TRPC4 inactivation confers a survival benefit in severe pulmonary arterial hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure with lumen-occluding neointimal and plexiform lesions. Activation of store-operated calcium entry channels promotes contraction and proliferation of lung vascular cells. TRPC4 is a ubiquitously expressed store-operated calcium entry channel, but its role in PAH is unknown. We tested the hypothesis that TRPC4 promotes pulmonary arterial constriction and occlusive remodeling, leading to right ventricular failure in severe PAH. Severe PAH was induced in Sprague-Dawley rats and in wild-type and TRPC4-knockout Fischer 344 rats by a single subcutaneous injection of SU5416 [SU (semaxanib)], followed by hypoxia exposure (Hx; 10% O2) for 3 weeks and then a return to normoxia (Nx; 21% O2) for 3 to 10 additional weeks (SU/Hx/Nx). Although rats of both backgrounds exhibited indistinguishable pulmonary hypertensive responses to SU/Hx/Nx, Fischer 344 rats died within 6 to 8 weeks. Normoxic and hypertensive TRPC4-knockout rats recorded hemodynamic parameters similar to those of their wild-type littermates. However, TRPC4 inactivation conferred a striking survival benefit, due in part to preservation of cardiac output. Histological grading of vascular lesions revealed a reduction in the density of severely occluded small pulmonary arteries and in the number of plexiform lesions in TRPC4-knockout rats. TRPC4 inactivation therefore provides a survival benefit in severe PAH, associated with a decrease in the magnitude of occlusive remodeling.

Figure 1

Rats of SD and F344 backgrounds develop severe pulmonary arterial hypertension. Eight weeks of SU/Hx/Nx exposure induced similar changes in RVSP (A), CI (B), LVSP (C), and the right ventricular hypertrophy index (D) in both strains of rats. For comparison, normotensive values from previous studies in SD rats (historic controls) are indicated by dotted lines. Data are expressed as means ± SEM. n = 4 per group.

Figure 2

TRPC4-KO rats exhibit a defect in ACh-induced calcium influx. A: Genotyping of TRPC4 WT, heterozygotic (Het), and KO F344 rats. B: Pulmonary artery segments were isolated and loaded with the Fluo-4 AM cytosolic calcium indicator. Endothelium was then imaged en face. Evaluation of endothelial cell shape revealed a distinct anatomical pattern among WT and TRPC4-KO vessels, with elongated endothelium in TRPC4-KO rats. C: Basal cytosolic Ca²⁺–dependent fluorescence recorded in open artery preparations of WT and TRPC4-KO rats revealed no difference in basal cytosolic calcium dynamic oscillations. Top panels: Representative tracings of dynamic cytosolic Ca²⁺ events occurring in individual cells over 2 minutes. Each line represents the ROI (5 μm diameter) from a different cell. Bottom panels: Total sites and events per field per minute (n = 4 per group). D: Average whole-field cytosolic Ca²⁺ signal stimulated by 2 μmol/L ACh in pulmonary artery endothelium of WT and TRPC4-KO rats over time, as well as peak Ca²⁺ response and steady-state signal after 120 seconds. Data are expressed as means ± SEM. n = 4. ^∗P < 0.05 versus WT.

Figure 3

TRPC4-KO rats exhibit a survival benefit that cannot be explained by improved hemodynamic response to SU/Hx/Nx. A: Survival over 12 weeks of SU/Hx/Nx exposure. SD rats (dotted line) serve as historic controls. TRPC4-KO rats (n = 12) exhibited a clear survival benefit versus WT rats (n = 6) after induction of severe pulmonary arterial hypertension. B: M-mode echocardiography of the left ventricle revealed a smaller LVEDV in WT rats after SU/Hx/Nx exposure. C–E: Development of pulmonary arterial hypertension was associated with decrements in CO (C), TAPSE (D), and PA-AT (E) in WT (n = 8) and KO (n = 12) rats. Echocardiography was performed at baseline and after 8 weeks of SU/Hx/Nx exposure. Data are expressed as means ± SEM. ^∗P < 0.05 versus control. ^†P < 0.0006 versus WT, Mantel–Cox log-rank test.

Figure 4

TRPC4 inactivation did not significantly impair either the maximal smooth muscle cell constriction or endothelium-dependent dilation. Pulmonary arterial ring responses to treatment with KCl (A), PE (B), time to peak contraction induced by PE (C), and ACh-induced dilation of the KCl contraction (D) in WT and TRPC4-KO rats under control and SU/Hx/Nx conditions. Data are expressed as means ± SEM. n = 4 per group. ^∗P < 0.05 versus control; ^†P < 0.05 versus WT SU/Hx/Nx group.

Figure 5

TRPC4 inactivation reduced the magnitude of the arteriopathy after SU/Hx/Nx exposure. A and B: Luminal occlusion density (OD) of the small pulmonary arteries (<50 μm) was graded in pulmonary hypertensive lungs of WT and TRPC4-KO rats. The frequency of severely (>50%) occluded vessels was higher in WT than in KO rats. C: Heath–Edwards classification of pulmonary hypertension-induced vascular lesions. D: Examples of severe fibrinoid necrosis and perivascular inflammation (grade VI, necrotizing arteritis) of the pulmonary arteries in WT rats. Grading of lesions was performed in 50 arbitrarily chosen, but consecutive, small pulmonary arteries per sample, in three samples per experimental group. Significance was analyzed by likelihood ratio and Pearson's P.^∗∗∗P < 0.001. Scale bar = 50 μm.

Figure 6

Both WT and TRPC4-KO rats exhibited cardiac fibrosis after SU/Hx/Nx exposure. A: Masson's trichrome staining of the RV and LV in WT and TRPC4-KO rats after 6 weeks of SU/Hx/Nx exposure; collagen fibers stain blue. Perivascular fibrosis (arrows) and interstitial fibrosis (asterisks) tended to be higher in both ventricles of the WT rats. Boxed regions correspond to the higher magnification images to the right. B: Quantification of the fibrotic zones in both ventricles revealed no significant difference between WT and TRPC4-KO hearts. Data are expressed as means ± SEM. n = 4 per group. Scale bar = 50 μm.