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

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

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

Excerpt of the article published in Frontiers in Physiology, 15, 29 April 2024, ISSN: 1664-042X, DOI: 10.3389/fphys.2024.1386296


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.

1 Introduction

Sigma1 receptor protein (Sigmar1) is a small molecular chaperone protein that is composed of 223 amino acids with a molecular mass of 25.3 KDa (Kekuda et al., 1996Seth et al., 1997Seth et al., 1998Mei and Pasternak, 2001). This protein has a single putative transmembrane domain, and the Sigmar1 gene consists of four exons and three introns with exon 3 being the shortest and exon 4 being the longest (Prasad et al., 1998). Sigmar1 was first identified as a subtype of opioid receptors using specific radiolabeled ligand binding assays (Martin et al., 1976), and later, it was cloned from guinea pig, rat, and mouse tissues (Hanner et al., 1996Kekuda et al., 1996Seth et al., 1997Seth et al., 1998). Human and animal studies have identified ubiquitous expression of Sigmar1 in almost all the body tissues, including brain, heart, liver, lung, kidney, skeletal muscle, stomach, spleen, thymus, and other regions (Kekuda et al., 1996Mei and Pasternak, 2001). Even though Sigmar1 is ubiquitously expressed in almost all body tissues, exploring its pathophysiological role in different organ systems has so far been limited to the brain, heart, kidney, and few other organs.

Sigmar1 has been extensively studied in the central nervous system, and the association of SIGMAR1 gene mutation has been demonstrated in numerous neurodegenerative diseases, such as, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, silver-like syndrome, amyotrophic lateral sclerosis, and certain psychiatric disorders (Mishina et al., 2005Mishina et al., 2008Aishwarya et al., 2021). Several studies from our group and others have uncovered Sigmar1’s involvement in cardiac function. Sigmar1 global knockout (Sigmar1−/−) mice showed cardiac hypertrophy, contractile dysfunction, perivascular and interstitial fibrosis in the heart, and a decline in mitochondrial respiratory function (Abdullah et al., 2018). Activating Sigmar1 using its non-selective ligand showed a protective role in heart failure and pressure-overload-induced cardiac injury models of rats and mice (Bhuiyan and Fukunaga, 2009Bhuiyan et al., 2010Tagashira et al., 2010Bhuiyan and Fukunaga, 2011). The ultrastructural analysis of the hearts from Sigmar1−/− mice showed accumulation of elongated-shaped mitochondria consistent with alteration in mitochondrial respiratory parameters and bioenergetics, which likely contributes to the impaired cardiac contractile function in the absence of Sigmar1 (Abdullah et al., 2018).

The subcellular distribution of Sigmar1 is highly tissue and cell-type dependent. It was initially thought that Sigmar1 is specifically localized in the mitochondria-associated endoplasmic reticulum membrane (MAM). However, later studies revealed that Sigmar1 is also present in the plasma membrane, ER, nuclear envelope, and in the lysosome (Dussossoy et al., 1999Hayashi and Su, 2007Su et al., 2009). The presence of Sigmar1 in the mitochondria was first identified by Klouz et al., 2002 in 2002 in the liver tissue section using colocalization of Sigmar1 with a mitochondrial marker. Recent studies confirmed Sigmar1’s localization in the mitochondria of cardiomyocytes using quantum dot-mediated immunolabeling of Sigmar1 using transmission electron microscopy (TEM), co-immunostaining with mitochondria-specific MitoTracker, and using subcellular fractionation assays (Abdullah et al., 2022aAishwarya et al., 2022a). Thus, Sigmar1 has a potential role in regulating mitochondrial respiration and bioenergetics that is essential to continue important cellular functions.

The existence of Sigmar1 protein in the vasculature of the thoracic aorta in rats was reported using Western blot analysis (Bhuiyan and Fukunaga, 2009). In pressure overload-induced cardiac injury and transverse aortic constriction (TAC) models, the expression of Sigmar1 was found to be significantly decreased in the thoracic aorta, leading to vascular remodeling. This reduced Sigmar1 expression was further associated with downregulation of Akt-eNOS signaling axis in the aorta, which was rescued by activating Sigmar1 using its ligands, e.g., DHEA and fluvoxamine (Bhuiyan and Fukunaga, 2009Bhuiyan et al., 2010Tagashira et al., 2010Bhuiyan et al., 2011aBhuiyan et al., 2011bTagashira et al., 2011Tagashira et al., 2013). This suggested the potential role of Sigmar1 in vascular system and remodeling. However, only a few studies to date have examined the role of Sigmar1 in the vasculature.

Activation of Sigmar1 using its agonist improved the barrier function of vascular endothelial cells and decreased its permeability to albumin and dextran (Motawe et al., 2020). Similarly, the Sigmar1 pharmacologic ligand, PRE084, enhanced endothelial integrity and barrier function after inflammatory induction in lymphatic endothelial cells (Motawe et al., 2021a). These studies also demonstrated Sigmar1’s involvement in mitochondrial bioenergetics regulation in vascular and lymphatic endothelial cells. A recent study showed that the opioid receptor agonist, oxycodone, preserves cardiac microvascular endothelial cell integrity and myocardial function after ischemia-reperfusion injury by binding to Sigmar1 (Ji et al., 2022). Pharmacologic inhibition of Sigmar1 using its antagonist, such as haloperidol metabolite-II valproate ester [(±)-MRJF22] exhibited an antiangiogenic effect, significantly reducing cell viability, migration, and tube formation in human retinal endothelial cells stimulated with vascular endothelial growth factor A (Olivieri et al., 2016). However, these previous studies were based only on the use of pharmacological ligands, and lacked genetic manipulation of Sigmar1 to establish its physiological roles in the vascular system.

The current study aims to explore the molecular and physiological roles of Sigmar1 associated with vascular tone, vascular remodeling, mitochondrial structure, and function using genetic mouse models. We used gender-matched Sigmar1 global knockout (Sigmar1−/−) and wild-type littermate control (Wt) mice for all our experiments.

3 Results

3.1 Sigmar1 expression in the vascular wall of human and mouse blood vessels

We previously reported the expression of Sigmar1 protein in the thoracic aorta lysates of rats in a pressure-overload-induced cardiac injury model using Western blot (Bhuiyan and Fukunaga, 2009). However, Sigmar1’s localization in different cells in the vascular wall lining has yet to be explored. In this study, we used human and mouse blood vessels to show Sigmar1’s expression in the vascular cells. We used formalin-fixed and paraffin-embedded human left anterior descending coronary artery (LAD) and mouse aortic root tissue sections to characterize Sigmar1 protein expression using immunohistochemical (IHC) staining. Brown precipitation from IHC (Figure 1A) showed that Sigmar1 is substantially expressed in both human and mouse arteries in vascular intima, media, and adventitial layers. A negative staining from global Sigmar1 knockout (Sigmar1−/−) mouse aorta confirmed the specificity of the Sigmar1 antibody used for IHC staining. Paraffin-fixed tissue sections of human LAD and mouse aortic roots were also used for immunofluorescence (IF) staining to delineate Sigmar1’s expression in all three layers of the vascular wall (green), including the innermost endothelial lining and medial vascular smooth muscle cells (Figure 1B). Both IHC and IF staining results confirmed the expression of Sigmar1 protein in the vascular wall at substantial density.

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.

3.2 Role of Sigmar1 in extracellular matrix remodeling and fibrosis in the vascular wall

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., 2018Aishwarya et al., 2022bRemex 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. Hematoxylin and eosin (H&E), Masson’s trichrome, Russell-movat pentachrome, and toluidine blue staining of aortic root showed aberrant organization of smooth muscle cells in the medial layer compared to Wt mice (Figures 2A–D). The nuclei disarray of vascular smooth muscle layer and abrupt organization of elastic fibers was observed in the absence of Sigmar1 (Figure 2A). Masson’s trichrome (Figure 2B), Russell-movat pentachrome (Figure 2C), and Toluidine blue (Figure 2D) staining showed higher collagen deposition in the vascular layer and perivascular area (blue and yellowish red respectively) causing higher fibrotic remodeling in Sigmar1−/− mice compared to Wt (Figures 2B–D). These staining images indicate that Sigmar1 has a potential role in vascular remodeling and vascular and perivascular fibrosis.

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.

3.3 Enhanced aortic stiffness and impaired vascular function in absence of Sigmar1

We next measured the in vivo pulse wave velocity (PWV) to determine vascular stiffness in the aorta of Wt and Sigmar1−/− mice. We used 4 months-old male and female mice to measure PWV using doppler ultrasound probe (VEVO 3100). This is an indirect measurement of arterial stiffness in mice. The time-transit method of PWV analysis (DuPont et al., 2021) showed a significant increase of aortic stiffness in Sigmar1 deficient mice compared to Wt. This indicated more rigid blood vessels in Sigmar1−/− mice (Figure 3A). Since Sigmar1 is expressed in the vascular wall and has a role in vascular remodeling, we then sought to examine the physiological function of Sigmar1 in the vascular system. To do so, we assessed vascular flow-mediated dilation (FMD) of the left femoral artery of Sigmar1−/− mice and compared that to Wt mice. This is a gold standard non-invasive method in the vascular field to determine vascular function in response to reactive hyperemia. FMD measurements showed that transient hindlimb ischemia for 1 min with an occlusion cuff in Sigmar1−/− mice did not induce femoral artery vasodilation upon reperfusion. The Wt mice showed FMD responses, especially at 90 s time point after removing the ischemic trigger, when the arterial diameter was the highest. Then, it gradually went back close to the baseline at 300 s time point. On the other hand, FMD responses were significantly blunted in Sigmar1 deficient mice (Figure 3B). Sigmar1−/− mice failed to respond to reactive hyperemia after removing the ischemic trigger, indicating profound vascular dysfunction in response to brief tissue ischemia.

Figure 3.
Sigmar1 deficiency causes increased pulse wave velocity (PWV) and decreased flow-mediated dilation (FMD) in mice. 
 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.

3.4 NO bioavailability and eNOS phosphorylation in Sigmar1−/− mice aorta

Nitric oxide (NO) has been a key determinant of vascular homeostasis that has a wide variety of functions in the vasculature regulating the physiological properties of the blood vessels. It maintains vascular tone, permeability, and has anti-inflammatory and anti-thrombotic properties (Palmer et al., 1987). Most of the vascular NO is produced by the endothelial cell using nitric oxide synthase (eNOS) enzyme and exerts its effects on the vascular smooth muscle cells (vSMCs), maintaining vessel tone (Jin and Loscalzo, 2010). Decreased bioavailability of NO has been associated with many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus (Forstermann et al., 2006) and is a determinant of vascular dysfunction. Therefore, we next measured the total bioavailability of NO in Wt and Sigmar1−/− mice in the plasma and tissue using an ozone-based hypersensitive chemiluminescence assay. The plasma NO metabolites were significantly decreased in the absence of Sigmar1 compared to Wt mice at 4 months of age (Figure 4A). The total NOx level in the vascularized gastrocnemius muscle also showed a significant decrease in the Sigmar1−/− mice (Figure 4A). Additionally, we observed a significant reduction of phos-eNOSser1177protein expression confirmed by Western blot of aorta tissue (Figure 4B) and immunohistochemical staining of aortic roots in Sigmar1−/− mice compared to Wt (Figure 4C). This suggested depletion in NO synthesis and bioavailability in the vascular system in the absence of Sigmar1 indicating vascular dysfunction in mice.

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.

3.5 TEM analysis of blood vessel wall of Sigmar1−/− mice

The aorta tissue isolated from Wt and Sigmar1−/− mice was analyzed for ultrastructural morphology of vascular wall cells using transmission electron microscopy (TEM). The vascular wall comprises three distinct layers: tunica intima containing a monolayer of endothelial cells, tunica media containing multiple layers of vascular smooth muscle cells, and outer adventitia containing nerve endings, fibroblasts, adipose tissue, etc. TEM images of the mouse aorta showed a detailed visual representation of vascular endothelial and smooth muscle cells (Figure 5) in the artery. 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., 2018Remex 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. These results indicate that Sigmar1 has a role in regulating mitochondrial shape and structure in vascular cells.

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.

3.6 Analysis of mitochondrial respirometry in ex-vivo aortic rings from Sigmar1−/− mice using Oroboros

Mitochondrial structural alteration is an indication of functional impairment as well, which is a hallmark of different cardiovascular diseases (Abdullah et al., 2018Alam et al., 2018Tyrrell et al., 2020Abdullah et al., 2022b). Since Sigmar1−/−mice showed elongated and clustered mitochondria in aortic endothelial cells and smooth muscle cells, we measured mitochondrial function using high-resolution mitochondrial respirometry in aortic rings from Wt and Sigmar1−/− mice (Figure 6). To determine mitochondrial fuel substrates-supported respiration in aortic rings, we injected NADH-generating substrates (donate electrons to complex I, CI), i.e., pyruvate, malate, and glutamate, followed by ADP addition to attain CI-supported oxidative phosphorylation (OXPHOS) state. Next, we injected succinate (activates complex II, CII) to measure respiration at the CI + CII-coupled OXPHOS state. FCCP was injected to uncouple mitochondrial respiration from OXPHOS to measure CI + CII-linked uncoupled respiration. Mitochondrial respiratory complex I inhibitor (Rotenone) and complex III inhibitor (Antimycin A) were injected at the end to measure non-mitochondrial residual oxygen flux (Figure 6A). These respirometry study protocol revealed significantly attenuated mitochondrial complex I supported basal respiration with impaired complex I + complex II-linked coupled and uncoupled respiration with non-significant changes in residual nonmitochondrial respiration in Sigmar1−/− mice aorta compared to Wt mice aorta (Figures 6B–F). This finding indicate that Sigmar1 is essential to maintain mitochondrial respiration in the vascular system.

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-vivoaortic 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.

4 Discussion

Sigma1 receptor (Sigmar1) is a multifunctional chaperone protein with important molecular and cellular functions, including ion channel regulation, lipid transportation, mitochondrial functions, and autophagy (Aishwarya et al., 2021). It plays a vital role in maintaining cardiovascular homeostasis by protecting hearts during heart failure and cardiac ischemia-reperfusion injury and regulating cardiac contractility and mitochondrial functions (Abdullah et al., 2018). Given the significant neuroprotective and cardioprotective roles of Sigmar1 that have been studied, the physiological roles of this protein in the vascular system remain elusive. This study confirmed its expression in the human and mouse arteries using histological and biochemical techniques. Functional assays indicated that global loss of Sigmar1 in mice leads to stiffer blood vessels, impaired response to reactive hyperemia upon the ischemic trigger, and decreased flow-mediated dilation (FMD) in the left femoral artery. This was consistent with decreased nitric oxide bioavailability and eNOS phosphorylation in global Sigmar1 deficient mice (Sigmar1−/−) compared to wildtype controls (Wt). The Sigmar1−/− mice also showed vascular reorganizations, altered mitochondrial structures, and decreased mitochondrial respiration in aorta. These findings indicate that Sigmar1 is a potential player in maintaining vascular homeostasis and mitochondrial function in the vascular tissue (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.

Sigmar1 was first identified in the thoracic aorta in a hypertrophy-induced vascular injury model of ovariectomized rats where the protein level of Sigmar1 was significantly decreased in the pressure overload-induced injured blood vessels (Bhuiyan et al., 2011b). Later, a few studies detected Sigmar1’s protein expression levels using Western blot analysis in the lymphatic endothelial cells (Motawe et al., 2021b) and human brain endothelial cells (Liu et al., 2022) to determine its protective role in endothelial barrier function. However, these ligand-dependent studies failed to show the protein expression level and distribution of Sigmar1 in vascular tissue using histological or immunostaining. We hereby identified Sigmar1 protein expression in both human and mouse vascular tissue using immunohistochemical and immunofluorescence staining (Figure 1). Sigmar1 protein is substantially expressed in all three vascular layers (innermost intima, middle smooth muscle cell layer media, and the outermost adventitia) in human left anterior descending coronary artery (LAD) and mouse aortic root. Sigmar1 protein expression is also detectable in the aorta in mice using Western blot. This study is the first to show this protein’s expression level and distribution in the vascular tissue in humans and mice.

Loss of Sigmar1 led to adverse cardiac remodeling, increased perivascular and interstitial collagen deposition, and increased fibrotic markers in the heart in our previous study (Abdullah et al., 2018). This evidence indicates that Sigmar1 has a potential role in tissue remodeling, resulting in significant fibrosis and collagen deposition. In this study, we observed remarkable vascular fibrosis in the aortic roots of Sigmar1−/− mice compared to Wt (Figure 2). 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., 2018Aishwarya et al., 2022bRemex 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., 2013Harvey et al., 2016Ding et al., 2020). Hypertension is closely associated with increased ECM content, especially the fibrillar collagen type, leading to vessel stiffness over time (Bashey et al., 1989). As Sigmar1−/− mice reported to have altered hemodynamics (Abdullah et al., 2018), which might be partially contributing to the ECM remodeling and higher collagen content in the vascular tissue in absence of Sigmar1.

Since ECM remodeling, collagen deposition, and vascular tissue reorganization are leading causes of vessel stiffness, it was worthy of measuring vascular stiffness in Sigmar1−/− mice. Using non-invasive pulse wave velocity (PWV), we determined the aortic stiffness indirectly following the previously used time-transit method (Williams et al., 2007DuPont et al., 2021). Sigmar1 deficient mice showed significantly higher aortic stiffness compared to Wt (Figure 3). Consistently, vascular functional analysis showed a decline in flow-mediated dilation (FMD) in left femoral artery in response to ischemic trigger indicating vascular dysfunction in absence of Sigmar1 (Figure 3). The Sigmar1−/− mice failed to increase vessel diameter in response to reactive hyperemia and this can be associated with stiffer blood vessels in these mice. FMD is also a non-invasive indirect assessment of endothelial function and can be used to examine endothelial dysfunction, which is an early event in the initiation of many vascular diseases like atherosclerosis (Matter et al., 2011). A decrease in FMD provides an index of vascular endothelium-derived nitric oxide (NO) function. It can indicate either insufficient production of endothelial NO or improper response of vascular SMCs to NO, leading to dysfunction in vessel dilation (Green et al., 2011). In line with this evidence, we also observed a significant decline in NO bioavailability in Sigmar1−/− mice compared to Wt controls (Figure 4). NO is an essential vasodilator molecule that regulates vascular tone, maintains vessel resistance, and overall vascular homeostasis. Decline in NO level can lead to stiffer vessel resistance and vascular dysfunction. Vascular NO is synthesized by the endothelial cells with the help of endothelial nitric oxide synthase enzyme (eNOS). Activation of Sigmar1 using its pharmacological agonist has previously been shown to have a protective effect on hypertrophy-mediated vascular injury in rats by upregulating AKT-eNOS signaling (Bhuiyan et al., 2011aBhuiyan et al., 2011b). When this signaling axis is activated in the vasculature, PI3K signaling pathway phosphorylates the AKT, which then phosphorylates eNOS at serine 1177, and synthesizes NO. In line with previous data, we observed a decrease in phos-eNOSser1177 level in the aorta lysate and aortic root tissue (Figure 4), indicating dysfunction in NO synthesis pathway.

Sigmar1 has long been known to regulate mitochondrial structure and functions in brain cells, cardiomyocytes, and skeletal muscles (Bernard-Marissal et al., 2015Abdullah et al., 2018Aishwarya et al., 2021Aishwarya et al., 2022b). Sigmar1 is a mitochondrial membrane protein regulating mitochondrial respiration, dynamics, bioenergetics, and architecture (Alam et al., 2018Abdullah et al., 2022aAishwarya et al., 2022a). Deletion of Sigmar1 globally in mice led to the accumulation of elongated shaped mitochondria, defective mitochondrial respiratory function, and altered mitochondrial dynamics in the heart, leading to impairment in ATP generation and heart contractile dysfunction (Abdullah et al., 2018). Our ultrastructural analysis of the aorta section showed accumulation of irregularly longer shaped mitochondria in aortic endothelial and smooth muscle cells in Sigmar1−/− mice (Figure 5). Analysis of mitochondrial respirometry parameters in ex-vivo aortic rings showed significant reduction in mitochondrial oxidative phosphorylation-linked respiration in absence of Sigmar1 (Figure 6). Mitochondrial dysfunctions are strongly associated with cardiovascular diseases, for example, atherosclerosis, ischemic heart disease, hypertension, and cardiomyopathy (Forte et al., 2021Ciccarelli et al., 2023). Mitochondrial dynamics-related abnormalities can lead to the development of cardiovascular diseases (Hubens et al., 2022). Impaired mitochondrial function causes a reduction in ATP production, generation of reactive oxygen species (ROS) and related signaling, apoptosis and cell survival, dysfunctional electron transport chain activities, and trigger inflammatory responses (Iglewski et al., 2010Lee et al., 2016Luongo et al., 2017Zhou and Tian, 2018). Mitochondrial electron transport chain complexes being the major sources of ROS generation, especially, complex I and II, dysfunction of the mitochondrial respiratory system can lead to an excess burden in mitochondrial and overall cellular oxidative stress (Madamanchi and Runge, 2007). Excessive ROS production can cause damage in mitochondrial DNA and oxidation of important proteins, lipids, and enzymes leading to mitochondrial and cellular dysfunction (Peng et al., 2019). Dysfunctional mitochondria, higher ROS, and reduced NO bioavailability can also signal for release of inflammatory cytokines and chemokines causing activation of inflammatory pathways (Qu et al., 2022Ciccarelli et al., 2023). These are underlying leading factors for many inflammatory and metabolic cardiovascular diseases like atherosclerosis. Thus, mitochondrial homeostasis is crucial for cardiovascular health, and Sigmar1, being an important player in regulating mitochondrial respiration and function, can be an attractive therapeutic target for mitochondrial dysfunction-related cardiovascular pathologies.

A limitation of the present study is the use of Sigmar1 global knockout mice in which the loss of Sigmar1 in other organs may also contribute to observed vascular dysfunction. In fact, hemodynamics measurements in the Sigmar1−/− mice in our previous study showed higher systolic blood pressure, mean arterial pressure, and left ventricular pressure compared to Wt mice (Abdullah et al., 2018). However, significant alterations in arterial stiffness, flow-mediated dilation, and vascular structural changes in the aorta of Sigmar1−/− mice demonstrate a potential pathophysiological role of Sigmar1 which should be delineated using both endothelial cell and smooth muscle cell specific Sigmar1 knockout mice.

5 Conclusion

This study has identified Sigmar1 protein expression level and distribution in the human and mouse arteries. Using Sigmar1 global knockout and wildtype control mice, we found the development of vascular dysfunction in the absence of Sigmar1 protein (Figure 7). Loss of Sigmar1 led to vascular reorganizations, ECM remodeling, increased arterial stiffness, and decreased flow-mediated dilation. This was consistent with a significant decline in total nitric oxide bioavailability in plasma and tissue and decreased phos-eNOSser1177 expression in aortic lysate. Finally, the ultrastructural analysis of aortic tissue showed an accumulation of elongated mitochondria in the vascular cells and decreased mitochondrial respirometry parameters in ex-vivo aortic rings from Sigmar1 deficient mice compared to Wt controls.