Mitochondria Amplify TRPV4‐dependent Calcium Dynamics within the Pulmonary Microvascular Endothelium

Abstract

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease common in critically ill patients marked by diffuse alveolar damage and increased pulmonary endothelial barrier permeability. A large body of evidence supports the role of mitochondrial dysfunction in ARDS, which has been shown to mediate endothelial permeability. For example, in cultured rat pulmonary microvascular endothelial cells (PMVECs), the mitochondrial complex I inhibitor rotenone increases endothelial permeability and in isolated perfused lung studies, rotenone increases the pulmonary vascular filtration coefficient. In both cell culture and isolated perfused lungs, the amphipathic quinone coenzyme Q1 (CoQ1) rescues rotenone‐dependent increases in endothelial permeability by bypassing complex I to restore mitochondrial bioenergetics.The transient receptor potential vanilloid protein 4 (TRPV4), a mechanically gated calcium channel, has also been implicated in mediating endothelial permeability by increasing intracellular calcium in response to high pressure ventilation. The phorbol ester 4α‐phorbol‐12,13‐didecanoate (4αPDD) provides a pharmacological means of TRPV4 activation and stimulates endothelial permeability responses in both cell culture and animal models of lung injury. Given that mitochondria actively participate in calcium buffering, we hypothesized that TRPV4‐dependent calcium signals may be compartmentalized by mitochondria. To test this hypothesis, we used confocal microscopy to measure calcium signals in PMVECs in response to TRPV4 activation via 4αPDD (5 μM) after treatment with vehicle, rotenone (0.5 μM), or rotenone in combination with CoQ1 (10 μM), and developed a sensitive, automated technique for intracellular calcium signal transient analysis.Using our novel analysis approach, we found that rotenone significantly decreased the number of calcium signal transients stimulated by 4αPDD over a 15 minute interval (mean ± SEM: 22 ± 4 rotenone vs 96 ± 14 vehicle, p <0.01), and that CoQ1 in combination with rotenone reversed this effect (60 ± 15, p = n.s. vs vehicle). Rotenone also dampened the amplitude of calcium responses to TRPV4, an effect that was partially restored by CoQ1. These findings support the hypothesis that mitochondria regulate TRPV4‐mediated calcium dynamics by amplifying calcium signals, and that mitochondrial dysfunction impairs this phenomenon. This suggests that mitochondrial regulation of endothelial calcium signaling is essential to the maintenance of barrier integrity and provides a possible therapeutic target in ARDS.Support or Funding InformationNIH K25HL136869 and NIH P01 HL066299‐17