Sigmar1 ablation leads to lung pathological changes associated with pulmonary fibrosis, inflammation, and altered surfactant proteins levels

Abstract

Sigma1 receptor protein (Sigmar1) is a small, multifunctional molecular chaperone protein ubiquitously expressed in almost all body tissues. This protein has previously shown its cardioprotective roles in rodent models of cardiac hypertrophy, heart failure, and ischemia-reperfusion injury. Extensive literature also suggested its protective functions in several central nervous system disorders. Sigmar1’s molecular functions in the pulmonary system remained unknown. Therefore, we aimed to determine the expression of Sigmar1 in the lungs. We also examined whether Sigmar1 ablation results in histological, ultrastructural, and biochemical changes associated with lung pathology over aging in mice. In the current study, we first confirmed the presence of Sigmar1 protein in human and mouse lungs using immunohistochemistry and immunostaining. We used the Sigmar1 global knockout mouse (Sigmar1−/−) to determine the pathophysiological role of Sigmar1 in lungs over aging. The histological staining of lung sections showed altered alveolar structures, higher immune cells infiltration, and upregulation of inflammatory markers (such as pNFκB) in Sigmar1−/− mice compared to wildtype (Wt) littermate control mice (Wt). This indicates higher pulmonary inflammation resulting from Sigmar1 deficiency in mice, which was associated with increased pulmonary fibrosis. The protein levels of some fibrotic markers, fibronectin, and pSMAD2 Ser 245/250/255 and Ser 465/467, were also elevated in mice lungs in the absence of Sigmar1 compared to Wt. The ultrastructural analysis of lungs in Wt mice showed numerous multilamellar bodies of different sizes with densely packed lipid lamellae and mitochondria with a dark matrix and dense cristae. In contrast, the Sigmar1−/− mice lung tissues showed altered multilamellar body structures in alveolar epithelial type-II pneumocytes with partial loss of lipid lamellae structures in the lamellar bodies. This was further associated with higher protein levels of all four surfactant proteins, SFTP-A, SFTP-B, SFTP-C, and SFTP-D, in the Sigmar1−/− mice lungs. This is the first study showing Sigmar1’s expression pattern in human and mouse lungs and its association with lung pathophysiology. Our findings suggest that Sigmar1 deficiency leads to increased pulmonary inflammation, higher pulmonary fibrosis, alterations of the multilamellar body stuructures, and elevated levels of lung surfactant proteins.

Deletion of Sigmar1 leads to increased arterial stiffness and altered mitochondrial respiration resulting in vascular dysfunction

“…In both cell types, it is notable that the mitochondrial shape is altered in the absence of Sigmar1. The mitochondria become markedly more elongated and clustered in Sigmar1 −/− mice, which is consistent with some previous findings in cardiac and lung ultrastructure ( Abdullah et al, 2018 ; Remex et al, 2023 ). In contrast, the Wt mouse aorta showed the rounder-shaped and dispersed distribution of mitochondria in the endothelial and smooth muscle cells.…”

“…We also showed reorganization of nuclear disarray in the medial layer, which is mostly vascular SMCs along with abrupt arrangement of elastic lamina in the aortic roots of Sigmar1 −/− mice compared to Wt ( Figure 2 ). This was consistent with our previous findings indicating Sigmar1 −/− mice develop fibrotic remodeling in the heart, lung, and skeletal muscle ( Abdullah et al, 2018 ; Aishwarya et al, 2022b ; Remex et al, 2023 ). Vascular fibrosis and extracellular matrix remodeling have been associated with many vascular pathologies, such as hypertension, atherosclerosis, and peripheral artery disease (PAD) ( Lan et al, 2013 ; Harvey et al, 2016 ; Ding et al, 2020 ).…”

“…Previous studies provided evidence of extracellular matrix remodeling and collagen deposition leading to fibrosis in different organs of Sigmar1 −/− mice, including the heart tissue, skeletal muscle, and pulmonary system ( Abdullah et al, 2018 ; Aishwarya et al, 2022b ; Remex et al, 2023 ). To assess whether Sigmar1 plays a role in vascular remodeling, we have used different histological staining techniques using paraffin-embedded aortic root tissue section from Sigmar1 −/− mice and compared with that of Wt littermate controls.…”

“…The vascular reorganization was assessed by hematoxylin and Eosin (H&E) staining using H&E kit (H-3502, Vector Laboratories) following the manufacturer’s protocol. Vascular and perivascular collagen deposition was analyzed by Masson’s trichrome, Russell-movat pentachrome, and toluidine blue staining of aortic roots following a previously published protocol ( Yurdagul et al, 2014 ; Molnar et al, 2017 ; Remex et al, 2023 ). All stained tissue sections were imaged under ×10 objective lens using an Olympus BX40 brightfield microscope in an investigator-blinded manner.…”

“…Total proteins from the tissue were extracted from Wt and Sigmar1 −/− mice aorta and prepared for Western blot to examine protein expression levels in the tissue following the previous protocol ( Abdullah et al, 2018 ; Remex et al, 2023 ). The whole aorta (aortic arch, thoracic, and abdominal aorta) was harvested from the mice and immediately snap-frozen.…”