Resistance to store depletion-induced endothelial injury in rat lung after chronic heart failure

Abstract

Rationale: In chronic heart failure, the lung endothelial permeability response to angiotensin II or thapsigargin-induced store depletion is ablated, although the mechanisms are not understood.

Objectives: To determine whether the ablated permeability response to store depletion during heart failure was due to impaired expression of store operated Ca2+ channels in lung endothelium.

Methods: Heart failure was induced by aortocaval fistula in rats. Permeability was measured in isolated lungs using the filtration coefficient and a low Ca2+/Ca2+ add-back strategy to identify the component of the permeability response dependent on Ca2+ entry.

Main results: In fistulas, right ventricular mass and left ventricular end diastolic pressure were increased and left ventricular shortening fraction decreased compared with shams. Thapsigargin-induced store depletion increased lung endothelial permeability in shams, but not in fistulas. Permeability increased in both groups after the Ca2+ ionophore A23187 or 14,15-epoxyeicosatrienoic acid, independent of store depletion. A diacylglycerol analog had no impact on permeability. Increased distance between the endoplasmic reticulum and the plasmalemmal membrane was ruled out as a mechanism for the loss of the permeability response to store depletion. Endothelial expression of the endoplasmic reticulum Ca2+ ATPase was not altered in fistulas compared with shams, whereas the store-operated canonical transient receptor potential channels 1, 3, and 4 were downregulated in extraalveolar vessel endothelium.

Conclusions: We conclude that the adaptive mechanism limiting store depletion-induced endothelial lung injury in the aortocaval model of heart failure involves downregulation of store-operated Ca2+ channels.

Figure 1.

Echocardiographic assessment of fistula patency and left ventricular function in the chronic heart failure (CHF) model. (A, C) Angio-mode Doppler images from representative sham and fistula animals, respectively. High-flow velocity limited to the abdominal aorta (a) was observed in shams but could also be seen in the inferior vena cava (vc) in fistulas. (B, D) M-mode echos in representative sham and fistula animals, respectively. Left ventricular dimensions were increased in the fistula group compared with shams (see Table 1 for detail).

Figure 2.

Effect of store depletion via thapsigargin on endothelial permeability. Baseline (BL) and final K_f (F) were compared after treatment with thapsigargin. Thapsigargin increased endothelial permeability in shams (open bars, n = 7) but not in fistulas (hatched bars, n = 5). *p < 0.05 versus BL. K_f = filtration coefficient.

Figure 3.

Effect of the Ca²⁺ entry agonists A23187 and 14,15–epoxyeicosatrienoic acid (14,15-EET) on endothelial permeability. (A) K_f was measured at baseline (BL) and after treatment (F) in shams (open bars) and fistulas (hatched bars); n = 5 in each group. A23187 significantly increased endothelial permeability in both groups. The 14,15-EET increased endothelial permeability in shams (n = 4) but not significantly in fistulas (n = 5) due to an exaggerated response in one of the fistula experiments. *p < 0.05 versus BL. (B) To show dependence of the 14,15-EET permeability response on Ca²⁺ entry, in fistulas (n = 5) we measured K_f using a low Ca²⁺/Ca²⁺ add-back strategy. The 14,15-EET increased endothelial permeability at low Ca²⁺, with further injury after Ca²⁺ add-back. *p < 0.05 versus BL.

Figure 4.

Distances between the endoplasmic reticulum and the endothelial plasmalemmal membrane in intact lungs from shams (n = 4) and fistulas (n = 3). Data points and horizontal lines represent individual and average measurements, respectively, in pulmonary artery (left) and septal microvessel (right) endothelial cells (EC). Although the endoplasmic reticulum and the plasma membrane tended to be more distant from each other at the basolateral aspect compared with that in the apical compartment of pulmonary artery endothelium, this trend was not significant. We found no significant differences between shams and fistulas at either site in either cell population.

Figure 5.

Immunostaining for transient receptor potential proteins TRPC1, TRPC3, TRPC4, and TRPC6/7 in rat lungs. Expression of each TRPC isoform in endothelium was confirmed in small muscular or partially muscularized extraalveolar vessels (A) and conduit artery (B) from shams and fistulas. Expression of TRPC1, TRPC3, and TRPC4 in endothelial cells was downregulated in small pulmonary arterioles after CHF, whereas TRPC6/7 expression was unchanged. TRPC4 expression in the medial layer of large conduit pulmonary vessels was increased in fistulas. Original magnification = 40×. See text for more detail.

Figure 6.

Expression of sarcoplasmic endoplasmic reticulum Ca²⁺ ATPase (SERCA) isoforms in rat lung. Expression of SERCA2 (left) and SERCA3 (right) in endothelium was confirmed in small muscular or partially muscularized extraalveolar vessels and conduit artery from shams (top row) and fistulas (bottom row). There were no apparent differences in endothelial SERCA expression after CHF. Original magnification = 40×.

Figure 7.

Effect of receptor-operated Ca²⁺ entry on endothelial permeability. Baseline (BL) and final (F) K_f were compared after treatment with 1-oleoyl-2-acetyl-sn-glycerol (OAG). With physiologic extracellular Ca²⁺, OAG increased endothelial permeability at 300 but not at 100 μmol/L (n = 3 in each group). Using the low Ca²⁺/Ca²⁺ add-back strategy, 300 μmol/L OAG (n = 3) was found to have no impact on endothelial permeability. See text for more detail. *p < 0.05 versus BL.