Abstract Rationale Extended synaptotagmins (ESyts) are endoplasmic reticulum-plasma membrane tethering proteins that play key roles in lipid transfer and plasma membrane (PM) repair. Although their functions are described in cellular membrane homeostasis, their role in lung physiology and response to chronic cigarette smoke exposure remains poorly understood. Here, ESyt family proteins deficiency was investigated to determine the role of ESyt proteins in lung plasma membrane integrity and extracellular matrix (ECM) homeostasis using a cigarette smoke exposure model. Methods Male and female C57BL/6J and ESyt1-3 triple knockout (KO) mice were exposed to cigarette smoke five times per week for nine months. Pulmonary function was assessed through forced oscillation and expiratory measurements, along with regular monitoring of body weight. Masson’s trichrome staining was used to evaluate collagen deposition in the lung tissue sections, and airspace enlargement was quantified by mean linear intercept analysis using ImageJ (Fiji) software. RNA sequencing was performed on A549 cells stably expressing scrambled or ESyt2 shRNA to identify ESyt2-dependent molecular pathways. Results ESyt1-3 triple KO mice exposed to either room air or cigarette smoke exhibited reduced weight gain compared to their wild-type littermates. Pulmonary function assessments revealed decreased forced expiratory volume (FEV0.2), and compliance, indicating a restrictive lung phenotype in ESyt1-3 triple KO mice. Additionally, the FEV0.2/FVC ratio was reduced in cigarette smoke-exposed ESyt1-3 triple KO mice. Analysis of Masson’s trichrome staining revealed increased collagen deposition in the lungs of cigarette smoke-exposed ESyt1-3 triple KO mice compared to lungs of room air-exposed wild-type and triple KO mice. Mean linear intercept measurements showed enlarged airspaces in ESyt1-3 triple KO mice under both room-air and smoke exposure. RNA sequencing identified 804 differentially expressed genes in cells expressing ESyt2 shRNA. Gene ontology analysis of ESyt2 shRNA expressing cells demonstrated that ECM organization as the most significantly affected process. Conclusion Loss of ESyt1-3 function leads to reduced lung compliance, restrictive physiology, and enhanced collagen deposition and airspace enlargement, consistent with emphysematous remodeling. At the cellular level, ESyt2 depletion alters signaling pathways related to ECM organization. Together, these findings suggest that loss of ESyt proteins disrupts membrane integrity and ECM homeostasis, compromising lung structural integrity under smoke-induced injury. This abstract is funded by: This work was supported by the Seed Grant from the Research Foundation of SUNY and the Office of the Assistant Secretary of Defense for Health Affairs through the Congressionally Directed Medical Research Programs under Award No: PR220774.
Surapaneni et al. (Fri,) studied this question.