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.

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.

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.

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.

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.

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.

Hypoxia-induced acidosis uncouples the STIM-Orai calcium signaling complex.

The endoplasmic reticulum Ca(2+)-sensing STIM proteins mediate Ca(2+) entry signals by coupling to activate plasma membrane Orai channels. We reveal that STIM-Orai coupling is rapidly blocked by hypoxia and the ensuing decrease in cytosolic pH. In smooth muscle cells or HEK293 cells coexpressing STIM1 and Orai1, acute hypoxic conditions rapidly blocked store-operated Ca(2+) entry and the Orai1-mediated Ca(2+) release-activated Ca(2+) current (I(CRAC)). Hypoxia-induced blockade of Ca(2+) entry and I(CRAC) was reversed by NH(4)(+)-induced cytosolic alkalinization. Hypoxia and acidification both blocked I(CRAC) induced by the short STIM1 Orai-activating region. Although hypoxia induced STIM1 translocation into junctions, it did not dissociate the STIM1-Orai1 complex. However, both hypoxia and cytosolic acidosis rapidly decreased Förster resonance energy transfer (FRET) between STIM1-YFP and Orai1-CFP. Thus, although hypoxia promotes STIM1 junctional accumulation, the ensuing acidification functionally uncouples the STIM1-Orai1 complex providing an important mechanism protecting cells from Ca(2+) overload under hypoxic stress conditions.

Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development.

Calcium signaling plays a central role in the regulation of bone cells, although uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared the mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1(-/-) mice lacked multinucleated osteoclasts. Yet, they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1(-/-) mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1(-/-) mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1(-/-) animals compared with controls; bone deposition was markedly decreased in the knockout mice. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1(-/-) and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation.

STIM protein coupling in the activation of Orai channels.

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca(2+) changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca(2+) entry through the Orai family of highly Ca(2+)-selective "store-operated" channels (SOCs). Dissecting the STIM-Orai coupling process is restricted by the abstruse nature of the ER-PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca(2+) release-activated Ca(2+) (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct-Orai1 coupling occurred in STIM1/STIM2(-/-) DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct-Orai1 and S2-ct-Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB's action, the locked STIMct-Orai complex provides a potentially useful probe to structurally examine coupling.

Rescue and worsening of congenital heart block-associated electrocardiographic abnormalities in two transgenic mice.

INTRODUCTION: Congenital heart block (CHB) is a passively acquired autoimmune disease considered to be due to the transfer of maternal autoantibodies, anti-SSA/Ro -SSB/La, to the fetus resulting in atrioventricular (AV) block and sinus bradycardia. We previously established a murine model for CHB where pups born to immunized wild-type (WT) mothers exhibited electrocardiographic abnormalities similar to those seen in CHB and demonstrated inhibition of L-type Ca channels (LTCCs) by maternal antibodies. Here, we hypothesize that overexpression of LTCC should rescue, whereas knockout of LTCC should worsen the electrocardiographic abnormalities in mice. METHODS AND RESULTS: Transgenic (TG) mice were immunized with SSA/Ro and SSB/La antigens. Pups born to immunized WT mothers had significantly greater sinus bradycardia and AV block compared to pups from nonimmunized WT. TG pups overexpressing LTCC had significantly less sinus bradycardia and AV block compared to their non-TG littermates and to pups born to immunized WT mothers. All LTCC knockout pups born to immunized mothers had sinus bradycardia, advanced degree of AV block, and decreased fetal parity. No sinus bradycardia or AV block were manifested in pups from control nonimmunized WT mothers. IgG from mothers with CHB children, but not normal IgG, completely inhibited intracellular Ca transient ([Ca](i)T) amplitude. CONCLUSIONS: Cardiac-specific overexpression of LTCC significantly reduced the incidence of AV block and sinus bradycardia in pups exposed to anti-SSA/Ro -SSB/La autoantibodies, whereas exposure of LTCC knockout pups to these autoantibodies significantly worsened the electrocardiographic abnormalities. These findings support the hypothesis that maternal antibodies inhibit LTCC and [Ca](i)T thus contributing to the development of CHB. Altogether, the results are relevant to the development of novel therapies for CHB.

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.

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.

Congenital heart block: identification of autoantibody binding site on the extracellular loop (domain I, S5-S6) of alpha(1D) L-type Ca channel.

Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have recently established that anti-SSA/Ro -SSB/La autoantibodies inhibit alpha(1D) L-type Ca current, I(Ca-L), and cross-react with the alpha(1D) Ca channel protein. This study aims at identifying the possible binding sites on alpha(1D) protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four alpha(1D) Ca channel protein domains I-IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera containing anti-Ro/La autoantibodies from 118 mothers with CHB children and from 15 mothers with anti-Ro/La autoantibodies but have healthy children, and from 28 healthy mothers without anti-Ro/La autoantibodies and healthy children were evaluated. Seventeen of 118 (14.4%) sera from mothers with CHB children reacted with the extracellular loop of domain I S5-S6 region (E1). In contrast, only 2 of 28 (7%) of sera from healthy mothers (-anti-Ro/La) and healthy children reacted with E1 loop and none (0 of 15) of sera from healthy mothers (+anti-Ro/La) and healthy children reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D reading establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera and purified IgG showed inhibition (44.1% and 49.8%, respectively) of the alpha(1D) I(Ca-L) expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel alpha(1D) subunit as a target for autoantibodies from a subset of mothers with CHB children. This novel finding provides insights into the potential development of therapeutic peptides that could bind to the pathogenic antibodies and prevent CHB.

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.

Silencing of Cav1.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA.

Cav1.2 (alpha1C) and Cav1.3 (alpha1D) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate alpha1D from alpha1C Ca currents (I(Ca-L)) in cardiomyocytes. Here, we established a physiological model to study alpha1D I(Ca-L) in native myocytes using RNA interference. Transfection of rat neonatal cardiomyocytes (RNC) with alpha1C specific siRNA resulted in low silencing efficiency (50-60%) at the mRNA and protein levels. The use of lentivirus shRNA resulted in 100% transfection efficiency and 92% silencing of the alpha1C gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total I(Ca-L) was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the cardiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of alpha1C and alpha1D Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings.

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.

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.