Activation of K_Ca channels downstream of TRPV4 amplify pulmonary edema formation

Study objective: In patients with acute respiratory failure (ARDS), mechanical ventilation is the only life-saving treatment. However, mechanical ventilation also can cause ventilator-induced lung injury (VILI) due to the excessive biomechanical forces acting on the distal lung and especially the microvascular endothelium, which in turn further exacerbate endothelial barrier failure thus initiating a vicious cycle leading to irreversible lung damage. The endothelial mechanosensitive transient receptor potential vanilloid 4 (TRPV4) is directly activated by mechanical force and leads to Ca2+ influx, which can activate Ca2+-activated K+ (KCa) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) KCa. We hypothesized that KCa channels acting downstream of TRPV4 may in turn amplify Ca2+ influx by increasing the electrochemical Ca2+ gradient and thus, promote stretch-induced barrier failure and aggravate lung injury in a positive feedback loop. Methods: Male C57Bl/6J wild-type mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the non-selective KCa antagonists apamin, charybdotoxin, or the selective IK1 antagonist TRAM34. In a second model, male wild-type mice were challenged with intratracheal HCl instillation (pH 1.5) and ventilated with a low tidal volume for 2h with or without TRAM34. Changes in endothelial Ca2+ concentration ([Ca2+]i) were monitored by real-time imaging of Fura-2 AM in isolated-perfused lungs in response to airway pressure elevation or in human pulmonary microvascular endothelial cells (HPMECs) in response to TRPV4 activation with or without inhibition of KCa channels. Analogously, changes in intracellular potassium concentration ([K+]i) and membrane potential ( Vm) were imaged in vitro. Results: In our established experimental lung injury models, pharmacological inhibition of KCa channels, in particular IK1 attenuated characteristic hallmarks of lung injury in response to high tidal volume ventilation or HCl instillation. Consistent with these findings, inhibition of IK1 channels in the ex vivo perfused lung reduced the sustained [Ca2+]i response to airway pressure elevation over time. In HPMECs, TRPV4-mediated Ca2+ influx induced a pronounced K+ efflux, which in turn caused membrane hyperpolarization. These effects could be prevented by different KCa inhibitors, with IK1 inhibition by TRAM34 showing the most pronounced protective effect. Antagonizing KCa channels as downstream mediators of TRPV4 also reduced endothelial [Ca2+]i transients in response to TRPV4 activation in vitro, indicating that KCa channels, and specifically IK1 serve as amplifiers by increasing the electrochemical gradient for TRPV4-mediated Ca2+ influx. Conclusion: KCa channels, specifically IK1 amplify TRPV4-mediated Ca2+ influx in lung endothelial cells and as such, establish a detrimental feedback that promotes barrier failure and drives the progression of VILI in overventilated lungs. WMK is supported by the German Research Foundation (DFG) (SFB-TR84: subprojects A02 & C09, SFB-1449 subproject B01, SFB 1470 subproject A04, KU1218/9-1, KU1218/11-1, and KU1218/12-1), the Berlin Institute of Health (BIH), the German Federal Ministry of Education and Research (BMBF) in the framework of the projects PROVID (01KI20160A) and SYMPATH (01ZX1906A), and the German Centre for Cardiovascular Research (DZHK). LM is supported by the German Centre for Cardiovascular Research (DZHK) (81X3100216). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.