No abstract

Background-In ␣1-AR knockout (␣1ABKO) mice that lacked cardiac myocyte ␣1-adrenergic receptor (␣1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte ␣1-ARs and whether the ␣1A, ␣1B, or both subtypes mediated protection. Therefore, we investigated ␣1A and ␣1B subtype-specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of ␣1-ARs in cardiac myocytes. Methods and Results-We cultured ␣1ABKO myocytes and reconstituted ␣1-AR signaling with adenoviruses expressing ␣1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H 2 O 2 and was measured by annexin V/propidium iodide staining. In ␣1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the ␣1A subtype, but not the ␣1B, rescued ␣1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined ␣1-AR activation of extracellular signal-regulated kinase (ERK). In ␣1ABKO hearts, aortic constriction failed to activate ERK, and in ␣1ABKO myocytes, expression of a constitutively active MEK1 rescued ␣1ABKO myocytes from norepinephrine-induced death. In addition, only the ␣1A-AR activated ERK in ␣1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked ␣1A survival signaling in ␣1ABKO myocytes. Conclusions-Our results demonstrate a direct protective effect of the ␣1A subtype in cardiac myocytes and define an ␣1A-ERK signaling pathway that is required for myocyte survival. Absence of the ␣1A-ERK pathway can explain the failure to activate ERK after aortic constriction in ␣1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.

Abstract-We previously identified an ␣1-AR-ERK (␣1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of ␣1-AR subtypes (␣1A and ␣1B) and how their localization influences ␣1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent ␣1-AR antagonist, we localized endogenous ␣1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify ␣1 subtype localization, we reconstituted ␣1 signaling in cultured ␣1A-and ␣1B-AR double knockout cardiac myocytes using ␣1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous ␣1-ARs and ␣1A-and ␣1B-GFP colocalized with LAP2 at the nuclear membrane. ␣1-AR nuclear localization was confirmed in vivo using ␣1-AR-GFP transgenic mice. The ␣1-signaling partners G␣q and phospholipase C␤1 also colocalized with ␣1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that ␣1-mediated activation of ERK was not inhibited by a membrane impermeant ␣1-blocker, suggesting ␣1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe ␣1-AR localization to caveolae, but we found that ␣1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear ␣1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes. (Circ Res. 2008;103:992-1000.)Key Words: ␣1-adrenergic receptors Ⅲ cardiac myocytes Ⅲ ERK C ardiovascular disease is the leading killer in the United States, accounting for 1.4 million deaths a year. Five million Americans experience heart failure, leading to 970 000 hospitalizations annually, a number that has tripled in the last 25 years. 1 In heart failure, increased activation of the sympathetic nervous system is correlated with pathophysiologic remodeling of the heart, 2 which has led to the therapeutic use of ␤-adrenergic receptor (AR) antagonists in heart failure. However, the general conclusion that inhibition of catecholamine activation of ARs is beneficial in heart failure is disputed by clinical trials with ␣1-AR antagonists. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) demonstrated that the ␣1-antagonist doxazosin increased the risk of heart failure by 80% and stroke by 26% leading to termination of the trial. 3,4 Similar detrimental effects were seen in the VasodilatorHeart Failure Trials (V-HeFT), in which prazosin was compared with other vasodilators for the prevention of death in heart failure. 5 All 3 ␣1-AR subtypes (A, B, and D) are expressed in the heart 6 -9 ; however, cardiac myocytes only express the ␣1A and ␣1B. 9 Using ␣1A-and ␣1B-AR double knockout mice (␣1ABKO), we demonstrated previously that ␣1-ARs are required for postnatal hypertrophy and adaptation to pathological stress. 9,10 In ␣1ABKO mice, we found that aortic constriction induced dilated cardiomyopathy that le...

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCRs oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.

BackgroundRecent studies indicate that α1‐adrenergic receptors (α1‐ARs) are cardioprotective by preventing cardiac myocyte death and augmenting contractility in heart failure. Although G‐protein‐coupled receptors are assumed to localize to and signal at the plasma membrane, we previously demonstrated that endogenous α1‐ARs localize to the nuclei in adult cardiac myocytes. However, the functional consequence of this nuclear localization remains unclear. Here, we attempted to reconcile nuclear localization of α1‐ARs with their physiologic function by examining α1‐AR‐induced contractility in adult cardiac myocytes.Methods and ResultsBy measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the α1A‐subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an α1A‐subtype nuclear localization mutant in cardiac myocytes lacking α1‐ARs failed to rescue nuclear α1A‐mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked α1A‐mediated phosphorylation of cTnI. These data indicate that α1‐AR signaling originates in the nucleus. Consistent with these observations, we localized the α1A‐subtype to the inner nuclear membrane, identified PKCα, δ, and ε in the nucleus, and found that α1‐ARs activate PKCδ in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCδ nuclear localization mutant blunted α1‐induced phosphorylation of cTnI.ConclusionsTogether, our data identify a novel, “inside‐out” nuclear α1A‐subtype/PKCδ/cTnI‐signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of α1‐ARs and α1‐AR‐mediated physiologic function.

The ruminant conceptus undergoes a period of elongation that is required for maternal recognition of pregnancy, prior to attaching to the endometrium. The purpose of these studies was to investigate the role of proline-rich 15 (PRR15) in the sheep conceptus by examining mRNA expression, protein localization, and the effect of PRR15 mRNA degradation. Conceptuses were collected on Days 11, 13, 15, 16, 17, 21, and 30 after mating. Quantitative RT-PCR showed expression of PRR15 mRNA corresponded with the process of trophoblast elongation, with peak expression occurring on Days 15 and 16. A recombinant ovine PRR15 was generated and used to create polyclonal antibodies against PRR15. Immunohistochemistry of a Day 15 conceptus indicated that PRR15 was localized predominantly in the nucleus of the trophectoderm and extraembryonic primitive endoderm. To test whether PRR15 was required during early conceptus development, RNA interference was employed. Blastocysts collected on Day 8 after mating were infected with a lentivirus expressing a short-hairpin RNA (shRNA) that targeted PRR15 mRNA for degradation, an shRNA containing a three-nucleotide mismatch to PRR15 mRNA, or a lentivirus expressing no shRNA. After infection, blastocysts were transferred into recipient ewes and collected back on Day 15 of gestation. Although the majority of the control and mismatched shRNA-treated conceptuses elongated and survived to Day 15, none of the embryos treated with the lentivirus expressing shRNA against PRR15 mRNA elongated, and most died. In conclusion, expression of PRR15 mRNA occurred during a narrow window of conceptus development, and degradation of PRR15 mRNA led to conceptus demise or abnormal development.

Background Fear and anxiety are important considerations in both acute and chronic pain. Effectively and efficiently measuring fear and anxiety associated with pain in healthcare settings is critical for identifying vulnerable patients. The length and administration time of current measures of pain‐related fear and anxiety inhibit their routine use, as screening tools and otherwise, suggesting the need for a shorter, more efficient instrument. Methods A 9‐item shortened version of the Fear of Pain Questionnaire – III ( FPQ ‐ III ), the Fear of Pain Questionnaire‐9 ( FPQ ‐9), was developed based upon statistical analyses of archival data from 275 outpatients with chronic pain and 275 undergraduates. Additionally, new data were collected from 100 outpatients with chronic pain and 190 undergraduates to directly compare the standard and short forms. Exploratory and confirmatory factor analyses, and other psychometric analyses, were conducted to examine and establish the FPQ ‐9 as a reliable and valid instrument. Results The original three‐factor structure of the FPQ ‐ III was retained in the shortened version; a confirmatory factor analysis produced good model fit ( RMSEA = 0.00, CFI = 1.00, TLI = 1.00, SRMR = 0.03). Results suggested a high degree of correlation between the original FPQ ‐ III and the new FPQ ‐9 ( r = 0.77, p < 0.001). Measures of internal consistency for FPQ ‐9 subscales were high; correlations with other pain and anxiety instruments suggested concurrent, convergent and divergent validity. Conclusions The FPQ ‐9 is a psychometrically sound alternative to longer instruments assessing fear and anxiety associated with pain, for use in both clinical and research situations that only allow brief screening. Significance The FPQ ‐9 has considerable potential for dissemination and utility for routine, brief screening, given its length (completion time ~2 min; scoring time ~1 min), reading level and psychometric properties.

Objectives: Dental utilization is an important determinant of oral health and wellbeing. The aim of this study was to evaluate potential associations between a variety of biopsychosocial factors and dental utilization in north-central Appalachia, USA, a region where oral health disparities are profound. Methods: This study used household-based data from the Center for Oral HealthResearch in Appalachia (COHRA1) study in north-central Appalachia, including 449 families with 868 adults. The generalized estimating equation (GEE) approach was used to determine the best-fitting predictor model for dental utilization among adult family members. Results:On average across West Virginia and Pennsylvania, having dental insurance was associated with greater dental utilization over a 3-year time period (OR = 2.20, 95% CI = 1.54, 3.14). When stratified by state, the association held for only West Virginia (OR = 2.41, 95% CI = 1.54, 3.79) and was nonsignificant for Pennsylvania residents (OR = 1.50, 95% CI = 0.80, 2.79). Individuals from Pennsylvania were more likely to utilize dental care and participants from West Virginia less so (2.31, 95% CI = 1.57, 3.40). Females from Pennsylvania were more likely than males to regularly