Prolonged tamoxifen-enriched diet is associated with cardiomyopathy and nutritional frailty in mice.

Tamoxifen (TAM) is required for gene recombination in the inducible Cre/lox system. The TAM-enriched diet is considered safe, with negligible impact on animal wellbeing. However, studies reporting the long-term effects of the TAM diet and its potential impact on experimental outcomes are scarce. We conducted a longitudinal study on mice exposed to a 4-week dietary TAM citrate supplementation. Several parameters were recorded, such as body weight, body composition, mortality, and cardiac function. The collagen1a2 (Col1a2) transgenic mouse was used to assess TAM-induced recombination in vivo in cardiac fibroblasts followed by myocardial infarction (MI). The impact of TAM on the MI outcome was also evaluated. The recombination efficiency and cytotoxic effect of the TAM active metabolite, 4-hydroxy-tamoxifen (4-OHT), were assessed in vitro. Mice exposed to a TAM diet showed body weight loss and a 10% increase in mortality (P = 0.045). The TAM diet decreased cardiac function and induced cardiac remodeling, indicated by decreased fractional shortening from 32.23% to 19.23% (P = 0.001) and left ventricular (LV) wall thinning. All measured parameters were reversed to normal when mice were returned to a normal diet. Infarcted Col1a2-CreER mice on the TAM regimen showed gene recombination in fibroblasts, but it was associated with a substantial increase in mortality post-surgery (2.5-fold) compared to the controls. In vitro, 4-OHT induced gene editing in fibroblasts; however, cell growth arrest and cytotoxicity were observed at high concentrations. In conclusion, prolonged exposure to the TAM diet can be detrimental and necessitates careful model selection and interpretation of the results.

Involvement of SAP97 anchored multiprotein complexes in regulating cardiorenal signaling and trafficking networks.

SAP97 is a member of the MAGUK family of proteins, but unlike other MAGUK proteins that are selectively expressed in the CNS, SAP97 is also expressed in peripheral organs, like the heart and kidneys. SAP97 has several protein binding cassettes, and this review will describe their involvement in creating SAP97-anchored multiprotein networks. SAP97-anchored networks localized at the inner leaflet of the cell membrane play a major role in trafficking and targeting of membrane G protein-coupled receptors (GPCR), channels, and structural proteins. SAP97 plays a major role in compartmentalizing voltage gated sodium and potassium channels to specific cellular compartments of heart cells. SAP97 undergoes extensive alternative splicing. These splice variants give rise to different SAP97 isoforms that alter its cellular localization, networking, signaling and trafficking effects. Regarding GPCR, SAP97 binds to the ß1-adrenergic receptor and recruits AKAP5/PKA and PDE4D8 to create a multiprotein complex that regulates trafficking and signaling of cardiac ß1-AR. In the kidneys, SAP97 anchored networks played a role in trafficking of aquaporin-2 water channels. Cardiac specific ablation of SAP97 (SAP97-cKO) resulted in cardiac hypertrophy and failure in aging mice. Similarly, instituting transverse aortic constriction (TAC) in young SAP97 c-KO mice exacerbated TAC-induced cardiac remodeling and dysfunction. These findings highlight a critical role for SAP97 in the pathophysiology of a number of cardiac and renal diseases, suggesting that SAP97 is a relevant target for drug discovery.

Mapping the Proximity Interaction Network of STIM1 Reveals New Mechanisms of Cytoskeletal Regulation.

Stromal interaction molecule 1 (STIM1) resides primarily in the sarco/endoplasmic reticulum, where it senses intraluminal Ca2+ levels and activates Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 that lead to cytoskeletal remodeling are not known. The proximity-labeling technique (BioID) can capture weak and transient protein-protein interactions, including proteins that reside in the close vicinity of the bait, but that may not be direct binders. Hence, in the present study, we investigated the STIM1 interactome using the BioID technique. A promiscuous biotin ligase was fused to the cytoplasmic C-terminus of STIM1 and was stably expressed in a mouse embryonic fibroblast (MEF) cell line. Screening of biotinylated proteins identified several high confidence targets. Here, we report Gelsolin (GSN) as a new member of the STIM1 interactome. GSN is a Ca2+-dependent actin-severing protein that promotes actin filament assembly and disassembly. Results were validated using knockdown approaches and immunostaining. We tested our results in neonatal cardiomyocytes where STIM1 overexpression induced altered actin dynamics and cytoskeletal instability. This is the first time that BioID assay was used to investigate the STIM1 interactome. Our work highlights the role of STIM1/GSN in the structure and function of the cytoskeleton.

Deficiency in ST2 signaling ameliorates RSV-associated pulmonary hypertension.

Pulmonary hypertension (PH) observed during respiratory syncytial virus (RSV) bronchiolitis is associated with morbidity and mortality, especially in children with congenital heart disease. Yet, the pathophysiological mechanisms of RSV-associated PH remain unclear. Therefore, this study aimed to investigate the pathophysiological mechanism of RSV-associated PH. We used a translational mouse model of RSV-associated PH, in which wild-type (WT) and suppression of tumorigenicity 2 (ST2) knockout neonatal mice were infected with RSV at 5 days old and reinfected 4 wk later. The development of PH in WT mice following RSV reinfection was evidenced by elevated right ventricle systolic pressure, shortened pulmonary artery acceleration time (PAT), and decreased PAT/ejection time (ET) ratio. It coincided with the augmentation of periostin and IL-13 expression and increased arginase bioactivity by both arginase 1 and 2 as well as induction of nitric oxide synthase (NOS) uncoupling. Absence of ST2 signaling prevented RSV-reinfected mice from developing PH by suppressing NOS uncoupling. In summary, ST2 signaling was involved in the development of RSV-associated PH. ST2 signaling inhibition may be a novel therapeutic target for RSV-associated PH.NEW & NOTEWORTHY We report that the pathogenic role of ST2-mediated type 2 immunity and mechanisms contribute to RSV-associated pulmonary hypertension. Inhibiting ST2 signaling may be a novel therapeutic target for this condition.

Cardiac-Specific Deletion of Orai3 Leads to Severe Dilated Cardiomyopathy and Heart Failure in Mice.

Background Orai3 is a mammalian-specific member of the Orai family (Orai1‒3) and a component of the store-operated Ca2+ entry channels. There is little understanding of the role of Orai channels in cardiomyocytes, and its role in cardiac function remains unexplored. Thus, we developed mice lacking Orai1 and Orai3 to address their role in cardiac homeostasis. Methods and Results We generated constitutive and inducible cardiomyocyte-specific Orai3 knockout (Orai3cKO) mice. Constitutive Orai3-loss led to ventricular dysfunction progressing to dilated cardiomyopathy and heart failure. Orai3cKO mice subjected to pressure overload developed a fulminant dilated cardiomyopathy with rapid heart failure onset, characterized by interstitial fibrosis and apoptosis. Ultrastructural analysis of Orai3-deficient cardiomyocytes showed abnormal M- and Z-line morphology. The greater density of condensed mitochondria in Orai3-deficient cardiomyocytes was associated with the upregulation of DRP1 (dynamin-related protein 1). Cardiomyocytes isolated from Orai3cKO mice exhibited profoundly altered myocardial Ca2+ cycling and changes in the expression of critical proteins involved in the Ca2+ clearance mechanisms. Upregulation of TRPC6 (transient receptor potential canonical type 6) channels was associated with upregulation of the RCAN1 (regulator of calcineurin 1), indicating the activation of the calcineurin signaling pathway in Orai3cKO mice. A more dramatic cardiac phenotype emerged when Orai3 was removed in adult mice using a tamoxifen-inducible Orai3cKO mouse. The removal of Orai1 from adult cardiomyocytes did not change the phenotype of tamoxifen-inducible Orai3cKO mice. Conclusions Our results identify a critical role for Orai3 in the heart. We provide evidence that Orai3-mediated Ca2+ signaling is required for maintaining sarcomere integrity and proper mitochondrial function in adult mammalian cardiomyocytes.

Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure.

PKD2 (polycystin-2, TRPP1), a TRP polycystin channel, is expressed in endothelial cells (ECs), but its physiological functions in this cell type are unclear. Here, we generated inducible, EC-specific Pkd2 knockout mice to examine vascular functions of PKD2. Data show that a broad range of intravascular flow rates stimulate EC PKD2 channels, producing vasodilation. Flow-mediated PKD2 channel activation leads to calcium influx that activates SK/IK channels and eNOS serine 1176 phosphorylation in ECs. These signaling mechanisms produce arterial hyperpolarization and vasodilation. In contrast, EC PKD2 channels do not contribute to acetylcholine-induced vasodilation, suggesting stimulus-specific function. EC-specific PKD2 knockout elevated blood pressure in mice without altering cardiac function or kidney anatomy. These data demonstrate that flow stimulates PKD2 channels in ECs, leading to SK/IK channel and eNOS activation, hyperpolarization, vasodilation and a reduction in systemic blood pressure. Thus, PKD2 channels are a major component of functional flow sensing in the vasculature.

FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model.

Observations in transgenic α-Klotho (Kl) mice (KlTg) defined the antiaging role of soluble Klotho (sKL130). A genetic translocation that elevates sKL levels in humans is paradoxically associated with increased circulating fibroblast growth factor 23 (FGF23) levels and the potential of both membrane KL (mKL135) and sKL130 to act as coreceptors for FGF23 activation of fibroblast growth factor receptors (FGFRs). Neither FGF23 expression nor the contributions of FGF23, mKL135, and sKL130 codependent and independent functions have been investigated in KlTg mice. In the current study, we examined the effects of Kl overexpression on FGF23 levels and functions in KlTg mice. We found that mKL135 but not sKL130 stimulated FGF23 expression in osteoblasts, leading to elevated Fgf23 bone expression and circulating levels in KlTg mice. Elevated FGF23 suppressed 1,25(OH)2D and parathyroid hormone levels but did not cause hypophosphatemic rickets in KlTg mice. KlTg mice developed low aldosterone-associated hypertension but not left ventricular hypertrophy. Mechanistically, we found that mKL135 and sKL130 are essential cofactors for FGF23-mediated ERK activation but that they inhibited FGF23 stimulation of PLC-γ and PI3K/AKT signaling. Thus, increased longevity in KlTg mice occurs in the presence of excess FGF23 that interacts with mKL and sKL to bias FGFR pathways.

TREK-1 protects the heart against ischemia-reperfusion-induced injury and from adverse remodeling after myocardial infarction.

The TWIK-related K+ channel (TREK-1) is a two-pore-domain potassium channel that produces background leaky potassium currents. TREK-1 has a protective role against ischemia-induced neuronal damage. TREK-1 is also expressed in the heart, but its role in myocardial ischemia-reperfusion (IR)-induced injury has not been examined. In the current study, we used a TREK-1 knockout (KO) mouse model to show that TREK-1 has a critical role in the cardiac I/R-induced injury and during remodeling after myocardial infarction (MI). At baseline, TREK-1 KO mice had similar blood pressure and heart rate as the wild-type (WT) mice. However, the lack of TREK-1 was associated with increased susceptibility to ischemic injury and compromised functional recovery following ex vivo I/R-induced injury. TREK-1 deficiency increased infarct size following permanent coronary artery ligation, resulting in greater systolic dysfunction than the WT counterpart. Electrocardiographic (ECG) analysis revealed QT interval prolongation in TREK-1 KO mice, but normal heart rate (HR). Acutely isolated TREK-1 KO cardiomyocytes exhibited prolonged Ca2+ transient duration associated with action potential duration (APD) prolongation. Our data suggest that TREK-1 has a protective effect against I/R-induced injury and influences the post-MI remodeling processes by regulating membrane potential and maintaining intracellular Ca2+ homeostasis. These data suggest that TREK-1 activation could be an effective strategy to provide cardioprotection against ischemia-induced damage.

Novel Paradigms Governing ß1-Adrenergic Receptor Trafficking in Primary Adult Rat Cardiac Myocytes.

The ß1-adrenergic receptor (ß1-AR) is a major cardiac G protein-coupled receptor, which mediates cardiac actions of catecholamines and is involved in genesis and treatment of numerous cardiovascular disorders. In mammalian cells, catecholamines induce the internalization of the ß1-AR into endosomes and their removal promotes the recycling of the endosomal ß1-AR back to the plasma membrane; however, whether these redistributive processes occur in terminally differentiated cells is unknown. Compartmentalization of the ß1-AR in response to ß-agonists and antagonists was determined by confocal microscopy in primary adult rat ventricular myocytes (ARVMs), which are terminally differentiated myocytes with unique structures such as transverse tubules (T-tubules) and contractile sarcomeres. In unstimulated ARVMs, the fluorescently labeled ß1-AR was expressed on the external membrane (the sarcolemma) of cardiomyocytes. Exposing ARVMs to isoproterenol redistributed surface ß1-ARs into small (∼225-250 nm) regularly spaced internal punctate structures that overlapped with puncta stained by Di-8 ANEPPS, a membrane-impermeant T-tubule-specific dye. Replacing the ß-agonist with the ß-blocker alprenolol, induced the translocation of the wild-type ß1-AR from these punctate structures back to the plasma membrane. This step was dependent on two barcodes, namely, the type-1 PDZ binding motif and serine at position 312 of the ß1-AR, which is phosphorylated by a pool of cAMP-dependent protein kinases anchored at the type-1 PDZ of the ß1-AR. These data show that redistribution of the ß1-AR in ARVMs from internal structures back to the plasma membrane was mediated by a novel sorting mechanism, which might explain unique aspects of cardiac ß1-AR signaling under normal or pathologic conditions.

New mouse model of pulmonary hypertension induced by respiratory syncytial virus bronchiolitis.

Pulmonary hypertension (PH) has been observed in up to 75% of infants with moderate to severe respiratory syncytial virus (RSV) bronchiolitis and is associated with significant morbidity and mortality in infants with congenital heart disease. The purpose of the present study was to establish a mouse model of PH secondary to RSV bronchiolitis that mimics the disease etiology as it occurs in infants. Neonatal mice were infected with RSV at 5 days of age and then reinfected 4 wk later. Serum-free medium was administered to age-matched mice as a control. Echocardiography and right ventricular systolic pressure (RVSP) measurements via right jugular vein catheterization were conducted 5 and 6 days after the second infection, respectively. Peripheral capillary oxygen saturation monitoring did not indicate hypoxia at 2-4 days post-RSV infection, before reinfection, and at 2-7 days after reinfection. RSV-infected mice had significantly higher RVSP than control mice. Pulsed-wave Doppler recording of the pulmonary blood flow by echocardiogram demonstrated a significantly shortened pulmonary artery acceleration time and decreased pulmonary artery acceleration time-to-ejection time ratio in RSV-infected mice. Morphometry showed that RSV-infected mice exhibited a significantly higher pulmonary artery medial wall thickness and had an increased number of muscularized pulmonary arteries compared with control mice. These findings, confirmed by RVSP measurements, demonstrate the development of PH in the lungs of mice infected with RSV as neonates. This animal model can be used to study the pathogenesis of PH secondary to RSV bronchiolitis and to assess the effect of treatment interventions. NEW & NOTEWORTHY This is the first mouse model of respiratory syncytial virus-induced pulmonary hypertension, to our knowledge. This model will allow us to decipher molecular mechanisms responsible for the pathogenesis of pulmonary hypertension secondary to respiratory syncytial virus bronchiolitis with the use of knockout and/or transgenic animals and to monitor therapeutic effects with echocardiography.

Elevated plasma catecholamines functionally compensate for the reduced myogenic tone in smooth muscle STIM1 knockout mice but with deleterious cardiac effects.

Aims: Stromal interaction molecule 1 (STIM1) has emerged as an important player in the regulation of growth and proliferation of smooth muscle cells. Therefore, we hypothesized that STIM1 plays a crucial role in the maintenance of vascular integrity. The objective of this study was to evaluate whether reduced expression of STIM1 could modify the structure and function of the vasculature, leading to changes in blood pressure (BP). Methods and results: Smooth muscle-specific STIM1 knockout (sm-STIM1 KO) in mice resulted in arteries with ∼80% reduced STIM1 protein expression as compared with control mice. Mesenteric vessels exposed to increasing transmural pressure revealed attenuated myogenic reactivity and reduced vasoconstrictor response to phenylephrine in sm-STIM1 KO arteries. BP monitored via telemetry in sm-STIM1 KO and matched controls did not reveal differences. However, heart rate was significantly increased in sm-STIM1 KO mice. Consistent with these findings, plasma catecholamine levels were higher in sm-STIM1 KO than in control mice. Increased sympathetic activity in sm-STIM1 KO mice was unmasked by apha1-adrenergic receptor inhibitor (prazosin) and by treatment with the ganglion-blocking agent, hexamethonium. Both treatments resulted in a greater reduction of BP in sm-STIM1 KO mice. Cytoskeleton of cultured smooth muscle cells was studied by immunocytochemistry using specific antibodies. Staining for actin and vinculin revealed significant alterations in the cytoskeletal architecture of cells isolated from sm-STIM1 KO arteries. Finally, although sm-STIM1 KO mice were protected from Ang II-induced hypertension, such treatment resulted in significant fibrosis and a rapid deterioration of cardiac function. Conclusions: STIM1 deletion in smooth muscle results in attenuated myogenic tone and cytoskeletal defects with detrimental effects on the mechanical properties of arterial tissue. Although BP is maintained by elevated circulating catecholamine, this compensatory stimulation has a deleterious long-term effect on the myocardium.

Identification of novel transplantable GPCR recycling motif for drug discovery.

ß1-Adrenergic receptor (ß1-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The ß1-AR like other G protein-coupled receptors (GPCRs) are endocytosed in response to intense agonist activation. Recycling of the agonist-internalized ß1-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine312 in the third intracellular loop of the ß1-AR. Progressive elongation of the ß1-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the ß1-AR. However, fusing a twenty amino acid peptide derived from the multiple cloning region of the mammalian expression vector pCDNA3 to the C-tail of the ß1-AR (ß1-AR[+20]) produced a chimeric ß1-AR that recycled rapidly and efficiently. The ß1-AR[+20] recycled in a type-1 PDZ and phospho-Ser312-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of ß1-AR[+20] generated a ß1-AR-EYFP chimera that was expressed on the membrane and recycled efficiently after agonist-induced internalization. This construct trafficked in a PDZ-SNX27/retromer-independent manner. We also fused EYFP to the N-terminus of the ß1-AR to created EYFP-WT ß1-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These ß1-AR-EYFP constructs would be useful for high throughput screening (HTS) programs to identify new entities that would interfere with the recycling of agonist internalized GPCR that traffic in PDZ-dependent vs. PDZ-independent roadmaps.

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

STIM1-dependent Ca(2+) microdomains are required for myofilament remodeling and signaling in the heart.

In non-excitable cells stromal interaction molecule 1 (STIM1) is a key element in the generation of Ca(2+) signals that lead to gene expression, migration and cell proliferation. A growing body of literature suggests that STIM1 plays a key role in the development of pathological cardiac hypertrophy. However, the precise mechanisms involving STIM-dependent Ca(2+) signaling in the heart are not clearly established. Here, we have investigated the STIM1-associated Ca(2+) signals in cardiomyocytes and their relevance to pathological cardiac remodeling. We show that mice with inducible, cardiac-restricted, ablation of STIM1 exhibited left ventricular reduced contractility, which was corroborated by impaired single cell contractility. The spatial properties of STIM1-dependent Ca(2+) signals determine restricted Ca(2+) microdomains that regulate myofilament remodeling and activate spatially segregated pro-hypertrophic factors. Indeed, mice lacking STIM1 showed less adverse structural remodeling in response to pressure overload-induced cardiac hypertrophy. These results highlight how STIM1-dependent Ca(2+) microdomains have a major impact on intracellular Ca(2+) homeostasis, cytoskeletal remodeling and cellular signaling, even when excitation-contraction coupling is present.

Atrophied cardiomyocytes and their potential for rescue and recovery of ventricular function.

Cardiomyocytes must be responsive to demands placed on the heart's contractile work as a muscular pump. In turn, myocyte size is largely dependent on the workload they perform. Both hypertrophied and atrophic myocytes are found in the normal and diseased ventricle. Individual myocytes become atrophic when encumbered by fibrillar collagen, such as occurs at sites of fibrosis. The mechanisms include: (a) being immobilized and subject to disuse with ensuing protein degradation mediated by redox-sensitive, proteolytic ligases of the ubiquitin-proteasome system and (b) dedifferentiated re-expressing fetal genes induced by low intracellular triiodothyronine (T3) via thyroid hormone receptor ß1. This myocyte-selective, low T3 state is a consequence of heterocellular signaling emanating from juxtaposed scar tissue myofibroblasts and their secretome with its de novo generation of angiotensin II. In a paracrine manner, angiotensin II promotes myocyte Ca(2+) entry and subsequent Ca(2+) overload with ensuing oxidative stress that overwhelms antioxidant defenses to activate deiodinase-3 and its enzymatic degradation of T3. In the failing heart, atrophic myocytes represent an endogenous population of viable myocytes which could be rescued to augment contractile mass, reduce systolic wall stress (afterload) and recover ventricular function. Experimental studies have shown the potential for the rescue and recovery of atrophic myocytes in rebuilding the myocardium--a method complementary to today's quest in regenerating myocardium using progenitor cells.

Myofibroblast secretome and its auto-/paracrine signaling.

Myofibroblasts (myoFb) are phenotypically transformed, contractile fibroblast-like cells expressing α-smooth muscle actin microfilaments. They are integral to collagen fibrillogenesis with scar tissue formation at sites of repair irrespective of the etiologic origins of injury or tissue involved. MyoFb can persist long after healing is complete, where their ongoing turnover of collagen accounts for a progressive structural remodeling of an organ (a.k.a. fibrosis, sclerosis or cirrhosis). Such persistent metabolic activity is derived from a secretome consisting of requisite components in the de novo generation of angiotensin (Ang) II. Autocrine and paracrine signaling induced by tissue AngII is expressed via AT1 receptor ligand binding to respectively promote: i) regulation of myoFb collagen synthesis via the fibrogenic cytokine TGF-ß1-Smad pathway; and ii) dedifferentiation and protein degradation of atrophic myocytes immobilized and ensnared by fibrillar collagen at sites of scarring. Several cardioprotective strategies in the prevention of fibrosis and involving myofibroblasts are considered. They include: inducing myoFb apoptosis through inactivation of antiapoptotic proteins; AT1 receptor antagonist to interfere with auto-/paracrine myoFb signaling or to induce counterregulatory expression of ACE2; and attacking the AngII-AT1R-TGF-ß1-Smad pathway by antibody or the use of triplex-forming oligonucleotides.

Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site.

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation.

During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1-dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1-/- mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3-dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release-activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.

Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle.

The Ca(2+)-sensing stromal interaction molecule (STIM) proteins are crucial Ca(2+) signal coordinators. Cre-lox technology was used to generate smooth muscle (sm)-targeted STIM1-, STIM2-, and double STIM1/STIM2-knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca(2+) homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre(+/-) littermates, sm-STIM1-KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm-STIM2-KO was without detectable phenotype, the STIM1/STIM double-KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm-STIM1-KO mice developed abnormally with distended, thinned morphology. While depolarization-induced aortic contraction was unchanged in sm-STIM1-KO mice, α1-adrenergic-mediated contraction was 26% reduced, and store-dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF-induced proliferation was greatly reduced (79%) in sm-STIM1-KO. Notably, the Ca(2+) store-refilling rate in STIM1-KO SMCs was substantially reduced, and sustained PDGF-induced Ca(2+) entry was abolished. This defective Ca(2+) homeostasis prevents PDGF-induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1-regulated Ca(2+) homeostasis is crucial for NFAT-mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.-Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle.