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. Author manuscript; available in PMC: 2015 Jan 1.
Published in final edited form as: Hypertension. 2013 Oct 21;63(1):173–180. doi: 10.1161/HYPERTENSIONAHA.113.01902

TRPC1 and TRPC6 Contribute to Hypoxic Pulmonary Hypertension through Differential Regulation of Pulmonary Vascular Functions RR

Yang Xia 1,2, Xiao-Ru Yang 1, Zhenzhen Fu 1, Omkar Paudel 1, Joel Abramowitz 3, Lutz Birnbaumer 3, James SK Sham 1,§
PMCID: PMC4102175  NIHMSID: NIHMS541912  PMID: 24144647

Abstract

Hypoxic pulmonary hypertension is characterized by increased vascular tone, altered vasoreactivity and vascular remodeling, which are associated with alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells. We have previously shown that classical transient receptor potential 1 and 6 (TRPC1 and TRPC6) are upregulated in pulmonary arteries of chronic hypoxic rats, but it is unclear whether these channels are essential for the development of pulmonary hypertension. Here we found that pulmonary hypertension was suppressed in TRPC1 and TRPC6 knockout (Trpc1−/− and Trpc6−/−) mice compared to wildtype after exposure to 10% O2 for 1 and 3 weeks. Muscularization of pulmonary microvessels was inhibited, but rarefaction was unaltered in hypoxic Trpc1−/− and Trpc6−/− mice. Small pulmonary arteries of normoxic wildtype mice exhibited vasomotor tone, which was significantly enhanced by chronic hypoxia. Similar vasomotor tone was found in normoxic Trpc1−/− pulmonary arteries, but the hypoxia-induced enhancement was blunted. In contrast, there was minimal vascular tone in normoxic Trpc6−/− pulmonary arteries, but the hypoxia-enhanced tone was preserved. Chronic hypoxia caused significant increase in serotonin-induced vasoconstriction; the enhanced vasoreactivity was attenuated in Trpc1−/− and eliminated in Trpc6−/− pulmonary arteries. Moreover, the effects of 3-week hypoxia on pulmonary arterial pressure, right ventricular hypertrophy and muscularization of microvessels were further suppressed in Trpc1−/−Trpc6−/− double-knockout mice. Our results therefore provide clear evidence that TRPC1 and TRPC6 participate differentially in various pathophysiological processes; and the presence of TRPC1 and TRPC6 are essential for the full development of hypoxic pulmonary hypertension in the mouse model.

Keywords: Chronic hypoxia, pulmonary hypertension, vasoreactivity, vascular remodeling, vasomotor tone

INTRODUCTION

Acute exposure to alveolar hypoxia triggers reversible hypoxic pulmonary vasoconstriction (HPV), whereas prolonged exposure, as occurs in high altitude inhabitants or in patients suffering from respiratory diseases, causes pulmonary hypertension (PH). PH is characterized by potentiated vascular tone, altered reactivity to agonists and profound vascular remodeling, eventually leading to right heart (RV) hypertrophy and failure1. Even though the mechanism of pathogenesis is complex, the intrinsic changes in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs) are the major determinants contributing to PASMC proliferation and vasoconstriction in chronic hypoxic pulmonary hypertension (CHPH)2. Cytosolic Ca2+ concentration ([Ca2+]i) is regulated by intracellular Ca2+ release and extracellular Ca2+ influx, which is gated by voltage-dependent Ca2+ channels and voltage-independent nonselective cation channels. To date, there is growing evidence supporting the pivotal role of multiple nonselective cation channels in acute3 and prolonged hypoxic responses4–6.

Transient receptor potential (TRP) proteins encode a large repertoire of nonselective cation channels in vascular smooth muscle cells7. We have previously identified the TRP channels of classical/canonical (TRPC), melastatin- and vanilloid-related subfamilies in rat PASMCs4,8. Functional studies show that TRPC1 and TRPC6 mediate store-operated (SOCE) and receptor-operated Ca2+ entry (ROCE), respectively4. Most importantly, chronic hypoxia (CH) upregulates the expression of TRPC1 and TRPC6, as well as the associated SOCE and ROCE in rat distal pulmonary arteries (PAs)4. A subsequent study confirmed the upregulation of TRPC1 and TRPC6 expression in the murine model of CHPH, and suggested that the process requires the full expression of hypoxia inducible factor-1α9. Abnormalities in TRPC expression have also been identified in various types of PH. For example, PASMC of idiopathic pulmonary arterial hypertension patients excessively expresses TRPC3 and TRPC6, resulting in augmented SOCE and proliferation10. In monocrotaline (MCT)-induced PAH, increased TRPC1 expression and SOCE contribute to the enhanced vasoconstriction to endothelin-1 (ET-1)11. Additionally, treatment of experimental PH with sildenafil and sodium tanshinone IIA sulfonate suppresses TRPC1 and TRPC6 expression12,13. All of the information hints that TRPC1 and TRPC6 are critically involved in CHPH, but leaves open the question of whether the altered expression and functions of these TRPC channels are essential for the development of the disease. The present study was undertaken to address these issues by using TRPC1 (Trpc1−/−), TRPC6 (Trpc6−/−) and TRPC1/TRPC6 (Trpc1−/−Trpc6−/−) null mice to examine how TRPC1 and TRPC6 channels affect vasomotor tone, agonist-induced vasoconstriction and pulmonary vascular remodeling, and whether genetic deletion of these two cation channels could prevent the animals from developing CHPH.

MATERIALS AND METHODS

Mouse model of chronic hypoxia-induced pulmonary hypertension

Trpc1−/− and Trpc6−/− mice (1:1, 129Sv:C57BL/6J background) were initially provided by the NIEHS's Comparative Medicine Branch14,15. Corresponding wild type (WT) mice of the same background were used as control. Subsequent generations of colonies and Trpc1−/−Trpc6−/− double knockout mice were maintained at Johns Hopkins University. Age-matched male WT and knockout mice (10–12 week old) were placed in a hypoxic chamber and exposed to 10% O2 for 1 or 3 weeks to induce hypoxic pulmonary hypertension16. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Johns Hopkins Animal Care and Use Committee. (See supplementary materials for detail methods)

RESULTS

TRPC expression in Trpc1−/−, Trpc6−/− mice and WT mice

Expression of TRPC subtypes in endothelium denuded PAs of Trpc1−/−, Trpc6−/−, and WT mice were compared by real-time RT-PCR. In WT PAs, the mRNA level of TRPC1 was the highest followed by TRPC6 and then TRPC3, while TRPC4, 5 and 7 expressions were minimal. Similar TRPC mRNA expression profiles were found in Trpc1−/− and Trpc6−/− PAs, except they were devoid of TRPC1 and TRPC6 mRNA, respectively (Figure 1A and B). Similar to previous observations in CH rats and mice4,9, TRPC1 and TRPC6 protein levels were significantly increased by 246±51% (n=6) and 103±38% (n=6), respectively, in PAs of WT mice exposed to 3-weeks of 10% O2 (Figure 1C and D). Comparable increases in TRPC1 (167±31%, n=3) and TRPC6 proteins (110±33%, n=6) were observed in PAs of CH Trpc6−/− and Trpc1−/− mice, respectively (Figure 1E and F). These results suggest that there is no compensatory shift in TRPC expression, and the hypoxic regulation of TRPC expression is unaltered in PAs of the knockout mice.

Figure 1.

Figure 1

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TRPC expression in PAs of WT, Trpc1−/−, and Trpc6−/− mice. A and B, TRPC mRNA normalized with 18S rRNA in PAs of normoxic WT and knockout mice (n=4 for each group). C, immunoblots of TRPC1 and TRPC6 proteins in PAs of normoxic and 3-week hypoxic WT mice. D, average values of TRPC1 and TRPC6 proteins normalized by β-actin in PAs of WT mice (n=6 samples for each group). E, immunoblots of TRPC1 and TRPC6 proteins in PAs of normoxic and CH Trpc6−/− mice (upper panels) and Trpc1−/− mice (lower panels), respectively. F, average values of TRPC1 and TRPC6 protein normalized by β-actin in PAs of Trpc6−/− mice (n = 3 samples in each group) and Trpc1−/− mice (n = 6 samples in each group), respectively. * and ** indicates P<0.05 and P<0.01 compared with normoxic controls.

Deletion of TRPC1 or TPRC6 suppresses CHPH

Trpc1−/−, Trpc6−/− mice and WT mice were exposed to 10% O2 for 0, 1, and 3 weeks. Right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (MPAP) were identical in the three types of mice under normoxic condition. Elevation of RVSP, MPAP, and RV mass index (RVMI, RV/(LV+S)) were evident in WT mice after 1-week of hypoxia exposure, and progressed to higher levels after 3 weeks (Figure 1). Compared to WT, the increase in RVSP, MPAP, and RVMI were significantly less in Trpc1−/− mice after 1-week and 3-week of hypoxia. The changes in RVSP and MPAP were suppressed and RV hypertrophy was virtually absent in Trpc6−/− mice after 1-week of hypoxia. However, PH and RVMI of Trpc6−/− mice progressed in subsequent weeks, with marginal reduction in MPAP (p=0.056) and RVMI (p = 0.116) after 3-weeks. Polycythemia was developed in CH WT, Trpc6−/− and Trpc1−/− mice, with the hematocrit of Trpc6−/− mice slightly higher than that of WT. Heart rate and mean systemic arterial pressure were similar in normoxic and CH WT and KO mice (Figure 2F and G). These results for the first time show that TRPC1 and TRPC6 are required for the full development of CHPH.

Figure 2.

Figure 2

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Hypoxia-induced pulmonary hypertension in Trpc1−/−, Trpc6−/− and WT mice. A, representative tracings of right ventricular (RV) pressure and pulmonary arterial ressure (PAP) recorded in Trpc1−/−, Trpc6−/− and WT mice exposed to normoxia or 10% O2 for 1 or 3 weeks. B, mean data of right ventricular systolic pressure, RVSP; C, RV to left ventricle and septum mass ratio, RV/(LV+S); D, mean PAP, MPAP; E, Hematocrit; F, heart rate; G: mean arterial pressure, MAP, n=14-33 in each group. * indicates P<0.05, normoxia vs. hypoxia for each genotype; + indicates P<0.05 vs. WT under different treatments.

TRPC1 and TRPC6 contribute to CH-induced pulmonary vascular remodeling

Morphological analysis showed that the percent distributions of non-muscularized, partially muscularized, and completely muscularized small vessels (<100 μm) were similar in normoxic WT, Trpc1−/− and Trpc6−/− mice (Figure 3A). Significant increase in the partially (normoxia=14.9±3.0%, CH=30.1±5.2%, P<0.01) and completely muscularized vessels (normoxia=4.3±0.9%, CH=11.9±2.2%, P<0.01) were observed in CH WT mice. In contrast, there was no significant reduction in the non-muscular vessels or increase in the partially muscularized vessels, except a marginal increase in the completely muscularized vessels of CH Trpc1−/− and Trpc6−/− mice. Furthermore, the vessel density was significantly reduced in CH WT mice, and similar reduction was observed in CH Trpc1−/− and Trpc6−/− mice (Figure 3B). These results clearly indicate that TRPC1 and TRPC6 are important contributing factors to CH-induced neomuscularization of small PAs, but have little influence on rarefraction of small PAs in CHPH.

Figure 3.

Figure 3

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Morphological analysis of pulmonary vascular remodeling. A, Proportion of non-(<25%), partially (25–75%) and completely (>75%) muscularized resistance pulmonary vessels (<100μm). B, Quantification of pulmonary vascular densities in lungs of normoxic and hypoxic Trpc1−/−, Trpc6−/− and WT mice. There were 4–6 mice in each group. * indicates P< 0.05, ** indicates P<0.01 and *** indicates P < 0.001 vs. WT or as specified.

TRPC1 and TRPC6 contribute differentially to pulmonary vascular tone

We devised a strategy to estimate vascular tone at different preloads by measuring the active and passive wall-tension of size-matched (ID ~200 μm) PA rings using a wire-myograph. Increasing vessel-width from the resting position (zero tension) in 50 μm steps was associated with progressive increase in wall-tension (Figure 4A and Supplementary Figure S1). Active tension at each point was determined by subtracting the total wall-tension by the passive tension measured after complete relaxation by Ca2+ removal and addition of papaverine (10 μM)(Figure 4B). The vessel-width versus tension relation shows significant active tone in normoxic WT PAs, and it increased with vessel-width in a quasi-linear manner (Figure 4C). The active tension was significantly enhanced in PAs of CH WT mice and abolished by Ca2+ removal. Stretch-activated spontaneous contractions were observed occasionally in CH WT PAs (Supplementary Figure S2). Compared to normoxic WT PAs, vascular tone was similar in Trpc1−/− PAs at the lower vessel-width, but it leveled-off when vessel-width was further increased (Figure 4D). In contrast, the active tone of Trpc6−/− PAs was significantly lower throughout the whole range of vessel-width tested (P<0.001). Moreover, the vascular tone was suppressed in both CH Trpc1−/− and Trpc6−/− PAs (Figure 4E). At Δ-width of 250 and 350 μm, where wall-tensions equivalent to transmural pressures of approximately 15 and 25 mmHg (see Methods in supplementary data), CH caused a significant increase in vascular tone in PAs of WT and Trpc6−/− mice (Figure 4F). But the enhancement was blunted in CH Trpc1−/− PAs. These results suggest that TRPC6 is important for maintaining the basal tone under normoxic condition, and TRPC1 plays a crucial role in the CH-induced enhancement of pulmonary vascular tone.

Figure 4.

Figure 4

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Pulmonary vascular tone in Trpc1−/−, Trpc6−/− and WT PAs. A, representative tracings of wall-tension generated by stepwise increase (50 μm) in vessel-width in PAs of normoxic (upper panel) and chronic hypoxic (CH, lower panel) mice in Ca2+-containing, Ca2+-free, and Ca2+-free plus papaverine solution. B, wall-tension versus vessel-width relations generated from small PAs isolated from normoxic and hypoxic Trpc1−/− (nor: n=22; hyp: n=21), Trpc6−/− (nor: n=20; hyp: n=20) and WT mice (nor: n=33; hyp: n=38) in Ca2+ containing (black), Ca2+-free (red), or Ca2+-free +10 μM papaverine solution (blue). C, active tone of normoxic and CH WT PAs, calculated by the difference between wall-tensions obtained in Ca2+ containing and Ca2+-free + papaverine solution. D and E, active tone of PAs from mice of different genotypes after normoxic and chronic hypoxic exposure, respectively, ** indicates P < 0.01vs. WT. F, active tone of PAs of normoxic and hypoxic mice at Δwidth of 250 μm (left panel) and 350 μm (right panel). ** and *** indicate P < 0.01 and P<0.001 respectively vs. normoxia, and + indicates P < 0.05 vs. WT.

TRPC1 and TRPC6 contribute to pulmonary vasoreactivity in CH PAs

5-HT elicited concentration-dependent contraction in endothelium-denuded PAs of normoxic WT and knockout mice, with similar maximum response (Emax) and sensitivity (−log EC50) except a slightly lower Emax in PAs of Trpc6−/− mice (Figure 5A–C). Consistent with previous reports, the maximal response elicited by 5-HT in CH WT PAs was augmented (normoxia: 117.8±2.08%, n=12; hypoxia: 146.8±4.4%, n=12, p<0.001) without significant change in EC50 (Figure 5B). In contrast, the CH-induced enhancement of Emax was completely eliminated (normoxia: 110.9±1.0%, n = 17; CH: 112.9±2.0 %, n=12, P = 0.594) and the sensitivity for 5-HT was slightly reduced in CH Trpc6−/− PAs. The 5-HT-induced maximal response was also significantly suppressed in CH Trpc1−/− PAs compared to WT. These data suggest that TRPC6 is critically involved in 5-HT-induced contractile responses in PA under CH; and TRPC1 contributes in part to the enhanced vasoreactivity to 5-HT in CH.

Figure 5.

Figure 5

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5-HT-induced contractile responses in endothelium denuded PAs of Trpc1−/−, Trpc6−/− and WT mice. A, concentrations-response curves of 5-HT in normoxic (left panel) and hypoxic PAs (right panel) from Trpc1−/− (nor: n=10; hyp: n=10), Trpc6−/− (nor: n=10; hyp: n=10) and WT mice (nor: n=12; hyp: n=12), * indicates P < 0.05 vs WT. B and C, average group data of the maximum response and –log EC50, respectively, calculated from concentration-response curves from A, ** indicates P < 0.01 and *** indicates P < 0.005.

TRPC1/TRPC6 double knockout further suppressed CHPH

Since TRPC1 and TRPC6 regulate different vascular functions, we further examined the effects of trpc1/trpc6 double-deletion on CHPH. RVSP, MPAP, and RVMI were all significantly lower in normoxic Trpc1−/−Trpc6−/− mice compared to WT, while heart rate and mean systemic arterial pressures were the same (Figure 6A–E). The double-knockout mice developed less severe PH compared to WT mice, as indicated by significantly lower RVSP (WT: 26.2±0.5; trpc1−/−trpc6−/−:21.3±0.6, p<0.001), MPAP (WT: 18.2±0.4; trpc1−/−trpc6−/−: 14.6±0.4, p<0.001), and RVMI (WT: 0.33±0.01; trpc1−/−trpc6−/−: 0.28±0.01, p<0.001). The RVSP and MPAP of CH double-knockout mice were also significantly lower than those of CH trpc1−/− or trpc6−/− mice (supplemental Figure S3A and B). Morphological analysis found that the neo-muscularization of small PAs was virtually absent in CH Trpc1−/−Trpc6−/− mice (Figure 6F). However, significant reduction of vessel density was still observed in lungs of CH Trpc1−/−Trpc6−/− mice (Figure 6G). In conjunction with the results observed in Trpc1−/− and Trpc6−/− mice, these data support the notion that TRPC1 and TRPC6 are major contributing factors in the pathogenic processes of CHPH.

Figure 6.

Figure 6

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Pulmonary and systemic hemodynamic parameters and pulmonary vascular remodeling in Trpc1−/−Trpc6−/− (DKO) and WT mice. A, mean group data of right ventricular systolic pressure, RVSP; B, mean PAP, MPAP; C, RV to left ventricle and septum mass ratio, RV/(LV+S); D, heart rate; and E, mean arterial pressure, MAP. n=9–33 in each group. F, Proportion of non- (<25%), partially (25–75%) and completely (>75%) muscularized resistance pulmonary vessels (<100μm). G, Quantification of pulmonary vascular densities after 3-week normoxic or hypoxic exposure in DKO and WT mice. n=4–9. * indicates P< 0.05, ** indicates P<0.01 and *** indicates P < 0.001 vs. WT or as specified.

DISCUSSION

In the present study, we used genetic mouse models to test the hypothesis that TRPC1 and TRPC6 are crucial for CHPH development, and to examine their roles in pulmonary vascular functions. Trpc1−/− and Trpc6−/− mice are suitable for the purposes because the expressions of TRPC subtypes in PA are unaltered, consistent with previous reports in systemic arteries of Trpc1−/− mice14 and in PAs of Trpc6−/− mice3 (but also see15); and their regulation by CH are similar to those in rats and WT mice4,9. Our results show that TRPC1 and TRPC6 participate differentially in the three salient features of PH: elevated pulmonary vasomotor tone, altered vascular reactivity, and vascular remodeling. Ablation of TRPC1 or TRPC6 has minimal effect on pulmonary circulation under normoxic conditions, but mitigates PH and RV hypertrophy induced by CH.

We established a strategy for studying vasomotor tone at different muscle-length in murine small PA, and much novel information has been revealed. In contrast to the lack of basal vasomotor tone in normoxic rat microvessels5,17, murine PA exhibits a small component of active tone. This active tone is myogenic and Ca2+ dependent, judging by the increase in magnitude with mechanical stretch and by its complete inhibition after Ca2+ removal. The vascular tone was enhanced after 3-week CH, similar to the de novo appearance of myogenic tone in PAs of CH rats5,17. Interestingly, the basal tone in normoxic PAs and the CH-enhanced vasomotor tone are apparently two separate components mediated by different mechanisms. Deletion of TRPC6 suppressed the basal tone in normoxic PAs, but did not interrupt the CH-induced elevation in vasomotor tone. The reduction in vasomotor tone of Trpc6−/− PAs is consistent with reports showing TRPC6 is mechanosensitive and mediates myogenic response18,19, but it is in contrast to the enhanced myogenic tone in Trpc6−/− cerebral arteries where compensatory upregulation of TRPC3 is evident15. Compared to TRPC6, deletion of TRPC1 has little effect on the vascular tone of normoxic PAs at the lower vessel-width, but eliminated the increase in active tone at the higher levels of mechanical stretch. This is consistent with findings that TRPC1 is mechanosensitive in non-vascular cells20, but it does not play a significant role in myogenic tone under normal physiological conditions14. More importantly, the disappearance of the enhanced tone in CH Trpc1−/− PA suggests that TRPC1 is recruited to facilitate the enhanced vascular tone under pathological conditions of CHPH. This is in concordance with previous reports suggesting that TRPC1 upregulation are responsible for the elevated basal tone in CH rat PAs and resting [Ca2+]i of hypoxic PASMCs4,9,12.

A wealth of data has been accumulated suggesting alterations of vasoreactivity of CH rat PAs in response to agonists. 5-HT elicited an enhanced contractile response in PAs of our CH WT mice, after normalization with maximal KCl-induced contraction to account for changes in other non-receptor dependent mechanisms. This is consistent with previous reports on CH rats and mice6,21. Moreover, the CH-enhanced 5-HT response was noticeably suppressed in Trpc1−/− PAs and virtually abolished in Trpc6−/− PAs, suggesting that TRPC1 plays a contributing role while TRPC6 is required for the enhanced response. The clear participation of TRPC6 and TRPC1 in 5-HT-induced contraction in the hypoxic but not the normoxic PA could be related to the upregulation of the TRPC channels, and it may also reflect changes in the signaling mechanism. 5-HT-induced pulmonary vasoconstriction is mediated primarily by 5-HT2A and to a lesser extent by 5-HT receptor in normoxic PAs21,22. 5-HT1B and 5-HT2B expressions are upregulated and the contribution of 5-HT1B to pulmonary vasoconstriction is augmented in CH PAs21,23. It will be interesting for future studies to investigate whether 5-HT1B and/or 5-HT2B receptors are preferentially coupled to the upregulated TRPC6 channels in CH PAs.

TRPC1 and TRPC6 both play a significant role in neo-muscularization of small PAs which was largely suppressed in CH Trpc1−/− and Trpc6−/− mice. This is consistent with the well-recognized roles of TRPC1 and TRPC6 in PASMC proliferation10,24. Lessening of muscularization may reduce PA vasomotor tone and reactivity, hence attenuates PH in CH Trpc1−/− and Trpc6−/− mice. It is noteworthy that CH caused a 30–40% reduction in the density of pulmonary microvessels that could lead to an increase in parallel resistance of pulmonary circulation and elevate PAP. Pulmonary vascular rarefaction is well documented in CH rats and mice, and is related to alterations in VEGF and other signaling pathways25,26. This process, however, is independent of TRPC1 and TRPC6, because deletion of either or both channels did not reverse the vascular regression.

The contributions of TRPC1 and TRPC6 to CHPH are different at various stages of the disease. For example, PH and RV hypertrophy was greatly suppressed in Trpc6−/− mice exposed for 1-week hypoxia; but the suppression was diminished after 3-week. The early suppression of PH in Trpc6−/− mice may suggest the TRPC6-dependent vasoreactivity is a major factor in the early development of PH. But it is more likely related to the important role of TRPC6 in HPV. It has been shown that acute hypoxia activates TRPC6 in PASMCs through DAG accumulation; and HPV is completely abolished in Trpc6−/− mice3,27. Since HPV occurs immediately after exposure to hypoxia and is blunted within a week after prolonged exposure to hypoxia28,29, the impact of TRPC6-mediated HPV on PAP and RV hypertrophy should be most prominent in the first week of CH and subside thereafter as PH progresses. This is congruent with the observations in the CH Trpc6−/− mice. TRPC1, on the other hand, is engaged in the development of the intrinsic vasomotor/myogenic tone which continues to affect PAP throughout CH exposure. Hence, PH was consistently suppressed in Trpc1−/− mice after 1- and 3-weeks of CH.

TRPC1 and TRPC6 double-deletion experiments provided further insights into the combined influence of these TRPC channels in pulmonary vasculatures. The hypotension of pulmonary circulation observed in normal Trpc1−/−Trpc6−/− mice demonstrates the importance of TRPC1 and TRPC6 in the regulation of pulmonary vascular tone, contrasting to the systemic circulation where MAP was unaltered. The dramatic attenuation of the CH-induced increase in RVSP, mean PA pressure and RVMI in Trpc1−/−Trpc6−/− mice, as opposed to the limited effects of single gene deletion, further suggests that the combined actions of the two channels have significantly larger influence than TRPC1 or TRPC6 alone. Targeting both (or multiple) TRPC channels simultaneously, hence, could be an effective approach for the treatment of PH. It has to be mentioned, however, that there are other TRPC1/TRPC6 independent mechanisms, such as vascular rarefaction, still remain effective, causing the residual elevation of PAP in CH Trpc1−/−Trpc6−/− animals.

PERSPECTIVES

This study showed that TRPC1 and TRPC6 are crucial for the regulation of vasomotor tone, vasoreactivity and neo-muscularization of pulmonary vasculatures. These vascular functions contribute at different stages of CHPH; and their participations are essential for the full manifestation of CHPH in the murine model. In view of their multifaceted contributions to PH, manipulation of TRPC functions may offer a promising therapeutic strategy for hypoxia-related PH.

NOVELTY AND SIGNIFICANCE.

1) What Is New?

  • -

    This is the first study using genetic models to examine the roles of TRPC1 and TRPC6 in pulmonary vascular functions and CHPH.

  • -

    We identified for the first time that TRPC1 and TRPC6 differentially regulate pulmonary vasomotor tone, vasoreactivity and neo-muscularization; their presence are essential for the full manifestation of CHPH.

2) What Is Relevant?

  • -

    Our results clearly indicate that TRPC1 and TRPC6 play crucial roles in CHPH. Manipulation of TRPC functions may offer a novel therapeutic strategy for hypoxia-related PH.

Acknowledgments

SOURCES OF FUNDING This work was supported by National Institutes of Health Grants (R01 HL071835 and R01 HL075134 to JSKS), and by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Project Z01-ES-101684 to LB).

Footnotes

CONFLICT OF INTEREST: None

REFERENCE

  • 1.Stenmark KR, Fagan KA, Frid MG. Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circ Res. 2006;99:675–691. doi: 10.1161/01.RES.0000243584.45145.3f. [DOI] [PubMed] [Google Scholar]
  • 2.Kuhr FK, Smith KA, Song MY, Levitan I, Yuan JX. New mechanisms of pulmonary arterial hypertension: role of Ca(2)(+) signaling. Am J Physiol Heart Circ Physiol. 2012;302:H1546–1562. doi: 10.1152/ajpheart.00944.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, Olschewski A, Storch U, Mederos y Schnitzler M, Ghofrani HA, Schermuly RT, Pinkenburg O, Seeger W, Grimminger F, Gudermann T. Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange. Proc Natl Acad Sci U S A. 2006;103:19093–19098. doi: 10.1073/pnas.0606728103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Lin MJ, Leung GP, Zhang WM, Yang XR, Yip KP, Tse CM, Sham JSK. Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells: a novel mechanism of hypoxic pulmonary hypertension. Circ Res. 2004;95:496–505. doi: 10.1161/01.RES.0000138952.16382.ad. [DOI] [PubMed] [Google Scholar]
  • 5.Yang XR, Lin AH, Hughes JM, Flavahan NA, Cao YN, Liedtke W, Sham JS. Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol. 2012;302:L555–568. doi: 10.1152/ajplung.00005.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Xia Y, Fu Z, Hu J, Huang C, Paudel O, Cai S, Liedtke W, Sham JS. TRPV4 Channel Contributes to Serotonin-Induced Pulmonary vasoconstriction and the Enhanced Vascular Reactivity in Chronic Hypoxic Pulmonary Hypertension. Am J Physiol Cell Physiol. 2013 doi: 10.1152/ajpcell.00099.2013. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Inoue R, Jensen LJ, Shi J, Morita H, Nishida M, Honda A, Ito Y. Transient receptor potential channels in cardiovascular function and disease. Circ Res. 2006;99:119–31. doi: 10.1161/01.RES.0000233356.10630.8a. [DOI] [PubMed] [Google Scholar]
  • 8.Yang XR, Lin MJ, McIntosh LS, Sham JSK. Functional expression of transient receptor potential melastatin- and vanilloid-related channels in pulmonary arterial and aortic smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2006;290:L1267–1276. doi: 10.1152/ajplung.00515.2005. [DOI] [PubMed] [Google Scholar]
  • 9.Wang J, Weigand L, Lu W, Sylvester JT, Semenza GL, Shimoda LA. Hypoxia inducible factor 1 mediates hypoxia-induced TRPC expression and elevated intracellular Ca2+ in pulmonary arterial smooth muscle cells. Circ Res. 2006;98:1528–1537. doi: 10.1161/01.RES.0000227551.68124.98. [DOI] [PubMed] [Google Scholar]
  • 10.Yu Y, Fantozzi I, Remillard CV, Landsberg JW, Kunichika N, Platoshyn O, Tigno DD, Thistlethwaite PA, Rubin LJ, Yuan JX. Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension. Proc Natl Acad Sci U S A. 2004;101:13861–13866. doi: 10.1073/pnas.0405908101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Liu XR, Zhang MF, Yang N, Liu Q, Wang RX, Cao YN, Yang XR, Sham JS, Lin MJ. Enhanced store-operated Ca(2)+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats. Am J Physiol Cell Physiol. 2012;302:C77–87. doi: 10.1152/ajpcell.00247.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lu W, Ran P, Zhang D, Peng G, Li B, Zhong N, Wang J. Sildenafil inhibits chronically hypoxic upregulation of canonical transient receptor potential expression in rat pulmonary arterial smooth muscle. Am J Physiol Cell Physiol. 2010;298:C114–123. doi: 10.1152/ajpcell.00629.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wang J, Jiang Q, Wan L, Yang K, Zhang Y, Chen Y, Wang E, Lai N, Zhao L, Jiang H, Sun Y, Zhong N, Ran P, Lu W. Sodium tanshinone IIA sulfonate inhibits canonical transient receptor potential expression in pulmonary arterial smooth muscle from pulmonary hypertensive rats. Am J Respir Cell Mol Biol. 2013;48:125–134. doi: 10.1165/rcmb.2012-0071OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Dietrich A, Kalwa H, Storch U, Mederos y Schnitzler M, Salanova B, Pinkenburg O, Dubrovska G, Essin K, Gollasch M, Birnbaumer L, Gudermann T. Pressure-induced and store-operated cation influx in vascular smooth muscle cells is independent of TRPC1. Pflugers Arch. 2007;455:465–477. doi: 10.1007/s00424-007-0314-3. [DOI] [PubMed] [Google Scholar]
  • 15.Dietrich A, Mederos YSM, Gollasch M, Gross V, Storch U, Dubrovska G, Obst M, Yildirim E, Salanova B, Kalwa H, Essin K, Pinkenburg O, Luft FC, Gudermann T, Birnbaumer L. Increased vascular smooth muscle contractility in TRPC6−/− mice. Mol Cell Biol. 2005;25:6980–6989. doi: 10.1128/MCB.25.16.6980-6989.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Sun H, Xia Y, Paudel O, Yang XR, Sham JS. Chronic hypoxia-induced upregulation of Ca2+-activated Cl- channel in pulmonary arterial myocytes: a mechanism contributing to enhanced vasoreactivity. J Physiol. 2012;590:3507–3521. doi: 10.1113/jphysiol.2012.232520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Broughton BR, Walker BR, Resta TC. Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries. Am J Physiol Lung Cell Mol Physiol. 2008;294:L797–806. doi: 10.1152/ajplung.00253.2007. [DOI] [PubMed] [Google Scholar]
  • 18.Welsh DG, Morielli AD, Nelson MT, Brayden JE. Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ Res. 2002;90:248–250. doi: 10.1161/hh0302.105662. [DOI] [PubMed] [Google Scholar]
  • 19.Spassova MA, Hewavitharana T, Xu W, Soboloff J, Gill DL. A common mechanism underlies stretch activation and receptor activation of TRPC6 channels. Proc Natl Acad Sci U S A. 2006;103:16586–16591. doi: 10.1073/pnas.0606894103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Maroto R, Raso A, Wood TG, Kurosky A, Martinac B, Hamill OP. TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol. 2005;7:179–185. doi: 10.1038/ncb1218. [DOI] [PubMed] [Google Scholar]
  • 21.Keegan A, Morecroft I, Smillie D, Hicks MN, MacLean MR. Contribution of the 5-HT(1B) receptor to hypoxia-induced pulmonary hypertension: converging evidence using 5-HT(1B)-receptor knockout mice and the 5-HT(1B/1D)-receptor antagonist GR127935. Circ Res. 2001;89:1231–1239. doi: 10.1161/hh2401.100426. [DOI] [PubMed] [Google Scholar]
  • 22.MacLean MR, Sweeney G, Baird M, McCulloch KM, Houslay M, Morecroft I. 5-Hydroxytryptamine receptors mediating vasoconstriction in pulmonary arteries from control and pulmonary hypertensive rats. Br J Pharmacol. 1996;119:917–930. doi: 10.1111/j.1476-5381.1996.tb15760.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Rondelet B, Van Beneden R, Kerbaul F, Motte S, Fesler P, McEntee K, Brimioulle S, Ketelslegers JM, Naeije R. Expression of the serotonin 1b receptor in experimental pulmonary hypertension. Eur Respir J. 2003;22:408–412. doi: 10.1183/09031936.03.00036203. [DOI] [PubMed] [Google Scholar]
  • 24.Sweeney M, Yu Y, Platoshyn O, Zhang S, McDaniel SS, Yuan JX. Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol. 2002;283:L144–155. doi: 10.1152/ajplung.00412.2001. [DOI] [PubMed] [Google Scholar]
  • 25.Lazarus A, Keshet E. Vascular endothelial growth factor and vascular homeostasis. Proc Am Thorac Soc. 2011;8:508–511. doi: 10.1513/pats.201102-021MW. [DOI] [PubMed] [Google Scholar]
  • 26.Li X, Zhang X, Leathers R, Makino A, Huang C, Parsa P, Macias J, Yuan JX, Jamieson SW, Thistlethwaite PA. Notch3 signaling promotes the development of pulmonary arterial hypertension. Nat Med. 2009;15:1289–1297. doi: 10.1038/nm.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Fuchs B, Rupp M, Ghofrani HA, Schermuly RT, Seeger W, Grimminger F, Gudermann T, Dietrich A, Weissmann N. Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6. Respir Res. 2011;12:20. doi: 10.1186/1465-9921-12-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.McMurtry IF, Petrun MD, Reeves JT. Lungs from chronically hypoxic rats have decreased pressor response to acute hypoxia. Am J Physiol. 1978;235:H104–109. doi: 10.1152/ajpheart.1978.235.1.H104. [DOI] [PubMed] [Google Scholar]
  • 29.Weissmann N, Nollen M, Gerigk B, Ardeschir Ghofrani H, Schermuly RT, Gunther A, Quanz K, Fink L, Hanze J, Rose F, Seeger W, Grimminger F. Downregulation of hypoxic vasoconstriction by chronic hypoxia in rabbits: effects of nitric oxide. Am J Physiol Heart Circ Physiol. 2003;284:H931–938. doi: 10.1152/ajpheart.00376.2002. [DOI] [PubMed] [Google Scholar]

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