Imaging Ca2+ Signals in Small Pulmonary Veins at Physiological Intraluminal Pressures

Abstract

Pulmonary veins (PVs) carry oxygen-rich blood from the lungs back to the left heart, thus serving an important function in the delivery of oxygen-rich blood to vital organs. However, most studies of pulmonary vasculature have focused on pulmonary arteries and capillaries under normal and disease conditions. Ca²⁺ signals are critical regulators of vascular function. Despite the critical physiological roles of PVs, Ca²⁺ signals in small intrapulmonary PVs have not been recorded under physiological conditions. Here, we describe a technique to record Ca²⁺ signal activity in mouse PVs isolated, cannulated and pressurized at 5 mmHg. By incorporating a Ca²⁺ indicator, we can study Ca²⁺ signals in the myocyte layer of small PVs using high-speed, spinning disk confocal imaging under physiological conditions. Our representative data indicates that the Ca²⁺ signals in small PV myocytes are mediated by openings of ryanodine receptor ion channels. This method will be of considerable interest to researchers in the field of pulmonary vascular physiology and disorders.

Figure 1:. Preparation and Ca²⁺ imaging of PVs.

Sequential images showing the identification and isolation of small PVs, incubation of fluo4-AM, cannulation and pressurization (5 mm Hg), spinning disk high-speed confocal imaging, and image analysis. The dashed rectangle in the image on the left represents the PV used for the study. Abbreviations: PA = pulmonary artery; RA = right atrium; PV = pulmonary vein; LA = left atrium.

Figure 2:. Analysis of Ca²⁺ imaging.

(A) Images of a field of view from a pressurized (5 mm Hg), fluo-4 loaded, small PV in the absence (left) or presence of ryanodine (5 μM; RyR inhibitor, right). The green rectangle denotes the area that was selected for autodetection Ca²⁺ signals. The + signs represent autodetected events within the frame. Red signs indicate signals with area of less than 3000 μm², whereas blue signs indicate signals that occupied an area of more than 3000 μm². Each + sign indicates one Ca²⁺ event or signal. (B) F/F0 traces of detected events from the selected area indicated in A, showing the activity of Ca²⁺ signals in the myocyte layer from pressurized small PVs under basal conditions (left) and in the presence of ryanodine (right). (C) Quantification of events per minute per μm² of Ca²⁺ signals in small PVs before and after the addition of ryanodine (1 PV per mouse; n=6 mice; **p < 0.01 vs Basal; paired t-test).