TRPC1, TRPC6and TRPV4Contribute To Hypoxic Pulmonary Hypertension Through Differential Regulation Of Pulmonary Vascular Functions

Rationale:Acute exposure to alveolar hypoxia triggers reversible hypoxic pulmonary vasoconstriction (HPV), whereas prolonged exposure to alveolar hypoxia, as occurs in high altitude sojourn or in patients suffering respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and sleep apnea, causes pulmonary hypertension (PH). Chronic hypoxic pulmonary hypertension (CHPH) is characterized by potentiated vascular tone [1,2], altered reactivity to agonists [3,4] and profound vascular remodeling [5,6], eventually resulting in right heart (RV) hypertrophy, failure and death.Complex as the underlying mechanism of chronic hypoxic pulmonary hypertension (CHPH) is, the onset of intrinsic changes in ionic balance and Ca2+homeostasis in pulmonary arterial smooth muscle cells (PASMCs) is a major trigger for the ongoing pathophysiological processes in CHPH, contributing to PASMCs proliferation, apoptosis, vasomotor tone and pulmonary vasoconstriction [4,7-9]. The intracellular Ca2+stores and extracellular Ca2+influx are the major resources mediating cytosolic Ca2+concentration ([Ca2+]i) in PASMCs. In general, in addition to voltage-gated Ca2+ channel (VDCC), extracellular Ca2+influx is regulated by a group of voltage-independent nonselective cation channels, such as store-operated Ca2+channel (SOCC) and receptor-operated Ca2+channels (ROCC). To date, there is a growing body of evidence supporting the pivotal role of multiple nonselective cation channels in acute [10,11] and prolonged hypoxia responses [12-15].Transient receptor potential (TRP) proteins, encoding a large repertoire of nonselective cation channels, are responsible for many voltage-independent Ca2+pathways in vascular smooth muscle cells (VSMCs)[16]. Fundamental insights into these channels arose from our previous works on the identification of various TRP subfamilies expression including classical/canonical (TRPC), melastatin-related (TRPM), vanilloid-related (TRPV) in PASMCs [12,17] and we elucidated TRPC1, TRPC6, TPRM8and TRPV4are the most abundant subtypes in each subfamily, respectively.Next, we further demonstrated chronic hypoxia (CH) upregulates the expression of TRPC1and TRPC6, as well as the associated SOCE and ROCE in rat distal pulmonary arteries (PAs). The upregulation was shown to be sufficient for sustaining elevated basal [Ca2+]i in conjunction with paralleled enhanced basal vascular tone using pharmacological inhibitors [12]. A subsequent study demonstrated the full expression of hypoxia inducible factor accounted for hypoxia-indeced upregulation [18]. Of note, the abnormality in TRPC expression has been identified beyond CHPH covering idiopathic pulmonary arterial hypertension (PAH) and monocrotaline (MCT)-induced PAH. The involvement of excessively expressed TRPCs refers to augmented SOCE [19,20] and enhanced vasoconstriction to endothelin-1(ET-1)[13]. Additionally, inhibition of TRPC1[21] and TRPC6[20] is capable of attenuating PASMCs proliferation. Sildenafil and Sodium Tanshinone IIA Sulfonate were functional in treating experimental PH via blockage of TRPCs upregulation [22,23]. Collectively, these studies implicate TRPC1and TRPC6are critically involved in CHPH, but leave open the question as to the altered expression and function of TRPC channels is essential for, just sufficient or even consequential in CHPH.On the other hand, TRPV4channels are highly expressed in endothelial and vascular smooth muscle cells of systemic and pulmonary vasculatures [24-29]. Accumulating evidence suggests that TRPV4plays important roles in the regulation of vascular functions, including vasorelaxiation[29-31][25,26,32], endothelial barrier integrity[33-37], regulations of SOCE[22,38,39] and ROCE[40]. TRPV4is a mechanosensitive channel in PASMCs. Its upregulation by CH is associated with enhanced basal and myogenic tone and genetic deletion of trpv4suppresses the development of CHPH. Previous studies show that serotonin (5-HT) activates an arachidonic acid-dependent non-selective cation current, which has pharmacological properties similar to TRPV4in PASMCs [41,42].Accordingly, we hypothesize that TRPV4of PASMCs may contribute to the agonist-induced vasoconstriction and participates in the regulation of pulmonary vascular reactivity in pulmonary hypertension.Materials and Methods:1. The present study is engaged to test whether TRPC1and TRPC6are essential for the development of CHPH as well as whether TRPV4contributes to agonist-induced vasoconstriction with the use of TRPC1(Trpc1-/-), TRPC6(Trpc6-/-), TRPC1/TRPC6(Trpc1-/-Trpc6-/-) and TRPV4(Trpv4-/-) null mice. Different genotypes of mice and age-matched wild-type (WT) mice were treated with10%O2for3-4weeks to generate CHPH.2. The expression of TRPC mRNA and protein normoxic and CH PAs were detected by Real-time PCR and Western Blot. 3. Direct pulmonary artery catheterizations through an open chest approach were performed to determine right ventricle systolic pressure (RVSP), mean pulmonary arterial pressure (MPAP) and cardiac output (CO).Pulmonary vascular resistance (PVR) was calculated by the equation of (MPAP-Pleft atrim)/CO, assuming the the end systolic pressure of left atrim is zero. Right heart hypertrophy was quantified by the mass ratio of right ventricle over left ventricle plus septum (RV/LV+S).4. Vasomotor tone and vasoconstriction to5-HT, ET-1and PE were measured with wire-myograph.5. Agarose inflated, paraffin embedded lungs were sectioned and stained with smooth muscle a-actin to assess the muscularization of pulmonary vasculature.6.[Ca2+]i of transient cultured PASMCs was monitored using the membrane-permeable Ca2+-sensitive fluorescent dye fluo3-AM and the fluorescent signal at>515nm was detected and data were collected online on a pClamp software package.Results:1. TRPC expression in Trpc1-/-, Trpc6-/-mice and WT mice.Trpc1-/-, Trpc6-/-mice and WT PAs share identical TRPC1-7expression profiles with the mRNA level of TRPC1was the highest followed by TRPC6and then TRPC3, while TRPC4,5and7expressions were minimal, except the specifically devoid gene in knockout mice. Moreover, TRPC1and TRPC6protein levels were significantly increased in PAs of WT mice exposed to3-weeks of10%O2. Comparable increases in TRPC1and TRPC6protein were also observed in PAs of CH Trpc6-/-and Trpc1-/-mice, respectively2. Genetic deletion of TRPC1or TPRC6suppresses CHPH.At baseline, different types of mice showed identical pattern of hemodynamic characters. Elevation of RVSP, MPAP, and RV/(LV+S) were evident in WT mice after1-week hypoxia exposure, and progressed to higher levels in prolonged treatment. Compared to WT mice, the increase in RVSP, MPAP, and RVMI were significantly less in Trpcl-/-mice after1-week and3-week hypoxia. In contrast, CHPH were dramatically suppressed in Trpc6-/-mice after1week of hypoxia, while progressed in subsequent weeks with marginal reduction in MPAP and RV/(LV+S) in3weeks. Interestingly, Trpcr-/-Trpc6-/-mice showed lower RVSP, MPAP, and RV/(LV+S) ratio at baseline, but not altered mean arterial pressure. Remarkably, when these mice were subjected to hypoxia for3-4weeks, they developed much less severe PH.3. TRPC1and TRPC6contribute to CH-induced pulmonary vascular remodeling.Similar profiles of muscularization were revealed among WT, Trpcl-/-and Trpc-/-mice under normoxic condition. Pulmonary vascular remodeling, characterized by increased portion of lung muscularized parenchymal vessels with concomitant decrease in non-muscularized vessels, was was observed in CH WT mice. Notably, pulmonary vascular morphometry of hypoxic Trpcl-/-and Trpc6-/-mice displayed a significantly less elevation of partially muscularized vessels and the attenuated decrease in non-muscularized vessels, while without affecting completely muscularized vessels as compared to hypoxic WT mice. Moreover, hypoxia-indecued PAs muscularization was virtually absent in hypoxic Trpc1-/-Trpc6-/-mice. On the other hand, the vessel density was significantly reduced in CH WT mice, and similar reduction was observed in CH Trpcl-/-, Trpc6-/-and Trpc1-/-Trpc6-/-mice.4. TRPC1and TRPC6contribute differentially to pulmonary vascular tone.In present study, we devised a new strategy to estimate vascular tone using wire-myograph. Active tension at each point, which was determined by subtracting the passive wall-tension measured after the vessel was completely relaxed, was abolished by the removal of extracellular Ca2+was significantly enhanced in PAs of CH WT mice. Ablation of TRPC1caused little effect on vasomotor tone in normoxia, but hypoxia-enhanced vascular tone was obliterated in Trpc-/-PAs. In contrast, the tone of Trpc6-/-PAs was significantly lower throughout the whole range of vessel-width tested in normoxia, while the enhancement triggered by CH remained.5. TRPC1and TRPC6contribute to pulmonary vasoreactivity in CH PAs.Comparable5-HT elicited concentration-dependent contractions were observed in WT, Trpcl-/-and Trpc6-/-PAs. The maximal response (Emax) elicited by5-HT in CH WT PAs was augmented and this augmentation was partially blunted in Trpc1-/-PAs, but totally abolished in Trpc6-/-PAs.6. Evaluation of TRPV4antagonists for the study of pulmonary vascular reactivity.Preincubation of PA rings with5μM TRPV4-selective antagonist HC-067047caused significant reduction in the maximal response and the EC50of PE. While identical dose-response curve was obtained in Trpv4-/-PAs and application of5μM HC-067047resulted in similar inhibition. Moreover, pretreatment of Trpv4-/-PAs with another TRPV4antagonist RN-1734(30μM) also significant inhibited the PE-induced contraction. We further determine the optimal concentration of HC-047067and RN-1734, and tested the vasorelaxant effect of the two antagonists on K+induced contraction. Only0.5μM HC-047067had no significant effect on K+-induced PA contraction.7. Contributions of TRPV4in pulmonary vasoreactivity to5-HT.Inhibition of TRPV4with0.5μM HC-067047or genetic deletion of trpv4had no noticeable effect on the Emax or EC50of PE and ET-1induced contraction in both EC+and EC-PAs. However, HC-067047caused a significant reduction in EC50of5-HT without affecting Emax, which was further confirmed in EC-Trpv4-/-PAs. 8. Contributions of TRPV4to5-HT-induced Ca2+response in PASMCs.5-HT elicited Ca2+response with a transient peak followed by a sustained Ca2+increase. The response was unaltered in Trpv4-/-PASMCs preincubated with0.5μM HC-067047. In contrast, HC-067047caused significant inhibition of both the peak and the sustained Ca2+response activated by5-HT in PASMCs of WT mice.1μM nifedipine alone or combined with HC-067047led to similar prohibition of5-HT response. Moreover, removal of extracellular Ca2+resulted in almost completely inhibition of5-HT response. Xestospongin C in line only caused a small reduction in the peak response.9. Chronic hypoxia-induced pulmonary hypertension in WT and Trpv4-/-mice.CHPH was developed after3-week hypoxia treatment, reflected as the increase in RVSP, MPAP, PVR and RV hypertrophy. Elevated RVSP, MPAP, PVR as well as RV/(LV+S) ratio induced by CH was abundantly alleviated in Trpv4-/-mice.10. Contribution of TRPV4in altered pulmonary vasoreactivity in CH mice.CH enhanced5-HT-induced Emax in EC+and more pronounced in EC-PAs of WT mice. HC-067047caused a small but significant suppression of Emax in EC+PAs, and the inhibition observed in EC-PAs was more profound.5-HT-induced contraction was also potentiated in CH Trpv4-/-PAs, but the enhancement was significantly less comparing to those of WT, when it is evaluated as%increase in maximal response.Conclusion:1. TRPC1is required for the full development of CHPH and TRPC6contributes to the early stage of CHPH.2. TRPC1and TRPC6are important contributing factors for CH-induced neomuscularization of small PAs, but have little influence on rarefraction of small PA in CHPH. 3. TRPC6is important for maintaining the basal tone under normoxic condition, and TRPC1is critically involved in the CH-induced enhancement of pulmonary vascular tone.4. TRPC6is essential for, whereas TRPC1contributes to, the enhanced5-HT response in CHPH.5. TRPV4in PASMCs participates in the5-HT-activated Ca2+mobilization and pharmaco-mechanical coupling for PA contraction in normoxia and moreover, plays a pivotal role in the enhanced pulmonary vasoreactivity to5-HT in CHPH.Collectively, TRPCl and TRPC6are crucial for the regulation of vasomotor tone, vasoreactivity and neo-muscularization of pulmonary vasculatures. TRPV4also contributes to serotonin-induced vasoconstriction in normoxia and CH. These vascular functions contribute at different stages of CHPH; and their participations are essential for the full manifestation of CHPH. In view of the multifaceted contributions to PH, manipulation of these TRP functions offers a promising therapeutic strategy for hypoxia-related PH.