The calcium transient coupled to the L-type calcium current attenuates cardiac alternans.

Cardiac action potential (AP) alternans have been linked to the development of arrhythmia. AP alternans may be driven by AP instabilities, Ca2+ transient (CaT) instabilities, or both. The mechanisms underlying CaT driven AP alternans is well-supported experimentally, but the ionic mechanism underlying alternans driven by AP instabilities remain incompletely understood. Here we used the Ca2+ buffer BAPTA to remove the CaT and generate a model of AP alternans driven primarily by AP instabilities. In isolated rabbit ventricle myocytes, AP alternans induced by rapid pacing were either critically damped and persisted over time, overdamped and ceased over seconds, or underdamped progressing to 2:1 capture. Control cells predominantly exhibited critically damped alternans. In contrast, removing CaT with BAPTA destabilized alternans formation in a concentration dependent manner. Importantly, alternans were easier to induce in CaT free cells as evidenced by a higher alternans threshold relative to control cells. While the L-type Ca2+ channel agonist Bay K 8644 had a minor effect on alternans formation in myocytes with conserved CaT, combining the agonist with BAPTA markedly promoted the formation of underdamped alternans and increased the alternans threshold more than four-fold as compared to controls. Our data support a mechanistic model in which AP alternans are a primary self-sustained event in which the CaT serves as a dampening cue that curbs alternans development, likely via a canonical negative feedback process involving Ca2+ induced inhibition of L-type Ca2+ current.

Role of Preservation Solution in Human Aneurysmatic Aorta Harvest and Transport: A Comparative Analysis of Different Solutions for Tissue Injury Protection.

INTRODUCTION: Human aortic tissues in vitro are tools to clarify the pathophysiological mechanisms of the cardiovascular system, cell culture, and transplants. Therefore, this study aims to analyze and compare the preservation of human aneurysmatic aortic tissues in three different solutions. METHODS: Six human abdominal aortic aneurysms were obtained from patients after surgical ablation. The aorta samples were incubated in different solutions - 0.9% normal physiological saline solution, Ringer's lactate solution, and histidine-tryptophan-ketoglutarate solution (Custodiol®). Segments were collected at 0, 6, 24, and 48 hours. Creatine kinase and nitrate/nitrite were quantified for each incubation time. The tissue's alpha-smooth muscle actin was analyzed by immunofluorescence. RESULTS: There was a significant increase in creatine kinase formation in the normal saline group at 0 and 48 hours and in the Ringer's lactate group at 0 and 48 hours (P=0.018 and P=0.028). The lower levels of creatine kinase and nitrate/nitrite and the aortic tissues' morphological integrity show that histidine-tryptophan-ketoglutarate has better tissue protection. These data suggest that histidine-tryptophan-ketoglutarate induces a protective effect on smooth muscle cells, with less tissue depletion in the aortic aneurysm. CONCLUSION: This study compared three preservation solutions with the potential for human abdominal aortic aneurysm tissue preservation. The histidine-tryptophan-ketoglutarate solution reduced tissue injury and improved tissue preservation in human abdominal aortic aneurysm tissue samples.

Third Trimester Stillbirth Associated With Hamartoma of Mature Cardiac Myocytes (HMCM).

Fetal primary cardiac tumors (FPCTs) are very rare. The majority of them correspond to cardiac rhabdomyomas, followed by other benign neoplasms or hamartomas. We describe the case of a third trimester female stillborn with an incidental autopsy finding of Hamartoma of Mature Cardiac Myocytes (HMCM), a rare benign cardiac tumor previously unreported in the fetal or neonatal period. The intrauterine demise occurred at 32 + 6 weeks gestation after an uneventful pregnancy. The fetal autopsy revealed a structurally normal heart with a small subendocardial nodule just below the membranous septum. Microscopically, the nodule was well-demarcated from the surrounding penetrating bundle of the conduction axis and the adjacent left ventricular myocardium and consisted of disorganized mature cardiac myocytes in a haphazard arrangement with patchy mild interstitial fibrosis, consistent with HMCM. Awareness that HMCM can occur in the fetus is important in order to consider it among the differential diagnosis of FPCTs.

The interaction between neutrophils and atrial myocytes in the occurrence and development of atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is one of the most prevalent sustained cardiac arrhythmias, strongly associated with neutrophils. However, the underlying mechanism remain unclear. This study aims to explore the interaction between neutrophils and atrial myocytes in the pathogenesis of AF. METHODS: Patch-clamp was employed to record the action potential duration (APD) and ion channels in HL-1 cells. Flow cytometry was used to assess the differentiation of neutrophils. The mRNA and protein levels of CACNA1C, CACNA2D, and CACNB2 in HL-1 cells were detected. RESULTS: High-frequency electrical stimulation resulted in a shortening of the APD in HL-1 cells. Flow cytometry demonstrated that neutrophils were polarized into N1 phenotype when cultured with stimulated HL-1 cells medium. Compared to control neutrophils conditioned medium (CM), cocultured with TNF-α knockout neutrophils CM prolonged APD and the L-type Ca (2+) channel (LTCC) of HL-1 cells. Additionally, the expression of CACNA2D, CACNB2 and CACNA1C in HL-1 cells were upregulated. Compared with CACNA1C siRNA-transfected HL-1 cells treated with TNF-α siRNA-transfected neutrophils CM, the APD and LTCC of CACNA1C siRNA-transfected HL-1 cells were shortened in control N1 neutrophil CM. The APD and LTCC of control HL-1 cells were also shortened in control N1 neutrophil CM, but prolonged in TNF-α siRNA-transfected neutrophils CM. CONCLUSION: These findings suggest that neutrophils were polarized into N1 phenotype in AF, TNF-α released from N1 neutrophils contributes to the pathogenesis of AF, via decreasing the APD and LTCC in atrial myocytes through down-regulation of CACNA1C expression.

Atherosclerosis on a Chip: A 3-Dimensional Microfluidic Model of Early Arterial Events in Human Plaques.

BACKGROUND: Realistic reconstruction of the in vivo human atherosclerotic environment requires the coculture of different cell types arranged in atherosclerotic vessel-like structures with exposure to flow and circulating cells, presenting challenges for disease modeling. This study aimed to develop a 3-dimensional tubular microfluidic model with quadruple coculture of human aortic smooth muscle cells, human umbilical cord vein endothelial cells, and foam cells to recreate a complex human atherosclerotic vessel in vitro to study the effects of flow and circulating immune cells. METHODS: We developed a coculture protocol utilizing BFP (blue fluorescent protein)-labeled human aortic smooth muscle cells, GFP (green fluorescent protein)-labeled human umbilical cord vein endothelial cells, and THP-1 macrophage-derived, Dil-labeled oxidized LDL (low-density lipoprotein) foam cells within a fibrinogen/collagen I-based 3-dimensional ECM (extracellular matrix). Perfusion experiments were conducted for 24 hours on both atherosclerotic vessels and healthy vessels (BFP-labeled human aortic smooth muscle cells and GFP-labeled human umbilical cord vein endothelial cells without foam cells). Additionally, perfusion with circulating THP-1 monocytes was performed to observe cell extravasation and recruitment. RESULTS: The resulting vessels displayed early lesion morphology, with a layered composition including an endothelium and media, and foam cells accumulating in the subendothelial space. The layered wall composition of both atherosclerotic and healthy vessels remained stable under perfusion. Circulating THP-1 monocytes demonstrated cell extravasation into the atherosclerotic vessel wall and recruitment to the foam cell core. The qPCR analysis indicated increased expression of atherosclerosis markers in the atherosclerotic vessels and adaptation of vascular smooth muscle cell migration in response to flow and the plaque microenvironment, compared with control vessels. CONCLUSIONS: The human 3-dimensional atherosclerosis model demonstrated stability under perfusion and allowed for the observation of immune cell behavior, providing a valuable tool for the atherosclerosis research field.

Endothelial TGF-ß Signaling Regulates Endothelial-Mesenchymal Transition During Arteriovenous Fistula Remodeling in Mice With Chronic Kidney Disease.

BACKGROUND: Arteriovenous fistulae (AVFs) are the preferred vascular access for hemodialysis in patients with end-stage kidney disease. Chronic kidney disease (CKD) is associated with endothelial injury, impaired AVF maturation, and reduced patency, as well as utilization. Because CKD is characterized by multiple pathophysiological processes that induce endothelial-to-mesenchymal transition (EndMT), we hypothesized that CKD promotes EndMT during venous remodeling and that disruption of endothelial TGF (transforming growth factor)-ß signaling inhibits EndMT to prevent AVF failure even in the end-stage kidney disease environment. METHODS: The mouse 5/6 nephrectomy and aortocaval fistula models were used. CKD was created via 5/6 nephrectomy, with controls of no (0/6) or partial (3/6) nephrectomy in C57BL/6J mice. AVFs were created in mice with knockdown of TGF-ßR1/R2 (TGF-ß receptors type 1/2) in either smooth muscle cells or endothelial cells. AVF diameters and patency were measured and confirmed by serial ultrasound examination. AVF, both murine and human, were examined using Western blot, histology, and immunofluorescence. Human and mouse endothelial cells were used for in vitro experiments. RESULTS: CKD accelerates TGF-ß activation and promotes EndMT that is associated with increased AVF wall thickness and reduced patency in mice. Inhibition of TGF-ß signaling in both endothelial cells and smooth muscle cells decreased smooth muscle cell proliferation in the AVF wall, attenuated EndMT, and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. Human AVF also showed increased TGF-ß signaling and EndMT. CONCLUSIONS: CKD promotes EndMT and reduces AVF patency. Inhibition of TGF-ß signaling, especially disruption of endothelial cell-specific TGF-ß signaling, attenuates EndMT and improves AVF patency in mouse AVF. Inhibition of EndMT may be a therapeutic approach of translational significance to improve AVF patency in human patients with CKD.

Crotonylation of NAE1 Modulates Cardiac Hypertrophy via Gelsolin Neddylation.

BACKGROUND: Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8 [neural precursor cell expressed developmentally downregulated protein 8]-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy. METHODS: Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. Tandem mass tag (TMT)-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 (lysine residue at site 238) crotonylation. RESULTS: The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy. CONCLUSIONS: Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

The cAMP/PKA signaling pathway conditions cardiac performance in experimental animals with metabolic syndrome.

Metabolic syndrome (MetS) increases the risk of coronary artery disease, but effects of this condition on the working myocardium remain to be fully elucidated. In the present study we evaluated the consequences of diet-induced metabolic disorders on cardiac function and myocyte performance using female mice fed with Western diet. Animals maintained on regular chow were used as control (Ctrl). Mice on the Western diet (WesD) had increased body weight, impaired glucose metabolism, preserved diastolic and systolic function, but increased left ventricular (LV) mass, with respect to Ctrl animals. Moreover, WesD mice had reduced heart rate variability (HRV), indicative of altered cardiac sympathovagal balance. Myocytes from WesD mice had increased volume, enhanced cell mechanics, and faster kinetics of contraction and relaxation. Moreover, levels of cAMP and protein kinase A (PKA) activity were enhanced in WesD myocytes, and interventions aimed at stabilizing cAMP/PKA abrogated functional differences between Ctrl and WesD cells. Interestingly, in vivo ß-adrenergic receptor (ß-AR) blockade normalized the mechanical properties of WesD myocytes and revealed defective cardiac function in WesD mice, with respect to Ctrl. Collectively, these results indicate that metabolic disorders induced by Western diet enhance the cAMP/PKA signaling pathway, a possible adaptation required to maintain cardiac function.

SUMOylation of cardiac myosin binding protein-C reduces its phosphorylation and results in impaired relaxation following treatment with isoprenaline.

Systolic and diastolic functions are coordinated in the heart by myofilament proteins that influence force of contraction and calcium sensitivity. Fine control of these processes is afforded by a variety of post-translation modifications that occur on specific proteins at different times during each heartbeat. Cardiac myosin binding protein-C is a sarcomeric accessory protein whose function is to interact transiently with actin, tropomyosin and myosin. Previously many different types of post-translational modification have been shown to influence the action of myosin binding protein-C and we present the first report that the protein can be modified covalently by the small ubiquitin like modifier protein tag. Analysis by mass spectrometry suggests that there are multiple modification sites on myosin binding protein-C for this tag and single point mutations did not serve to abolish the covalent addition of the small ubiquitin like modifier protein. Functionally, our data from both model human embryonic kidney cells and transfected neonatal cardiac myocytes suggests that the modification reduces phosphorylation of the filament protein on serine 282. In cardiac myocytes, the hypo-phosphorylation coincided with a significantly slower relaxation response following isoprenaline induced contraction. We hypothesise that this novel modification of myosin binding protein-C represents a new level of control that acts to alter the relaxation kinetics of cardiac myocytes.

Restoring Atrial T-Tubules Augments Systolic Ca Upon Recovery From Heart Failure.

BACKGROUND: Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. METHODS: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. RESULTS: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. CONCLUSIONS: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.

Human induced pluripotent stem cells for live cell cycle monitoring and endogenous gene activation.

The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.

AMPK Attenuation of ß-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of ß-Arrestin-1-ser330.

BACKGROUND: ß-adrenergic receptor (ß-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of ß-AR remains unclear. METHODS: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates ß-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the ß-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between ß-adrenergic insult and ß-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from ß-arrestin-1-S330A/S330D mutation and ß-adrenergic insult. RESULTS: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to ß-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted ß-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing ß-arrestin-1-S330D (active form) inhibited the ß-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. ß-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the ß-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS: AMPK phosphorylation of ß-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting ß-AR/cAMP/PKA activation. Subsequently, ß-arrestin-1 Ser330 phosphorylation blocks ß-AR-induced cardiac inflammasome activation and remodeling.

Sorting differentiated mammalian cells using deterministic lateral displacement microfluidic devices.

Separation of differentiated and undifferentiated cells without labeling is required for cell analyses and clinical application of cultured differentiated cells in vitro. To proceed with the passive separation of differentiated cells inside a clean bench, we developed a system of deterministic lateral displacement (DLD) microfluidic devices and applied this system to sort differentiated cells in vitro. The fluid flow is driven by compressed air to the buffer. Priming and sorting can be completed by air pressure control. We use this system to separate C2C12 mononuclear myocytes from multinuclear myotubes. Additionally, using a DLD microfluidic channel of Dc = 20 µm, multinuclear myotubes can be effectively sorted as larger particles. We prepared differentiated adipocytes from mouse embryonic fibroblast (MEF) cells and sorted those containing lipid droplets. The diameters of these sorted adipocytes considered larger particles, exceeded 20 µm, similar to the Dc of the DLD microfluidic channel. Differentiated cell sorting by cell size will contribute to single-cell analyses and in vitro tissue model preparation for drug discovery.

Generation of musculoskeletal cells from human urine epithelium-derived presomitic mesoderm cells.

BACKGROUND: Numerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally, we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation. RESULTS: Selective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice, as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover, UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro. CONCLUSIONS: UiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders.

Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

BACKGROUND: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. METHODS: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. RESULTS: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. CONCLUSIONS: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

L-Wnk1 Deletion in Smooth Muscle Cells Causes Aortitis and Inflammatory Shift.

BACKGROUND: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. METHODS: AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. RESULTS: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-ß (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-ß blockade using neutralizing anti-TGF-ß antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals. CONCLUSION: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.