Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions.

Fenja Knoepp,Kerstin Ramser,Anders Andersson,Norbert Weissmann,Johan Borg,Joel Wahl

doi:10.3390/s18103238

Abstract

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K+-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia—or the subsequent change in the cellular redox-state—inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O2-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H2O2. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system (p-values from 0.00% to 16.30%) compared to the open system (p-value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

Highlights

Hypoxic pulmonary vasoconstriction (HPV) is a local response of the pulmonary vasculature that diverts blood from poorly to well-oxygenated lung areas
It is well established that precapillary pulmonary arterial smooth muscle cells (PASMCs) are the sensor and effector cells in HPV, since they constrict upon exposure to acute hypoxia—thereby narrowing the diameter of resistance pulmonary arteries [1,2,3]
The design of the microfluidic system drawn in a CAD program was based on (1) the number of of needed the microfluidic system drawn in a CAD program was based on (1) theand number of entrancesThe anddesign outlets (for inflow, outflow, patch-pipette, reference electrode entrances and outlets needed and (2) the shape of the dishes used for PASMC cultivation

Summary

Introduction

Hypoxic pulmonary vasoconstriction (HPV) is a local response of the pulmonary vasculature that diverts blood from poorly to well-oxygenated lung areas. Decreased HPV (e.g., during pneumonia, sepsis, anesthesia or liver failure) can lead to life-threatening hypoxemia, whereas prolonged and global alveolar hypoxia (e.g., at high altitudes or in patients suffering from chronic lung diseases) results in exaggerated HPV This chronic and global vasoconstriction can lead to an irreversible pathological remodeling of the pulmonary vasculature, resulting in pulmonary hypertension (PH) and —due to the constant work-overload—to right heart failure [1,2,3]. The pulmonary-specific isoform 2 of the mitochondrial complex IV subunit 4 (Cox4i2) has been identified as primary oxygen (O2 )-sensor in PASMCs. O2 -sensing via Cox4i2 initiates acute HPV by promoting hypoxia-induced ROS-release preferentially at complex III of the electron transport chain [2]. The t-test is often used in Raman measurements and it is able to handle small sample sizes [22]

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