Transient Receptor Potential Channel 4 Encodes a Vascular Permeability Defect and High-Frequency Ca2+ Transients in Severe Pulmonary Arterial Hypertension
2016; Elsevier BV; Volume: 186; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2016.02.002
ISSN1525-2191
AutoresMichael Francis, Ningyong Xu, Chun Zhou, Troy Stevens,
Tópico(s)Ion Channels and Receptors
ResumoThe canonical transient receptor potential channel 4 (TRPC4) comprises an endothelial store–operated Ca2+ entry channel, and TRPC4 inactivation confers a survival benefit in pulmonary arterial hypertension (PAH). Endothelial Ca2+ signals mediated by TRPC4 enhance vascular permeability in vitro, but the contribution of TRPC4-dependent Ca2+ signals to the regulation of endothelial permeability in PAH is poorly understood. We tested the hypothesis that TRPC4 increases vascular permeability and alters the frequency of endothelial Ca2+ transients in PAH. We measured permeability in isolated lungs, and found that TRPC4 exaggerated permeability responses to thapsigargin in Sugen/hypoxia-treated PAH rats. We compared endothelial Ca2+ activity of wild-type with TRPC4-knockout rats using confocal microscopy, and evaluated how Ca2+ signals were influenced in response to thapsigargin and sequential treatment with acetylcholine. We found that thapsigargin-stimulated Ca2+ signals were increased in PAH, and recovered by TRPC4 inactivation. Store depletion revealed bimodal Ca2+ responses to acetylcholine, with both short- and long-duration populations. Our results show that TRPC4 underlies an exaggerated endothelial permeability response in PAH. Furthermore, TRPC4 increased the frequency of endothelial Ca2+ transients in severe PAH, suggesting that TRPC4 provides a Ca2+ source associated with endothelial dysfunction in the pathophysiology of PAH. This phenomenon represents a new facet of the etiology of PAH, and may contribute to PAH vasculopathy by enabling inflammatory mediator flux across the endothelial barrier. The canonical transient receptor potential channel 4 (TRPC4) comprises an endothelial store–operated Ca2+ entry channel, and TRPC4 inactivation confers a survival benefit in pulmonary arterial hypertension (PAH). Endothelial Ca2+ signals mediated by TRPC4 enhance vascular permeability in vitro, but the contribution of TRPC4-dependent Ca2+ signals to the regulation of endothelial permeability in PAH is poorly understood. We tested the hypothesis that TRPC4 increases vascular permeability and alters the frequency of endothelial Ca2+ transients in PAH. We measured permeability in isolated lungs, and found that TRPC4 exaggerated permeability responses to thapsigargin in Sugen/hypoxia-treated PAH rats. We compared endothelial Ca2+ activity of wild-type with TRPC4-knockout rats using confocal microscopy, and evaluated how Ca2+ signals were influenced in response to thapsigargin and sequential treatment with acetylcholine. We found that thapsigargin-stimulated Ca2+ signals were increased in PAH, and recovered by TRPC4 inactivation. Store depletion revealed bimodal Ca2+ responses to acetylcholine, with both short- and long-duration populations. Our results show that TRPC4 underlies an exaggerated endothelial permeability response in PAH. Furthermore, TRPC4 increased the frequency of endothelial Ca2+ transients in severe PAH, suggesting that TRPC4 provides a Ca2+ source associated with endothelial dysfunction in the pathophysiology of PAH. This phenomenon represents a new facet of the etiology of PAH, and may contribute to PAH vasculopathy by enabling inflammatory mediator flux across the endothelial barrier. The current model of the endothelium extends beyond the concept of a homogeneous cell monolayer in contact with the blood.1Aird W.C. Phenotypic heterogeneity of the endothelium, I: structure, function, and mechanisms.Circ Res. 2007; 100: 158-173Crossref PubMed Scopus (1212) Google Scholar, 2Aird W.C. Endothelial cell heterogeneity.Cold Spring Harb Perspect Med. 2012; 2: a006429Crossref Scopus (463) Google Scholar, 3Ochoa C.D. Wu S. Stevens T. 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Ca2+ channels and pulmonary endothelial permeability: insights from study of intact lung and chronic pulmonary hypertension.Microcirculation. 2006; 13: 725-739Crossref PubMed Scopus (49) Google Scholar Recently, our laboratory found that genetic knockout of the canonical transient receptor potential channel 4 (TRPC4), an ion channel that is involved in the formation of a store-operated Ca2+ entry complex, confers a substantial survival benefit in severe PAH.29Alzoubi A. Almalouf P. Toba M. O'Neill K. Qian X. Francis M. Taylor M.S. Alexeyev M. McMurtry I.F. Oka M. Stevens T. TRPC4 inactivation confers a survival benefit in severe pulmonary arterial hypertension.Am J Pathol. 2013; 183: 1779-1788Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar This phenomenon is associated with a reduction of both vascular lesion density and number,29Alzoubi A. Almalouf P. Toba M. O'Neill K. Qian X. Francis M. Taylor M.S. Alexeyev M. McMurtry I.F. Oka M. Stevens T. TRPC4 inactivation confers a survival benefit in severe pulmonary arterial hypertension.Am J Pathol. 2013; 183: 1779-1788Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar and maintenance of cardiac output.29Alzoubi A. Almalouf P. Toba M. O'Neill K. Qian X. Francis M. Taylor M.S. Alexeyev M. McMurtry I.F. Oka M. Stevens T. TRPC4 inactivation confers a survival benefit in severe pulmonary arterial hypertension.Am J Pathol. 2013; 183: 1779-1788Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar We identified ongoing endothelial Ca2+ transients in wild-type and TRPC4-deficient rats, and found that TRPC4 potentiates tissue-wide Ca2+ responses to acetylcholine (ACh). Furthermore, we found that PAH impairs endothelial-dependent vasodilation in response to ACh. However, the precise role of TRPC4 as a subunit of a functional store-operated Ca2+ entry complex involved in regulating localized endothelial Ca2+ signals and its impact on lung vascular permeability in PAH remain unknown. Therefore, we tested the hypothesis that TRPC4 underlies a permeability defect and encodes localized, high-frequency endothelial Ca2+ signaling events in severe PAH. All experimental procedures were performed in accordance with and approved by the Institutional Animal Care and Use Committee of the University of South Alabama (Mobile, AL). Severe occlusive PAH in rats was induced by a single s.c. 20 mg/kg injection of the vascular endothelial growth factor receptor type II inhibitor SU5416 (Cayman Chemical, Ann Arbor, MI) at 7 weeks after birth, followed by exposure to 3 weeks of normobaric hypoxia (10% O2) and then reexposure to normoxia (21% O2) for 2 to 3 additional weeks (PAH). TRPC4-knockout Fischer rats were generated by Transposagen Biopharmaceuticals (Lexington, KY), as part of the Knockout Rat Consortium (Trpc4tm1Bni, targeted mutation 1, Bernd Nilius), and were bred and genotyped both at Transposagen Biopharmaceuticals and at the University of South Alabama. Control rats were exposed to normoxia for 6 to 8 weeks. All rats were male and aged 12 to 16 weeks. Rat tail snips were collected according to the guidelines of the University of South Alabama Animal Care and Use Committee. DNA was extracted from tail snips, as described previously,29Alzoubi A. Almalouf P. Toba M. O'Neill K. Qian X. Francis M. Taylor M.S. Alexeyev M. McMurtry I.F. Oka M. Stevens T. TRPC4 inactivation confers a survival benefit in severe pulmonary arterial hypertension.Am J Pathol. 2013; 183: 1779-1788Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar and 2 μL of the resulting DNA solution was subjected to PCR analysis using three primers (primer A, 5′-GTGTTGGTCTCCATTACTTCAGCT-3′; primer B, 5′-ATTCTTCCCTTTGAGCCCACT-3′; and transposon primer, 5′-CTGACCTAAGACAGGGAATT-3′) in a total volume of 20 μL containing 1× GoTaq Green PCR master mix (Promega, Madison, WI) and 1 μmol/L of each primer. The cycling parameters were denaturation at 94°C for 5 minutes; then 30 cycles of 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 1 minute; and extension at 72°C for 7 minutes. Animals were anesthetized using 65 mg pentobarbital (Nembutal; Sigma-Aldrich, St. Louis, MO)/kg body weight. Once a surgical plane was achieved, defined by the absence of a withdrawal reflex after toe and tail pinch, animals were intubated and ventilated, a sternotomy was performed, and pulmonary artery and left ventricular catheters were inserted. Blood was taken by heart puncture from the right ventricle. Heart and lungs were removed en bloc and suspended in a humidified chamber where mechanical ventilation and blood flow was established. Rat lungs were perfused at constant flow (40 mL/minute per kg body weight) with buffer (in mmol/L: 119.0 NaCl, 4.7 KCl, 1.17 MgSO4, 1.0 Na2HPO4, 1.18 KH2PO4, 2.2 NaHCO3, and 5.5 glucose) containing 4% bovine serum albumin/6% autologous blood and physiological (2.2 mmol/L) CaCl2 at pH 7.30 to 7.40 at 38°C, and mean pulmonary artery pressure measurements were recorded. Filtration coefficient (Kf) values were measured as previously described, using zone 3 conditions.30Alvarez D.F. Gjerde E.A. Townsley M.I. Role of EETs in regulation of endothelial permeability in rat lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L445-L451Crossref Scopus (48) Google Scholar Baseline Kf was calculated as the rate of weight gain obtained 13 to 15 minutes after a 10 cm H2O increase in pulmonary venous pressure, normalized per 100 g predicted wet lung weight. A dose of 75 nmol/L thapsigargin was added to the perfusate reservoir and circulated for 10 minutes, and a second Kf was determined after the treatment in both the wild-type and TRPC4-knockout groups under normotensive and pulmonary arterial hypertensive conditions. Intrapulmonary branches of the left pulmonary artery were dissected from the lung in cold HEPES/bicarbonate-buffered physiological saline solution. Pulmonary arteries (approximately 5 mm in length) were dissected from surrounding connective tissue, cut longitudinally, and mounted in a custom viewing chamber. Tissue preparations were loaded with 1 mL of physiological saline solution (in mmol/L: 2 CaCl, 134 NaCl, 6 KCl, 1 MgCl, 10 HEPES, and 10 glucose) containing 10 μmol/L Fluo-4 fluorescent Ca2+ indicator dye and 0.03% Pluronic for 35 minutes at 25°C. After a 5-minute wash and a 10-minute equilibration period, tissues were mounted in a custom chamber with 1 mL of buffer and viewed on a Perkin Elmer RS-3 spinning disk inverted confocal microscope (Perkin Elmer, Waltham, MA). Excitation and emission wavelengths were 488 and 510 nm, respectively. Fluorescence intensity recordings were captured at ×20 magnification. Each recording began with an unstimulated 3-minute interval, followed by stimulation with 1 μmol/L thapsigargin, and a 3-minute treatment sampling interval, then 1 μmol/L ACh and a final 3-minute treatment sampling interval. Stimulation was performed with volume swaps using 0.5 mL of drug solution at 2 μmol/L for either ACh or thapsigargin, swapped with 0.5 mL of viewing buffer for an effective concentration of 1 μmol/L. Image sequences were acquired with Perkin Elmer Ultraview software version 1.0.0.9 at 9.8 frames per second. Ca2+ events were analyzed offline with LC_pro custom analysis software version 12.18.11 (linear base) as a plugin for ImageJ software version 1.49v (NIH, Bethesda, MD) and statistical processing using custom analysis scripts with R software version 3.1.2 ("Pumpkin Helmet"; http://www.r-project.org).31Abramoff M.D. Magalhaes P.J. Ram S.J. Image processing with ImageJ.Biophotonics Int. 2004; 11: 36-42Google Scholar, 32Francis M. Qian X. Charbel C. Ledoux J. Parker J.C. Taylor M.S. Automated region of interest analysis of dynamic Ca2+ signals in image sequences.Am J Physiol Cell Physiol. 2012; 303: C236-C243Crossref PubMed Scopus (51) Google Scholar Treatment with thapsigargin and ACh caused significant movement of the endothelial cell plane, because of the vasodilation effect of both drugs. We therefore used StackReg version 07.07.11, ImageJ-based image stack registration software, to compensate for this motion. Image sequences of endothelial Ca2+ activity en face were registered by selecting the anchor frame to be the last frame before drug treatment, and the stackreg plugin was set to the translation setting for continuous, unidirectional linear motion compensation throughout each sequence. All data were analyzed using the R statistical processing program. For Fulton index and pulmonary artery pressure measurements, data were analyzed using a two-tailed t-test. Filtration coefficient, cell number, modal Ca2+ event number, and event duration measurements were analyzed using a two-way analysis of variance with Tukey's post hoc test for pairwise comparisons. To determine the effect of pulmonary arterial hypertension on endothelial barrier responses to store depletion, we induced severe pulmonary hypertension in wild-type and TRPC4-knockout littermates with Sugen/hypoxia treatment, and measured whole lung permeability. Consistent with previous reports, both wild-type and TRPC4-knockout animals developed severe pulmonary arterial hypertension, with right ventricular hypertrophy.29Alzoubi A. Almalouf P. Toba M. O'Neill K. Qian X. Francis M. Taylor M.S. Alexeyev M. McMurtry I.F. Oka M. Stevens T. TRPC4 inactivation confers a survival benefit in severe pulmonary arterial hypertension.Am J Pathol. 2013; 183: 1779-1788Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 33Jiang B. Deng Y. Suen C. Taha M. Chaudhary K.R. Courtman D.W. Stewart D.J. Marked strain-specific differences in the SU5416 rat model of severe pulmonary arterial hypertension.Am J Respir Cell Mol Biol. 2016; 54: 461-468Crossref Scopus (67) Google Scholar Fulton index values computed as the weight of the right ventricle divided by the weight of the left ventricle and septum were 0.76 ± 0.04 for wild-type hypertensive and 0.71 ± 0.04 for TRPC4-knockout hypertensive groups, compared with 0.26 ± 0.02 for wild-type normotensive animals (P < 0.01) (Figure 1A). Isolated perfused lungs were prepared as previously described and equilibrated so that a baseline isogravimetric period was established, where lung pressures and weight remained stable.30Alvarez D.F. Gjerde E.A. Townsley M.I. Role of EETs in regulation of endothelial permeability in rat lung.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L445-L451Crossref Scopus (48) Google Scholar Under these conditions, mean pulmonary artery pressure was 9 ± 1 and 48 ± 6.23 cm H2O in normotensive and pulmonary arterial hypertensive wild-type lungs, respectively (P < 0.01). Mean pulmonary artery pressures were comparable between hypertensive wild-type (48 ± 6.23) and TRPC4-knockout rats (43 ± 4.69) (Figure 1B). Next, baseline permeability was measured. Baseline filtration coefficients were not significantly different for hypertensive animals (wild-type: 0.21 ± 0.04; TRPC4-knockout: 0.32 ± 0.06) compared with normotensive animals (wild-type: 0.17 ± 0.05; TRPC4-knockout: 0.24 ± 0.03) (Figure 1C). To bypass upstream signal cascades and deplete intracellular Ca2+ stores directly, thapsigargin (75 nmol/L) was added to the recirculating bath for 10 minutes, and a second filtration coefficient measurement was obtained (Figure 1C). Thapsigargin treatment did not significantly increase filtration coefficient absolute values in normotensive wild-type animals (0.36 ± 0.12), although Kf values expressed as fold-change over baseline were significantly greater than the predicted value of 1 (2 ± 0.13; P < 0.01), suggesting a comparable sensitivity to endothelial permeability evoked by activation of store-operated Ca2+ entry between Fischer and Sprague-Dawley rats.27Chetham P.M. Babál P. Bridges J.P. Moore T.M. Stevens T. Segmental regulation of pulmonary vascular permeability by store-operated Ca2+ entry.Am J Physiol. 1999; 276: L41-L50PubMed Google Scholar Thapsigargin did not increase the filtration coefficient in normotensive lungs from TRPC4-knockout (0.3 ± 0.03) rats at baseline. However, thapsigargin significantly increased the filtration coefficient in wild-type animals with pulmonary arterial hypertension (0.57 ± 0.08) (P < 0.01 versus baseline). Thapsigargin-dependent increases in filtration coefficient values were abolished in TRPC4-knockout hypertensive rats (0.36 ± 0.06). Thus, TRPC4 underlies thapsigargin-induced increases in permeability in the hypertensive pulmonary circulation. Next, endothelial cell morphology and Ca2+ signaling in excised pulmonary artery segments were examined en face using confocal microscopy and the Ca2+ indicator dye, Fluo-4. Notably, arteries from PAH rats were markedly thicker on gross observation en face compared with normotensive controls. Opened pulmonary artery segments loaded with Fluo-4 revealed an atypical polygonal appearance of the pulmonary endothelium of hypertensive animals compared with normotensive wild-type and TRPC4-knockout controls (Figure 2A). However, endothelial morphology was preserved in hypertensive animals harboring a TRPC4 deletion. There was no significant difference in endothelial cell number per 133 × 133-μm confocal viewing field in hypertensive wild-type and TRPC4-knockout animals (141 ± 42 and 154 ± 47, respectively) compared with normotensive wild-type (173 ± 20) and TRPC4-knockout (205 ± 2) controls (Figure 2B). We next examined whether adaptation to pulmonary arterial hypertension altered endothelial Ca2+ signaling. To determine whether endothelial Ca2+ event frequency and duration were affected by PAH, baseline Ca2+ was analyzed for 3 minutes and then 1 μmol/L thapsigargin was applied for an additional 3 minutes (Supplemental Movie S1). Transient increases in Ca2+ (events) were detected and counted by automated region-of-interest analysis using LC_Pro, a plugin for ImageJ (Figure 3). Event duration was defined by the difference in time between half-maximal peak values on either side of a detected signal peak. Histograms of the number of events versus event duration during baseline measurement periods (basal) for wild-type, TRPC4-knockout, wild-type hypertensive, and TRPC4-knockout hypertensive groups are shown in Figure 3A. Figure 3B shows histograms for the corresponding measurement period after 1 μmol/L thapsigargin stimulation. We quantified the number of events at the most frequent (modal) value of signal duration for both unstimulated and thapsigargin-stimulated treatment intervals (Figure 3C). In normotensive wild-type and TRPC4-knockout pulmonary arteries, 1 ± 0.58 and 3.5 ± 1.25 Ca2+ events occurred at the modal signal duration, whereas this number was significantly increased in wild-type PAH pulmonary arteries, 19 ± 10.3 (P < 0.05). In PAH, TRPC4 inactivation partially restored the number of Ca2+ events at the modal signal duration (10 ± 3.8) (Figure 3C). Thus, basal Ca2+ events were more prominent in pulmonary endothelium from PAH arteries. After treatment with 1 μmol/L thapsigargin, the number of Ca2+ events in wild-type normotensive pulmonary arteries expanded to 10.3 ± 9.3, whereas a significant expansion (55 ± 14.6) occurred in wild-type PAH pulmonary arteries (Figure 3C). TRPC4-knockout cell responses in normotensive (12.5 ± 3.7) and PAH (25.7 ± 3.5) conditions were not significantly different from wild-type controls. In oscillating cells, we measured the duration of Ca2+ events (eg, half-maximal time of an increase and corresponding decrease in Ca2+). Event duration values were not significantly different among groups at baseline (Figure 3D), indicating that the only parameter changed by PAH at baseline was the number of Ca2+ signal transient events (Figure 3C). However, PAH significantly reduced the duration of Ca2+ events in response to 1 μmol/L thapsigargin (13.8 ± 9.9 seconds) relative to normotensive control (41.8 ± 34.7 seconds) (Figure 3D). Thus, the endothelium of PAH pulmonary arteries responds to thapsigargin uniquely, with an increased frequency of events occurring at an attenuated duration. These data suggest that TRPC4 regulates the number of short-duration endothelial Ca2+ signals, and its activation is associated with increased vascular permeability. To determine the effect of TRPC4 in regulating endothelial Ca2+ entry after physiological stimuli, we next examined the Ca2+ signals initiated by ACh subsequent to store depletion with thapsigargin. Histograms of the number of detected ACh-induced Ca2+ events versus event duration reveal bimodal population distributions for wild-type, TRPC4-knockout, wild-type hypertensive, and TRPC4-knockout hypertensive groups (Figure 4A). ACh induced both long-duration (>30 seconds) and short-duration (<30 seconds) responses in normotensive and PAH pulmonary artery endothelium. Figure 4B illustrates representative short- and long-duration ACh-induced Ca2+ signals, which were characterized by a rapid increase in Ca2+. Long-duration signals exhibited a sustained increase and gradual decrease in Ca2+ levels. ACh stimulation increased the number of short- and long-duration Ca2+ events differentially after store depletion (Figure 4C). The wild-type PAH group displayed a decreased number of long-duration events (3.02 ± 1.6) relative to wild-type controls (44.6 ± 38) (Figure 4C). Furthermore, TRPC4-knockout PAH animals exhibited a restoration of the number of long-duration responses (52.1 ± 42) to control levels. The number of short-duration events was not significantly changed with respect to animal group (Figure 4C). TRPC4-knockout significantly increased the duration of short ACh responses in both normotensive and hypertensive conditions (Figure 4D). Notably, TRPC4 inactivation significantly decreased the duration of normotensive long-duration responses (50.5 ± 11.04) relative to wild-type (87.8 ± 16.73) (Figure 4D). Together, these data reveal that ACh stimulation after store depletion with thapsigargin elicits two populations of Ca2+ responses with respect to response duration. Only the long-duration responses are abolished in PAH, suggesting that PAH inhibits TRPC4-dependent potentiation of endothelial Ca2+ responses. Herein, we used isolated lung studies and confocal microscopy of endothelial Ca2+ activity to determine lung vascular permeability and the frequency of TRPC4-dependent pulmonary artery endothelial Ca2+ transients in PAH. We used a model of severe hypertension in Fischer rats that uniquely recapitulates key features of the human PAH, including occlusive vascular lesions and early death because of right-sided heart failure.29Alzoubi A. Almalouf P. Toba M. O'Neill K. Qian X. Francis M. Taylor M.S. Alexeyev M. McMurtry I.F. Oka M. Stevens T. TRPC4 inactivation confers a survival benefit in severe pulmonary art
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