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. 2024 Apr 29:15:1386296.
doi: 10.3389/fphys.2024.1386296. eCollection 2024.

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

Naznin Sultana Remex  1 , Chowdhury S Abdullah  2 , Richa Aishwarya  2 , Gopi K Kolluru  2 , James Traylor  2 , Mohammad Alfrad Nobel Bhuiyan  3 , Christopher G Kevil  1   2 , A Wayne Orr  1   2 , Oren Rom  1   2 , Christopher B Pattillo  1 , Md Shenuarin Bhuiyan  1   2
Affiliations

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

Naznin Sultana Remex et al. Front Physiol. .

Abstract

Sigmar1 is a ubiquitously expressed, multifunctional protein known for its cardioprotective roles in cardiovascular diseases. While accumulating evidence indicate a critical role of Sigmar1 in cardiac biology, its physiological function in the vasculature remains unknown. In this study, we characterized the expression of Sigmar1 in the vascular wall and assessed its physiological function in the vascular system using global Sigmar1 knockout (Sigmar1-/-) mice. We determined the expression of Sigmar1 in the vascular tissue using immunostaining and biochemical experiments in both human and mouse blood vessels. Deletion of Sigmar1 globally in mice (Sigmar1-/-) led to blood vessel wall reorganizations characterized by nuclei disarray of vascular smooth muscle cells, altered organizations of elastic lamina, and higher collagen fibers deposition in and around the arteries compared to wildtype littermate controls (Wt). Vascular function was assessed in mice using non-invasive time-transit method of aortic stiffness measurement and flow-mediated dilation (FMD) of the left femoral artery. Sigmar1-/- mice showed a notable increase in arterial stiffness in the abdominal aorta and failed to increase the vessel diameter in response to reactive-hyperemia compared to Wt. This was consistent with reduced plasma and tissue nitric-oxide bioavailability (NOx) and decreased phosphorylation of endothelial nitric oxide synthase (eNOS) in the aorta of Sigmar1-/- mice. Ultrastructural analysis by transmission electron microscopy (TEM) of aorta sections showed accumulation of elongated shaped mitochondria in both vascular smooth muscle and endothelial cells of Sigmar1-/- mice. In accordance, decreased mitochondrial respirometry parameters were found in ex-vivo aortic rings from Sigmar1 deficient mice compared to Wt controls. These data indicate a potential role of Sigmar1 in maintaining vascular homeostasis.

Keywords: SIGMAR1; flow mediated dilatation (FMD); mitochondria; physiological function; vascular function.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

FIGURE 1
FIGURE 1
Sigmar1 is expressed in the vascular walls of mouse and human blood vessels. Paraffin-fixed mouse aortic roots and human left anterior descending coronary artery (LAD) tissue sections were used for immunohistochemical and immunofluorescence staining to determine the protein expression level of Sigmar1 in the vasculature. (A) immunohistochemical staining of human LAD and mouse aortic root tissue sections from wildtype mice (Wt) using anti-Sigmar1 antibody showing Sigmar1 protein expression in all three vascular layers-intima, media, and adventitia (Shown in brown precipitates). The aortic root tissue section from the Sigmar1 global knockout mouse (Sigmar1−/−) was used as a negative control for showing the specificity of anti-Sigmar1 antibody. (B) Immunofluorescent staining of human LAD and mouse aortic roots showing Sigmar1 protein expression in the blood vessel layers. Anti-Sigmar1 (green) co-immunostained with endothelial marker CD31 (red) or smooth muscle cell marker αSMA (red). Negative staining for Sigmar1 in the Sigmar1−/− aortic root was used as a negative control. Images were taken from two biological replicates, and at least 10 to 12 microscopic fields were analyzed. N = 2 mice per group (1 male and 1 female per group). Scale bar 100 μm.
FIGURE 2
FIGURE 2
Sigmar1 modulates extracellular matrix remodeling and vascular reorganization in the blood vessel wall. Histological staining of paraffin-fixed mouse aortic root tissue sections visually represents blood vessel walls from wildtype (Wt) and Sigmar1−/− mice. (A) Hematoxylin and Eosin (H&E) staining of aortic root showing reorganization of smooth muscle cell layer determined by nuclei disarray and thick extracellular matrix (ECM) in the adventitial layer in the absence of Sigmar1 compared to Wt. (B) Masson’s trichrome staining of mouse aortic roots in the Sigmar1−/− mice shows a remarkably higher amount of collagen deposition in the ECM and medial layer disarray. (C) Russell-Movat pentachrome staining of formalin-fixed aortic root tissue sections shows abrupt organization of elastic lamina in the medial layer and higher fibrosis in the Sigmar1−/− mice blood vessel wall. (D) Toluidine blue staining of aortic root microsections showing altered blood vessel reorganizations and thick ECM layer in the Sigmar1−/− mice aortic root, indicating Sigmar1’s potential role in vascular tissue reorganization and ECM remodeling. Aortic root tissues of both genotypes were collected from male and female littermate mice between the ages of 3–4 months-old. N = 3 mice per group (Wt; 1 male and 2 female mice, Sigmar1−/−; 2 males and 1 female mice). At least 10 to 12 microscopic fields per sample were analyzed in an investigator-blinded fashion. Scale bar 50 μm.
FIGURE 3
FIGURE 3
Sigmar1 deficiency causes increased pulse wave velocity (PWV) and decreased flow-mediated dilation (FMD) in mice. (A) Vascular stiffness was measured as PWV in the aorta of male Wt and Sigmar1−/− mice at 3 months of age using echocardiogram. The transit time was identified from the M mode of pulse wave Doppler ultrasound velocity measurements, and the distance was measured from the B mode images from echocardiography data. The time-transit quantification for PWV identified significantly higher vascular stiffness in mice aorta in Sigmar1 null mice compared to Wt. N = 7 (Wt) and 8 (Sigmar1−/−). p values were determined by unpaired t-test. (B) Vascular function was measured by non-invasive FMD of left femoral artery in Wt and Sigmar1−/− mice at 4 months of age using both male and female mice. The left femoral artery underwent transient ischemia for a minute, and then the trigger was removed. Arterial diameter was the highest at 90s post-ischemic timepoint, and FMD response gradually went back down close to the baseline. However, the Sigmar1−/− mice showed significantly decreased FMD response indicating notable vascular dysfunction in response to reactive hyperemia. N = 5 mice per group (Wt; 3 male and 2 female mice) and 6 (Sigmar1−/−; 3 male and 3 female mice). The recorded loops were analyzed by Vevo LAB analysis software. Data were compared between groups using two-way ANOVA followed by Sidak’s multiple comparisons. A value of p < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 4
FIGURE 4
Sigmar1 deletion leads to decreased total nitric oxide metabolite (NOx) levels in the plasma and decreased phos-eNOSser1177 protein levels in mice. Plasma, gastrocnemius muscle, aorta, and aortic roots were collected from 4 to 5 months-old Wt and Sigmar1−/− mice. (A) Total NOx level was measured using a very sensitive ozone-based chemiluminescent assay in blood plasma and highly vascularized gastrocnemius muscle. Sigmar1 null mice showed a significant decrease in NOx level both in plasma and in the skeletal muscle compared to the Wt mice. N = 4 mice per group (2 male and 2 female mice). (B) Sigmar1 deficient mice also showed significant decrease in phos-eNOSser1177 protein level in the aortic tissue lysate compared to that of Wt control mice. phos-eNOSser1177 expression was normalized by total eNOS protein level. Sigmar1 Western blot confirmed complete deletion of the protein from Sigmar1−/− mice aorta, and β-Actin was used as a housekeeping control. N = 6 mice per group; 3 male and 3 female mice. (C) Immunohistochemical staining of phos-eNOSser1177 in aortic root was remarkably reduced in Sigmar1−/− mice aortic roots. N = 3 mice per group; 2 male and 1 female mice. Scale bar 25 μm. p values were determined by non-parametric Mann-Whitney U and Kruskal-Wallis test. A value of p < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.
FIGURE 5
FIGURE 5
The ultrastructural analysis of mouse aortic tissue shows accumulation of elongated mitochondria in the vascular endothelial and smooth muscle cells in Sigmar1 null mice. Aorta tissues were collected from 4 months-old Wt and Sigmar1−/− mice and analyzed for ultrastructural alterations using transmission electron microscopy (TEM). (A) TEM images of vascular endothelial cells in the aorta showed longer mitochondrial shapes and sizes in the absence of Sigmar1, whereas Wt endothelial cells showed normal, regular mitochondrial structure. Scale bar: 2 μm. (B) Representative TEM image of vascular smooth muscle cells from Sigmar1−/− mice aorta showed accumulation of elongated mitochondria around the nucleus. Wt mice showed circular mitochondria with dispersed distribution in the aortic smooth muscle cell. N = 3 mice per group; 2 male and 1 female mice. Scale bar: 1 μm. EC: Endothelial cell, SM: Smooth muscle cell, EL: Elastic lemina, M: Mitochondria, N: Nucleus.
FIGURE 6
FIGURE 6
Sigmar1 deficiency results in reduced mitochondrial respiration and function in mouse aorta measured by high-resolution respirometry. Aorta tissues, including the aortic arch, thoracic, and abdominal aorta (until the bifurcation of iliac arteries) were isolated from 3 months-old male and female Wt and Sigmar1−/− mice. The aortas were immediately cut into small rings of 2–3 mm and placed in the oxygraph chamber to measure mitochondrial oxygen flux in ex-vivo aortic rings. Different inhibitors, substrates, and uncouplers were used to measure oxygen flux of mitochondrial respiratory chain complexes in high-resolution respirometry. (A) The mitochondrial oxygen flux of Sigmar1−/− mice aortic rings was substantially decreased compared to Wt mice aortas. (B–F) oxygen flux at the basal, Complex I coupled, complex I + II coupled, and Complex I + II uncoupled states were significantly reduced in the aortic rings from Sigmar1−/− mice compared to Wt mice. The residual oxygen consumption rate was comparable between the two groups. All oxygen flux values were normalized by the wet weights of the aorta tissue. N = 5 mice per group; 3 male and 2 female mice. p values were determined by the Mann-Whitney U test. A value of p < 0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
FIGURE 7
FIGURE 7
Schematic diagram showing how Sigmar1 deficiency leads to vascular dysfunction. Absence of Sigmar1 contributes to decreased flow mediated dilation, decreased eNOS phosphorylation and nitric oxide bioavailability, increased vascular fibrosis and aortic stiffness in mice. This also leads to altered mitochondrial shape and structure causing mitochondrial dysfunction in global Sigmar1 mice.
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