normoxic control rats (n=5) received DCA for 12 to 15 days; neither SBP nor pulmonary hemodynamics (PVR, 0.100.03 mm Hg min 1 mL 1) were affected.

PASMC Electrophysiology

IK and current density were markedly reduced in CH rats but significantly increased in CH-DCA rats. (Figure 4). As predicted, compared to normoxic controls (12.70.6 pF), the CH-PHT group experienced an increase in PASMC capacitance, which measures cell size. This increase was reversed in the CH-DCA group (13.50.4 pF). 4-AP (5 mmol/L) inhibited 50% of IK in normoxic control rats, indicating that it is transported by Kv channels, but IK in CH rats was insensitive to 4-AP. (Figure 5A). Sensitivity to 4-AP was restored in the CH-DCA rats.

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Figure 4.

Left, Pa SMC IK density average data plotted against voltages under the patch-clamp protocol. The decrease in current density brought on by CH over the course of long-term DCA treatment is reversed across the entire membrane potential spectrum examined. Bottom: A sample raw trace of IK for each group under study. Capacitance for the control, CH-PHT, and CH-DCA PA SMCs in the cells displayed was 12.8, 15.9, and 13.6 pF, respectively.

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Figure 5.Data from each study group’s average results on the effects of perfusion with 4-AP (A), hypoxic solution (B), or DCA (1 mol/L) on PASMC IK (current density) (regression protocol). In normoxic PASMCs, an A, 4-AP-sensitive current is present. CH-PHT rats lose this current, and CH-DCA rats partially restore it. B, In a manner similar to that of A, acute hypoxia-sensitive current in normoxic PASMCs is lost in CH-PHT, with a tendency for recovery (P=0.06) in CH-DCA mice. C, Low-dose DCA enhances IK in the CH group when administered short-term to PASMCs, but not in the normoxic or CH-DCA groups.

IK was markedly reduced by acute hypoxia in normoxic PASMCs but not in CH PASMCs. In CH-DCA PASMCs, there was a significant trend toward recovery of the O2-sensitive component of IK (P=0.06).(Figure 5B). Low-dose DCA (1 mol/L) given over a short period of time increased IK in CH PASMCs within 5 minutes of application, but neither normoxic nor CH-DCA rats’ IK was affected. (Figure 5C).

Immunoblotting

The expression of other Kv channels and the large-conductance calcium-sensitive K+ channels (BKCa) did not change significantly in CH PAs despite the fact that Kv1.5 and Kv2.1 were significantly downregulated. (Figure 6). Although DCA therapy had no effect on Kv1.5 expression, it partially reversed Kv2.1 downregulation.(Figure 6). Figure 6 also demonstrates that, despite appearing to be slightly higher in the CH rats compared to the normoxic rats, the expression of PDK (isoform 1) did not change further with DCA.

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Figure 6.data from immunoblotting on isolated PAs. Below each gel are background-corrected intensities that have been normalized to Ponceau-S values. Kv1.5 and Kv2.1 expression is reduced by CH, but the other K+ channels that were tested are not significantly impacted. Long-term administration of DCA partially restores Kv2.1’s downregulation, but has no appreciable effects on the expression of other K+ channels or PDK1.

CHO Cell Electrophysiology

CHO cells free of infection lacked Kv current. (Figure 7). The successfully infected CHO cells were identified by their green fluorescence, and it was discovered that they had a Kv2.1 current, which is an early activating, noninactivating, and voltage-dependent current. (Figure 7). On the CHO cells used as a control, DCA had no impact. In contrast, in the infected CHO cells, 1 mol/L DCA quickly activated the Kv2.1 current. the tyrosine kinase inhibitor genistein (100 mol/L, Sigma) was used as a pretreatment.)22 without affecting the basal Kv2.1 current, prevented the effects of DCA on IK. (Figure 7).

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Figure 7. CHO cells lacking GFP and Kv2.1 exhibit only a voltage-insensitive outward current and no baseline fluorescence, whereas CHO cells expressing GFP and Kv2.1 exhibit both a bright green fluorescence and a voltage- and 4-AP-sensitive current. One micromole per liter of DCA activates the Kv2.1 current. Genistein pretreatment reduces the effects of DCA..

PDH Activity

Phosphorylation of PDH by the PDK directly controls its activity. Because there wasn’t enough PA tissue, we looked at the RV. At least in the RV, there was no difference in the total PDH activity or the active/total PDH activity between the three groups: active/total normoxic, 0.720.11; CH, 0.630.12; CH-DCA, 0.790.13; P=NS.

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Discussion

We demonstrate how DCA both stops and stops established CH-PHT. DCA’s advantageous effects on hemodynamics (Figure 2) and the PA remodeling (Figure 3) parallel effects on the expression and operation of Kv channels (Figures 5 to 7). In the short term, DCA specifically activates Kv2.1 channels expressed in hypoxic CHO cells and raises IK in recently isolated PASMCs from rats with CH-PHT. Long-term administration of DCA prevents the downregulation of Kv channels, particularly Kv2.1, caused by CH. (Figure 6). We suggest that the capacity of DCA to reverse CH-PHT results from its treatment’s effects on the expression and operation of Kv channels. To the best of our knowledge, this is the first study to measure PAP in the more physiological closed-chest rat model using catheters with micromanometer tips. The pressures are higher than in the commonly used open-chest models because the chest is closed. The restricted frequency response and damping that are characteristics of fluid-filled catheters are also avoided by these catheters.

Numerous studies on the effects of DCA on LV performance have produced mixed results.,13–15,23 Its impact on the pulmonary circulation, however, has not been previously investigated. Our discovery that DCA has no impact on the LVEDP or SBP raises the possibility that the drug’s effects are confined to the pulmonary circulation.

The information is consistent with the theory that the downregulation and inhibition of Kv channels may contribute to the etiology of CH-PHT. A particular downregulation of PA Kv1.5 in human primary pulmonary hypertension is linked to the onset of the condition or at the very least a predisposition to it..24 Smirnov et al5 first demonstrated that Kv channel function is suppressed and PASMCs are depolarized in rats with CH-PHT. Later research revealed a link between CH-PHT and decreased Kv1.2 and Kv1.57 and Kv2.1.4 There is still debate over whether or not the disease’s etiology is connected to these modifications in Kv function and expression. Our discovery that DCA reversed CH-PHT by regaining Kv2.1 expression and function supports the idea that Kv channel deficiency plays a causal role in the pathogenesis of this type of experimental PHT.

We demonstrate that DCA increases Kv2.1 expression. (Figure 6), a channel that helps rat PASMCs determine their membrane potential.20 The downregulation of Kv1.5 and Kv2.1 in CH, despite the fact that immunoblotting is only a semiquantitative technique(Figure 6) confirms previously released information.4,7 Given that other channels are unaffected, these are most likely not nonspecific effects. (Figure 6). But more importantly, the immunoblotting results concur with those from our electrophysiology study. The fact that the CH rats’ significantly reduced 4-AP IK sensitivity (Figure 5A) is consistent with the noticed reduction in the expression of the 4-AP-sensitive channels Kv1.5 and Kv2.1.3 1 mmol/L 4-AP’s insignificant effects on normoxic IK (Figure 5A), However, suggests that Kv2.1 might not be as significant a contributor to the IK as Kv1.5, which is very sensitive to 4-AP. Partial 4-AP sensitivity recovery(Figure 5A) The partial recovery of the Kv2.1 expression is likely the functional result in the CH-DCA PASMCs. Additionally, the CH rats’ decreased sensitivity to acute hypoxia (Figure 5B) is most likely partially caused by the disappearance of oxygen-sensitive channels like Kv1.5 and Kv2.1. Similarly, albeit not statistically significant (P=0.06), the trend in CH-DCA PASMCs to recover their responsiveness to acute hypoxia Figure 5B), suggests that the increased expression of Kv2.1 has functional significance. The range of current-density data and voltage dependence of the IK in PASMCs reported in this study is consistent with that found in the literature.25–28 Because there is regional diversity in the expression of K+ channels within the pulmonary circulation, variations in the enzymatic dispersion techniques and the particular vascular sections used can help to explain variation in these values.28

We demonstrate that DCA can increase IK in the short term much more quickly and at much lower doses than previously described (mol/L versus mmol/L), indicating novel and previously unrecognized properties of DCA in addition to its long-term effects. Rozanski et al al10 demonstrated that 4 hours of incubation with 1.5 mmol/L DCA increased IK in cultured cardiomyocytes from infarcted rat hearts. Pyruvate mimicked this and a PDH blocker inhibited it, indicating that the effects of DCA were brought on by the drug’s metabolic actions.10 We can now demonstrate that DCA activates K+ current in CH PASMCs in just 5 minutes at a dose of just 1 mol/L. (Figure 5C). These findings are in line with DCA acting via additional kinases that could activate Kv currents.29 The molecular target of DCA in PASMCs cannot be identified because the IK in vascular SMCs is an ensemble of various K+ channels. Therefore, we demonstrated using a CHO expression system that DCA specifically activates Kv2.1 through a tyrosine kinase mechanism. (Figure 7). Because Kv2.1 regulates membrane potential in PASMCs, its activation and upregulation might be able to explain how DCA affects hemodynamics.

Despite the fact that we demonstrated that DCA, at least at the dose used, had no impact on PA PDK-1 expression (Figure 6) and RV PDH activity (Figure 7), We cannot rule out metabolic or redox effects on PASMCs. There is good information about PDK diversity and tissue-specific regulation of PDH.30,31 Additionally, it is unknown whether the energetics of vascular SMCs parallel those of the heart because they have not been thoroughly studied..12 However, our electrophysiological data present a unique mechanism that might work in conjunction with a redox process. We propose that the tyrosine kinase-dependent mechanism by which DCA opens Kv2.1 results in membrane hyperpolarization and a reduction in [Ca2+]i. Both vasorelaxation and PASMC growth are inhibited by these actions.

Given that it has already been used in small, short-term human studies without significant toxicity, DCA is a very alluring drug to be studied in human PHT.13–15 To the best of our knowledge, no other medications currently being used in clinical trials have the ability to open Kv channels. Kv channel function and expression may be able to be restored by DCA, which would be advantageous for the treatment of pulmonary vascular diseases.

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Acknowledgments

The Alberta Heritage Foundation for Medical Research, the Canadian Institutes for Health Research, the Heart and Stroke Foundation of Canada, and the Canadian Foundation for Innovation all provided funding to Drs. Michelakis and Archer.

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