Twik-2-/- mouse demonstrates pulmonary vascular heterogeneity in intracellular pathways for vasocontractility

Abstract

We have previously shown Twik-2^-/- mice develop pulmonary hypertension and vascular remodeling. We hypothesized that distal pulmonary arteries (D-PAs) of the Twik-2^-/- mice are hypercontractile under physiological venous conditions due to altered electrophysiologic properties between the conduit and resistance vessels in the pulmonary vascular bed. We measured resting membrane potential and intracellular calcium through Fura-2 in freshly digested pulmonary artery smooth muscles (PASMCs) from both the right main (RM-PA) and D-PA (distal) regions of pulmonary artery from WT and Twik-2^-/- mice. Whole segments of RM-PAs and D-PAs from 20 to 24-week-old wildtype (WT) and Twik-2^-/- mice were also pressurized between two glass micropipettes and bathed in buffer with either arterial or venous conditions. Abluminally-applied phenylephrine (PE) and U46619 were added to the buffer at log increments and vessel diameter was measured. All values were expressed as averages with ±SEM. Vasoconstrictor responses did not differ between WT and Twik-2^-/- RM-PAs under arterial conditions. Under venous conditions, Twik-2^-/- RM-PAs showed an increased sensitivity to PE with a lower EC50 (P = 0.02). Under venous conditions, Twik-2^-/- D-PAs showed an increase maximal vasoconstrictor response to both phenylephrine and U46619 compared to the WT mice (P < 0.05). Isolated PASMCs from Twik-2 ^-/- D-PA were depolarized and had higher intracellular calcium levels compared to PASMCs from RM-PA of both WT and Twik-2^-/- mice. These studies suggest that hypercontractile responses and electrophysiologic properties unique to the anatomic location of the D-PAs may contribute to pulmonary hypertensive vasculopathy.

Keywords: Potassium channels; pulmonary artery smooth muscle cells; pulmonary hypertension; vasocontractility.

Figure 1

Resting membrane potential of freshly dispersed PASMCs. PASMCs digested and dispersed from right main (RM‐PA; proximal) vessel location and intralobar (D‐PA; distal) vessel location from both wildtype and Twik‐2^−/− mice. (P* < 0.05 between distal Twik‐2^−/− and distal WT cells; P** < 0.01 between proximal and distal PASMCs.) n = 5 animals per genotype.

Figure 2

Baseline F340/380 ratio of cultured PASMCs from right main (proximal) vessel location and intralobar (distal) vessel location from both wildtype and TWIK‐2 ^−/− mice. (P** < 0.01 between all distal and proximal cells, P* < 0.05 between distal WT and distal Twik‐2^−/− PASMCs.) n = 5 animals per genotype, 12–15 cells per animal, per location. Overall anatomic variation between {Ca²⁺} approached statistical significance (P = 0.06).

Figure 3

RM‐PA from Twik‐2^−/− and WT mice on different backgrounds in arterial (systemic)buffer. (A) Measurement of vessel diameter with increasing pressures evaluating for myogenic tone, which was not present. (B) Dose titration response curve to phenylephrine n = 5–6 animals per group.

Figure 4

RM‐PA from Twik‐2^−/− and WT mice in PA(venous) conditions buffer. (A) Phenylephrine dose titration curve showing increased vasoreactivity in the TWIK‐2^−/−. (B) Combined phenylephrine dose titration curve of the RM‐PA in venous and arterial conditions. (Fig 3A and 4A) (C) EC 50 for RM‐PA in PA conditions (venous.)

Figure 5

Distal‐PA >from Twik‐2^−/− and WT mice in PA conditions (venous) buffer. (A) Phenylephrine dose titration curve showing altered vasoreactivity in the TWIK‐2 KO (*P < 0.05), particularly at the higher concentrations of phenylephrine, and overall effect (***P < 0.001). (B) EC50 for WT and Twik‐2^−/− D‐PA vessels in response to phenylephrine. n = 5–6 animals per group.

Figure 6

D‐PA from Twik‐2^−/− and WT mice in PA conditions(venous) buffer. A U46619 dose titration curve showing altered vasoreactivity in the Twik‐2^−/− D‐PA (P < 0.05), particularly at the higher concentrations of U46619. n = 5 animals per group.