Assessing the interrelationship between asthma and obesity self-management behaviors.

Asthma and obesity are common coexisting conditions with increasing prevalence and substantial morbidity. This study examines the inter-relationship between illness and treatment beliefs in asthma and obesity and how they influence self-management behaviors. Overweight and obese adults ≥ 18 years with asthma were recruited from primary care and pulmonary practices in New York, NY and Denver, CO (n = 219). Path analysis was used to examine the relationship between asthma, weight and exercise-related illness and medication beliefs and SMB. Necessity beliefs about asthma medications and diet were associated with better medication adherence and healthier dietary behaviors (ß = 0.276, p = < 0.001, ß = 0.148, p = 0.018 respectively) whereas concerns about these self-care activities were associated with poorer adherence and worse dietary behaviors (ß = - 0.282, p < 0.001, ß = - 0.188, p = 0.003 respectively). We found no statistically significant association of exercise behaviors with any other weight or asthma illness or treatment beliefs. Our study demonstrates that necessity and concerns about treatment are associated with adherence in asthma and obesity. The lack of association of exercise behaviors with any asthma or weight related beliefs may reflect limited awareness of the impact of weight on asthma and warrants additional research.

The parabrachial to central amygdala pathway is critical to injury-induced pain sensitization in mice.

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PBN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. However, the functional significance of this pathway in the modulation of the somatosensory component of pain was recently challenged by studies showing that spinal nociceptive neurons do not target CeA-projecting PBN cells and that manipulations of this pathway have no effect on reflexive-defensive somatosensory responses to peripheral noxious stimulation. Here, we showed that activation of CeA-projecting PBN neurons is critical to increase both stimulus-evoked and spontaneous nociceptive responses following an injury in male and female mice. Using optogenetic-assisted circuit mapping, we confirmed a functional excitatory projection from PBN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increased the expression of the neuronal activity marker Fos in CeA-projecting PBN neurons and that chemogenetic inactivation of these cells decreased behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we showed that chemogenetic activation of CeA-projecting PBN neurons is sufficient to induced bilateral hypersensitivity without injury. Together, our results indicate that the PBN→CeA pathway is a key modulator of pain-related behaviors that can increase reflexive-defensive and affective-motivational responses to somatosensory stimulation in injured states without affecting nociception under normal physiological conditions.

The parabrachial to central amygdala circuit is a key mediator of injury-induced pain sensitization.

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PbN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. At the behavioral level, the PbN→CeA pathway has been proposed to serve as a general alarm system to potential threats that modulates pain-related escape behaviors, threat memory, aversion, and affective-motivational (but not somatosensory) responses to painful stimuli. Increased sensitivity to previously innocuous somatosensory stimulation is a hallmark of chronic pain. Whether the PbN→CeA circuit contributes to heightened peripheral sensitivity following an injury, however, remains unknown. Here, we demonstrate that activation of CeA-projecting PbN neurons contributes to injury-induced behavioral hypersensitivity but not baseline nociception in male and female mice. Using optogenetic assisted circuit mapping, we confirmed a functional excitatory projection from PbN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increases the expression of the neuronal activity marker c-Fos in CeA-projecting PbN neurons and chemogenetic inactivation of these cells reduces behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we show that chemogenetic activation of CeA-projecting PbN neurons is sufficient to induce bilateral hypersensitivity without injury. Together, our results demonstrate that the PbN→CeA pathway is a key modulator of pain-related behaviors that can amplify responses to somatosensory stimulation in pathological states without affecting nociception under normal physiological conditions. Significance Statement: Early studies identified the spino-ponto-amygdaloid pathway as a major ascending circuit conveying nociceptive inputs from the spinal cord to the brain. The functional significance of this circuit to injury-induced hypersensitivity, however, remains unknown. Here, we addressed this gap in knowledge using viral-mediated anatomical tracers, ex-vivo electrophysiology and chemogenetic intersectional approaches in rodent models of persistent pain. We found that activation of this pathway contributes to injury-induced hypersensitivity, directly demonstrating a critical function of the PbN→CeA circuit in pain modulation.

Intercalated disc protein Xinß is required for Hippo-YAP signaling in the heart.

Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinß, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinß in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinß mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinß knock-out mice-indicating a functional and genetic interaction between Xinß and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinß modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.

Pathways linking health literacy, health beliefs, and cognition to medication adherence in older adults with asthma.

BACKGROUND: Limited health literacy is associated with low adherence to asthma controller medications among older adults. OBJECTIVE: We sought to describe the causal pathway linking health literacy to medication adherence by modeling asthma illness and medication beliefs as mediators. METHODS: We recruited adults aged 60 years and older with asthma from hospital and community practices in New York, New York, and Chicago, Illinois. We measured health literacy and medication adherence using the Short Test of Functional Health Literacy in Adults and the Medication Adherence Rating Scale, respectively. We used validated instruments to assess asthma illness and medication beliefs. We assessed cognition using a cognitive battery. Using structural equation modeling, we modeled illness and medication beliefs as mediators of the relationship between health literacy and adherence while controlling for cognition. RESULTS: Our study included 433 patients with a mean age of 67 ± 6.8 years. The sample had 84% women, 31% non-Hispanic blacks, and 39% Hispanics. The 36% of patients with limited health literacy were more likely to have misconceptions about asthma (P < .001) and asthma medications (P < .001). Health literacy had a direct effect (ß = 0.089; P < .001) as well as an indirect effect on adherence mediated by medications concerns (ß = 0.033; P = .002). Neither medication necessity (ß = 0.044; P = .138) nor illness beliefs (ß = 0.007; P = .143) demonstrated a mediational role between health literacy and adherence. CONCLUSIONS: Interventions designed to improve asthma controller medication adherence in older adults may be enhanced by addressing concerns about medications in addition to using communication strategies appropriate for populations with limited health literacy and cognitive impairments.

Localization and function of Xinα in mouse skeletal muscle.

The Xin repeat-containing proteins were originally found in the intercalated discs of cardiac muscle with implicated roles in cardiac development and function. A pair of paralogous genes, Xinα (Xirp1) and Xinß (Xirp2), is present in mammals. Ablation of the mouse Xinα (mXinα) did not affect heart development but caused late-onset adulthood cardiac hypertrophy and cardiomyopathy with conductive defects. Both mXinα and mXinß are also found in the myotendinous junction (MTJ) of skeletal muscle. Here we investigated the structural and functional significance of mXinα in skeletal muscle. In addition to MTJ and the contact sites between muscle and perimysium, mXinα but not mXinß was found in the blood vessel walls, whereas both proteins were absent in neuromuscular junctions and nerve fascicles. Coimmunoprecipitation suggested association of mXinα with talin, vinculin, and filamin, but not ß-catenin, in adult skeletal muscle, consistent with our previous report of colocalization of mXinα with vinculin. Loss of mXinα in mXinα-null mice had subtle effects on the MTJ structure and the levels of several MTJ components. Diaphragm muscle of mXinα-null mice showed hypertrophy. Compared with wild-type controls, mouse extensor digitorum longus (EDL) muscle lacking mXinα exhibited no overt change in contractile and relaxation velocities or maximum force development but better tolerance to fatigue. Loaded fatigue contractions generated stretch injury in wild-type EDL muscle as indicated by a fragmentation of troponin T. This effect was blunted in mXinα-null EDL muscle. The results suggest that mXinα play a role in MTJ conductance of contractile and stretching forces.

Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms.

Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, expression and developmental regulation of myofilament proteins of mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues using a lacZ reporter gene driven by a cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images, and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in precise synchrony with that in the heart. However, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically pre-set developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart.

The intercalated disk protein, mXinalpha, is capable of interacting with beta-catenin and bundling actin filaments [corrected].

Targeted deletion of mXinalpha results in cardiac hypertrophy and cardiomyopathy with conduction defects (Gustafson-Wagner, E., Sinn, H. W., Chen, Y.-L., Wang, D.-Z., Reiter, R. S., Lin, J. L.-C., Yang, B., Williamson, R. A., Chen, J. N., Lin, C.-I., and Lin, J. J.-C. (2007) Am. J. Physiol. 293, H2680-H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXinalpha and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXinalpha directly interacts with beta-catenin. The beta-catenin-binding site on mXinalpha was mapped to amino acids 535-636, which overlaps with the known actin-binding domains composed of the Xin repeats. The overlapping nature of these domains provides insight into the molecular mechanism for mXinalpha localization and function. Purified recombinant glutathione S-transferase- or His-tagged mXinalpha proteins are capable of binding and bundling actin filaments, as determined by co-sedimentation and electron microscopic studies. The binding to actin was saturated at an approximate stoichiometry of nine actin monomers to one mXinalpha. A stronger interaction was observed between mXinalpha C-terminal deletion and actin as compared with the interaction between full-length mXinalpha and actin. Furthermore, force expression of green fluorescent protein fused to an mXinalpha C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXinalpha. These results suggest a model whereby the C terminus of mXinalpha may prevent the full-length molecule from binding to actin, until the beta-catenin-binding domain is occupied by beta-catenin. The binding of mXinalpha to beta-catenin at the adherens junction would then facilitate actin binding. In support of this model, we found that the actin binding and bundling activity of mXinalpha was enhanced in the presence of beta-catenin.

Loss of mXinalpha, an intercalated disk protein, results in cardiac hypertrophy and cardiomyopathy with conduction defects.

The intercalated disk protein Xin was originally discovered in chicken striated muscle and implicated in cardiac morphogenesis. In the mouse, there are two homologous genes, mXinalpha and mXinbeta. The human homolog of mXinalpha, Cmya1, maps to chromosomal region 3p21.2-21.3, near a dilated cardiomyopathy with conduction defect-2 locus. Here we report that mXinalpha-null mouse hearts are hypertrophied and exhibit fibrosis, indicative of cardiomyopathy. A significant upregulation of mXinbeta likely provides partial compensation and accounts for the viability of the mXinalpha-null mice. Ultrastructural studies of mXinalpha-null mouse hearts reveal intercalated disk disruption and myofilament disarray. In mXinalpha-null mice, there is a significant decrease in the expression level of p120-catenin, beta-catenin, N-cadherin, and desmoplakin, which could compromise the integrity of the intercalated disks and functionally weaken adhesion, leading to cardiac defects. Additionally, altered localization and decreased expression of connexin 43 are observed in the mXinalpha-null mouse heart, which, together with previously observed abnormal electrophysiological properties of mXinalpha-deficient mouse ventricular myocytes, could potentially lead to conduction defects. Indeed, ECG recordings on isolated, perfused hearts (Langendorff preparations) show a significantly prolonged QT interval in mXinalpha-deficient hearts. Thus mXinalpha functions in regulating the hypertrophic response and maintaining the structural integrity of the intercalated disk in normal mice, likely through its association with adherens junctional components and actin cytoskeleton. The mXinalpha-knockout mouse line provides a novel model of cardiac hypertrophy and cardiomyopathy with conduction defects.

Tropomyosin and caldesmon regulate cytokinesis speed and membrane stability during cell division.

The contractile ring and the cell cortex generate force to divide the cell while maintaining symmetrical shape. This requires temporal and spatial regulation of the actin cytoskeleton at these areas. We force-expressed misregulated versions of actin-binding proteins, tropomyosin and caldesmon, into cells and analyzed their effects on cell division. Cells expressing proteins that increase actomyosin ATPase, such as human tropomyosin chimera (hTM5/3), significantly speed up division, whereas cells expressing proteins that inhibit actomyosin, such as caldesmon mutants defective in Ca(2+)/calmodulin binding (CaD39-AB) and in cdk1 phosphorylation sites (CaD39-6F), divide slowly. hTM5 and hTM5/3-expressing cells lift one daughter cell off the substrate and twist. Furthermore, CaD39-AB- and CaD39-6F-expressing cells are sensitive to hypotonic swelling and show severe blebbing during division, whereas hTM5/3-expressing cells are resistant to hypotonic swelling and produce membrane bulges. These results support a model where Ca(2+)/calmodulin and cdk1 dynamically control caldesmon inhibition of tropomyosin-activated actomyosin to regulate division speed and to suppress membrane blebs.

Requirement of reversible caldesmon phosphorylation at P21-activated kinase-responsive sites for lamellipodia extensions during cell migration.

Caldesmon is believed to be one of the key regulators for actin dynamics and thereby cell polarity, membrane extension, and cell motility. We have shown previously that stress fiber formation and cell movement are severely impaired in the cells expressing human fibroblast caldesmon fragment defective in Ca2+/CaM binding sites. Both Ser458 and Ser489, adjacent to the Ca2+/CaM-binding sites, are phosphorylated by p21-activated kinase (PAK) in vitro. Here we report that Ser458 is phosphorylated in response to cell movement. We substituted Ser458 and Ser489 on C-terminal caldesmon (CaD39) with alanine or glutamic acid to mimic under-phosphorylated (CaD39-PAKA) or constitutively phosphorylated (CaD39-PAKE) caldesmon. In vitro, CaD39-PAKE, but not CaD39-PAKA, fails to inhibit myosin ATPase activity and exhibits reduced binding to Ca2+/CaM. When stably expressed in Chinese Hamster Ovary cells, both CaD39-PAKA and CaD39-PAKE incorporate into stress fibers and localize to the leading edge of the migrating cell. Expression of CaD39-PAKE, but not CaD39-PAKA, fails to protect stress fibers from cytochalasin depolymerization. However, both mutations inhibit cell polarization and lead to defects in membrane extension and cell migration. We conclude that phosphorylation of caldesmon by PAK is a dynamic process required to regulate actin dynamics and membrane protrusions in wound-induced cell migration.

1274 full-open reading frames of transcripts expressed in the developing mouse nervous system.

As part of the trans-National Institutes of Health (NIH) Mouse Brain Molecular Anatomy Project (BMAP), and in close coordination with the NIH Mammalian Gene Collection Program (MGC), we initiated a large-scale project to clone, identify, and sequence the complete open reading frame (ORF) of transcripts expressed in the developing mouse nervous system. Here we report the analysis of the ORF sequence of 1274 cDNAs, obtained from 47 full-length-enriched cDNA libraries, constructed by using a novel approach, herein described. cDNA libraries were derived from size-fractionated cytoplasmic mRNA isolated from brain and eye tissues obtained at several embryonic stages and postnatal days. Altogether, including the full-ORF MGC sequences derived from these libraries by the MGC sequencing team, NIH_BMAP full-ORF sequences correspond to approximately 20% of all transcripts currently represented in mouse MGC. We show that NIH_BMAP clones comprise 68% of mouse MGC cDNAs > or =5 kb, and 54% of those > or =4 kb, as of March 15, 2004. Importantly, we identified transcripts, among the 1274 full-ORF sequences, that are exclusively or predominantly expressed in brain and eye tissues, many of which encode yet uncharacterized proteins.

Caldesmon mutant defective in Ca(2+)-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility.

Despite intensive in vitro studies, little is known about the regulation of caldesmon (CaD) by Ca(2+)-calmodulin (Ca(2+)-CaM) in vivo. To investigate this regulation, a mutant was generated of the C-terminal fragment of human fibroblast CaD, termed CaD39-AB, in which two crucial tryptophan residues involved in Ca(2+)-CaM binding were each replaced with alanine. The mutation abolished most CaD39-AB binding to Ca(2+)-CaM in vitro but had little effect on in vitro binding to actin filaments and the ability to inhibit actin/tropomyosin-activated heavy meromyosin ATPase. To study the functional consequences of these mutations in vivo, we transfected an expression plasmid carrying CaD39-AB cDNA into Chinese hamster ovary (CHO) cells and isolated several clones expressing various amounts of CaD39-AB. Immunofluorescence microscopy revealed that mutant CaD39-AB was distributed diffusely throughout the cytoplasm but also concentrated at membrane ruffle regions. Stable expression of CaD39-AB in CHO cells disrupted assembly of stress fibers and focal adhesions, altered cell morphology, and slowed cell cycle progression. Moreover, CaD39-AB-expressing cells exhibited motility defects in a wound-healing assay, in both velocity and the persistence of translocation, suggesting a role for CaD regulation by Ca(2+)-CaM in cell migration. Together, these results demonstrate that CaD plays a crucial role in mediating the effects of Ca(2+)-CaM on the dynamics of the actin cytoskeleton during cell migration.

Isolation and sequencing of a novel tropomyosin isoform preferentially associated with colon cancer.

BACKGROUND & AIMS: Nonmuscle human tropomyosin (hTM) isoforms have distinct functions and may play important roles in various disease processes. METHODS: In an attempt to identify colon epithelial tropomyosin isoform, a complementary DNA library prepared from a human colon cancer cell line T84 was screened by an oligonucleotide probe complementary to messages of all known hTM isoforms. A novel clone called TC22 was obtained. The amino acid sequence of TC22 isoform is identical to isoform 5 (hTM5) apart from the C terminal domain, amino acids 222-247 coding the exon 9. RESULTS: Northern blot analysis showed that TC22 message is expressed in transformed epithelial cell lines and tumor tissues but not in normal epithelial cells. We developed a monoclonal antibody specific to TC22 isoform (TC22-4). By Western blot and immunoperoxidase assays, we analyzed 105 colonic specimens (fresh frozen and formalin fixed) from 96 patients with colon polyps (hyperplastic) or adenomas with or without dysplasia and cancer. Twenty-one of 22 (95%) of colon cancer specimens showed the presence of TC22, compared with only 1 of the 17 normal colon specimens and none of the 13 hyperplastic polyps (P < 0.0001). As assayed by immunoperoxidase staining, TC22 expression progressively increased in benign adenomatous polyps (35%) and polyps with mild and severe dysplasia (57% and 100%, respectively). CONCLUSIONS: We cloned and sequenced a novel hTM isoform, TC22, which is strongly associated with colonic neoplasia and carcinoma. TC22 may provide a useful biomarker for surveillance of colon cancer.