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Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-β2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-β2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-β2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-β2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis. [ABSTRACT FROM AUTHOR]

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease (1,2), but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) (3-5) or pattern (for example, Brugada's syndrome) (6) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, kruppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current (7). Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis., Sudden cardiac death from ventricular arrhythmias is the principal cause of mortality from heart disease worldwide and remains a major unresolved public health problem. The incidence of sudden cardiac death [...]

The ubiquitinproteasome system degrades most intracellular proteins, including misfolded proteins. Proteasome functional insufficiency (PFI) has been observed in proteinopathies, such as desminrelated cardiosympathy, and implicated in many common diseases, including dilated cardiomyopathy and ischemic heart disease. However, the pathogenic role of PFI has not been established. Here we created inducible Tg mice with cardiomyocyterestricted overexpression of proteasome 28 subunit α (CR-PA28αOE) to investigate whether upregulation of the 11S proteasome enhances the proteolytic function of the proteasome in mice and, if so, whether the enhancement can rescue a bona fide proteinopathy and protect against ischemia/reperfusion (I/R) injury. We found that CR-PA28αOE did not alter the homeostasis of normal proteins and cardiac function, but did facilitate the degradation of a surrogate misfolded protein in the heart. By breeding mice with CR-PA28αOE with mice representing a well-established model of desminrelated cardiomyopathy, we demonstrated that CR-PA28αOE markedly reduced aberrant protein aggregation. Cardiac hypertrophy was decreased, and the lifespan of the animals was increased. Furthermore, PA28αknockdown promoted, whereas PA28α overexpression attenuated, accumulation of the mutant protein associated with desminrelated cardiomyopathy in cultured cardiomyocytes. Moreover, CR-PA28αOE limited infarct size and prevented postreperfusion cardiac dysfunction in mice with myocardial I/R injury. We therefore conclude that benign enhancement of cardiac proteasome proteolytic function can be achieved by CR-PA28αOE and that PFI plays a major pathogenic role in cardiac proteinopathy and myocardial I/R injury., Introduction The ubiquitin-proteasome system (UPS) mediates the targeted degradation of abnormal and most normal intracellular proteins in the cell and generally includes 2 main steps: ubiquitination of a specific protein [...]

The neuropeptide hormone arginine-vasopressin (AVP) is well known to exert its antidiuretic effect via the vasopressin V2 receptor (V2R), whereas the role of the vasopressin Vla receptor (V1aR) in the kidney remains to be clarified. Previously, we reported decreased plasma volume and blood pressure in V1a receptor-deficient ([V1aR.sup.-/-]) mice (Koshimizu T, Nasa Y, Tanoue A, Oikawa R, Kawahara Y, Kiyono Y, Adachi T, Tanaka T, Kuwaki T, Mod T. Proc Natl Acad Sci USA 103: 7807-7812, 2006). In this study, we investigated the role of V 1 aR in urine concentration, renal function, and the renin-angiotensin system (RAS) using [V1aR.sup.-/-] mice. Urine volume of [V1aR.sup.-/-] mice was greater than that of wild-type mice, particularly when water was loaded, while the glomerular filtration rate (GFR), urinary NaCl excretion, AVP-dependent cAMP generation, V2R, and aquaporin 2 (AQP2) expression in the kidney were lower, indicating that the diminished GFR and V2R-AQP2 system led to impaired urinary concentration in [V1aR.sup.-/-] mice. Since the GFR and V2R-AQP2 system are regulated by RAS, we analyzed renin and angiotensin II in [V1aR.sup.-/-] mice and found that the plasma renin and angiotensin II were decreased. The expression of renin in granule cells was decreased in [V1aR.sup.-/-] mice, which led to a decreased level of plasma renin. In addition, the expression of renin stimulators such as neuronal nitric oxide synthase and cyclooxygenase-2 in macula densa (MD) cells, where V1aR was specifically expressed, was decreased in [V1aR.sup.-/-] mice. These data indicate that AVP regulates body fluid homeostasis and GFR via the V1aR in MD cells by activating RAS and subsequently the V2R-AQP2 system. renin-angiotensin system

[[Arg.sup.8]]-vasopressin (Avp), a nonapeptide hormone, is known to regulate blood pressure, water balance, and a variety of behaviors such as anxiety, aggression, and bonding. Although some evidence that Avp modifies ethanol consumption and some of the effects of ethanol on behavior have been reported, the role of Avp in alcohol consumption and preference is poorly understood. The Avp1a receptor (Avpr1a) is ubiquitously expressed in the central nervous system. To determine the role of Avp signaling on the behavioral effects of alcohol, we examined voluntary ethanol consumption in mice with targeted disruptions of the Avpr1a knockout (Avpr1a KO) gene. Avpr1a KO mice displayed both increased ethanol consumption and preference compared with wild-type (WT) mice. Enhanced ethanol consumption was dramatically and reversibly reduced by treatment with N-methyl-D-aspartic acid antagonists. Basal glutamate release was elevated around the striatum in Avpr1a KO mice. Elevation of extracellular glutamate was also produced in WT mice by local application of an Avpr1a antagonist though a dialysis probe, and this elevation was quickly reversed by stopping the perfusion. These results suggest that Avp can inhibit the release of glutamate from the presynaptic terminal via the Avp1a receptor and that elevation of glutamate levels owing to loss of the inhibitory effect via Avp-Avpr1a signaling may play an important role in the preference for ethanol. [[Arg.sup.8]]-vasopressin

Attenuation of early restenosis after percutaneous coronary intervention (PCI) is important for the successful treatment of coronary artery disease. Some clinical studies have shown that hypertension is a risk factor for early restenosis after PCI. These findings suggest that [[alpha].sub.1]-adrenergic receptors ([[alpha].sub.1]-ARs) may facilitate restenosis after PCI because of [[alpha].sub.1]-AR's remarkable contribution to the onset of hypertension. In this study, we examined the neointimal formation after vascular injury in the femoral artery of [[alpha].sub.1A]-knockout ([[alpha].sub.1A]-KO), [[alpha].sub.1B]-KO, [[alpha].sub.1D]-KO, [[alpha].sub.1A]-/[[alpha].sub.1B]-AR double-KO ([[alpha].sub.1AB]-KO), and wild-type mice to investigate the functional role of each [[alpha].sub.1]-AR subtype in neointimal formation, which is known to promote restenosis. Neointimal formation 4 wk after wire injury was significantly (P < 0.05) smaller in [[alpha].sub.1AB]-KO mice than in any other group of mice, while blood pressures were not altered in any of the groups of mice after wire injury compared with those before it. These results suggest that lack of both [[alpha].sub.1A]- and [[alpha].sub.1B]-ARs could be necessary to inhibit neointimal formation in the mouse femoral artery. [[alpha].sub.1]-adrenergic receptor: vascular injury; smooth muscle cell: femoral artery of mouse; wire injury doi:10.1152/ajpheart.00626.2006

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved [Ca.sup.2+] sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte [Ca.sup.2+] transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of [Ca.sup.2+] waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node | [Ca.sup.2+] imaging | genetic sensor | heart development | fluorescent [Ca.sup.2+] sensor

ANG II type 2 receptor (A[T.sub.2]) is upregulated in failing hearts, but its effect on myocyte contractile function is not known. We measured fractional cell shortening and intracellular [Ca.sup.2+] concentration transients in left ventricular myocytes derived from transgenic mice in which ventricle-specific expression of A[T.sub.2] was driven by the myosin light chain 2v promoter. Confocal microscopy studies confirmed upregulation of A[T.sub.2] in the ventricular myocytes and partial colocalization of A[T.sub.2] with A[T.sub.1]. Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/l extracellular [Ca.sup.2+] concentration, 25[degrees]C) and study of contractile reserve at faster pacing rates (1-5 Hz) revealed [Ca.sup.2+]-dependent contractile dysfunction in myocytes from A[T.sub.2] transgenic mice. Comparison of two transgenic lines suggested a dose-dependent relationship between magnitude of contractile dysfunction and level of A[T.sub.2] expression. Second, activity of the [Na.sup.+]/[H.sup.+] exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to [beta]-adrenergic versus ANG II stimulation differed. Both lines showed impaired contractile response to [beta]-adrenergic stimulation. ANG II elicited an increase in contractility and intracellular [Ca.sup.2+] in wild-type myocytes but caused a negative inotropic effect in myocytes from A[T.sub.2] transgenic mice. In contrast with [beta]-adrenergic response, the depressed response to ANG II was related to level of A[T.sub.2] overexpression. The depressed response to ANG II was also present in myocytes from young transgenic mice before development of heart failure. Thus chronic overexpression of A[T.sub.2] has the potential to cause [Ca.sup.2+]- and pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to ANG II. contractility; calcium ion transients; pH

Yan, Xinhua, Robert L. Price, Masaharu Nakayama, Kenta Ito, Adam J. T. Schuldt, Warren J. Manning, Atsushi Sanbe, Thomas K. Borg, Jeffrey Robbins, and Beverly H. Lorell. Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice. Am J Physiol Heart Circ Physiol 285: H2179-H2187, 2003. First published July 17, 2003; 10.1152/ajpheart.00361.2003.-The angiotensin II type 2 [(AT.sub.2)] receptor is upregulated in the left ventricle in heart failure, but its pathophysiological roles in vivo are not understood. In the present study, [AT.sub.2] receptors were expressed in transgenic (TG) mice using the ventricular-specific myosin light-chain (MLC-2v) promoter. In TG compared with nontransgenic (NTG) mice, in vivo left ventricular (LV) systolic pressure and peak +dP/dt were depressed while LV diastolic pressure was elevated (P < 0.05). Echocardiography showed severely depressed LV fractional shortening, increased systolic and diastolic dimensions, and wall thinning (P < 0.05). Confocal and electron microscopy studies revealed an increase in the size of myocytes and interstitial spaces as well as an increase in interstitial collagen, disruption of the Z-band, and changes in cytochrome c localization. The changes were most prominent in the highest-expressing TG line, which implies a dose-response relationship. [AT.sub.2] overexpression was also directly associated with the increase of phosphotTlated protein levels of PKC-[alpha], PKC-[beta], and p70S6 kinase. These data demonstrate that ventricular myocytespecific expression of [AT.sub.2] receptors promotes the development of dilated cardiomyopathy and heart failure in vivo. myosin light-chain promoter; cardiac disease; myocyte