Qinchuan Wang

Chapter 1: roles of caldesmon in cell motility and actin cytoskeleton remodeling.

Caldesmon (CaD) is a multimodular protein that regulates contractility and actin cytoskeleton remodeling in smooth muscle and nonmuscle cells. A single gene (CALD1) encodes high molecular mass CaD (h-CaD) and low molecular mass CaD (l-CaD) by alternative splicings. The h-CaD exclusively expresses in smooth muscle, whereas the l-CaD ubiquitously expresses in all cell types except skeletal muscle. The h-CaD/l-CaD ratio could be a marker for monitoring differentiating and pathological states of smooth muscles. The l-CaD associates with stress fibers and membrane ruffles in nonmuscle cells and with the actin core of podosomes in highly motile/invasive cells. Together with tropomyosin, CaD stabilizes actin filaments and inhibits actin-tropomyosin-activated myosin ATPase activity. This inhibition can be effectively released by Ca(2+)-calmodulin and/or by phosphorylation with various kinases. Through its interactions with a spectrum of actin-binding proteins, CaD modulates dynamics of cortical actin networks and stress fibers, which are essential to cell motility and cytoskeleton rearrangement. Regulation of CaD level and its activity may provide a novel strategy for gene therapy.

The Intercalated Disc Protein, mXinα, Is Capable of Interacting with β-Catenin and Bundling Actin Filaments

Targeted deletion of mXinα 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 mXinα and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXinα directly interacts with β-catenin. The β-catenin-binding site on mXinα 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 mXinα localization and function. Purified recombinant glutathione S-transferase- or His-tagged mXinα 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 mXinα. A stronger interaction was observed between mXinα C-terminal deletion and actin as compared with the interaction between full-length mXinα and actin. Furthermore, force expression of green fluorescent protein fused to an mXinα C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXinα. These results suggest a model whereby the C terminus of mXinα may prevent the full-length molecule from binding to actin, until the β-catenin-binding domain is occupied by β-catenin. The binding of mXinα to β-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 mXinα was enhanced in the presence of β-catenin.

Identification of cis Elements in the Cardiac Troponin T Gene Conferring Specific Expression in Cardiac Muscle of Transgenic Mice

To investigate the underlying mechanism regulating cardiac gene expression, transgenic mice carrying the rat cardiac troponin T proximal promoter (-497 bp from the transcriptional start site) fused to a LacZ or chloramphenicol acetyltransferase (CAT) reporter gene were analyzed. The LacZ expression pattern throughout development was very similar to that of the endogenous cardiac troponin T gene. Within this promoter, a high degree of sequence homology was found at 2 sites, modules D (-335 to -289 bp) and F (-249 to -209 bp). Both regions contain at least a TCTG(G/C) direct repeat and an A/T-rich site, whereas only the F module has a muscle enhancer factor 2 (MEF2)-like motif. No significant decrease in CAT transgene expression was observed when only the MEF2 core sequence was mutated. However, when the MEF2 core sequence and its flanking TCTGG site were mutated (Mut5), CAT transgene expression was significantly decreased in the heart, and ectopic expression of the transgene was also observed. When mutations were introduced into this promoter to destroy all upstream TCTG(G/C) direct repeats in the D module (MutD), CAT expression remained cardiac specific, but the expression level was dramatically decreased. Relaxation of cardiac-specific transgene expression became even more severe in transgenic mice carrying double mutations (Mut[D+5]). In addition, CAT activity in the heart was nearly abolished. These results suggest that D and F modules have an additive function in determining the level of expression in the heart and only the F module confers cardiac-specific expression.

Essential Roles of an Intercalated Disc Protein, mXinβ, in Postnatal Heart Growth and Survival

Rationale: The Xin repeat-containing proteins mXin&agr; and mXin&bgr; localize to the intercalated disc of mouse heart and are implicated in cardiac development and function. The mXin&agr; directly interacts with &bgr;-catenin, p120-catenin, and actin filaments. Ablation of mXin&agr; results in adult late-onset cardiomyopathy with conduction defects. An upregulation of the mXin&bgr; in mXin&agr;-deficient hearts suggests a partial compensation. Objective: The essential roles of mXin&bgr; in cardiac development and intercalated disc maturation were investigated. Methods and Results: Ablation of mXin&bgr; led to abnormal heart shape, ventricular septal defects, severe growth retardation, and postnatal lethality with no upregulation of the mXin&agr;. Postnatal upregulation of mXin&bgr; in wild-type hearts, as well as altered apoptosis and proliferation in mXin&bgr;-null hearts, suggests that mXin&bgr; is required for postnatal heart remodeling. The mXin&bgr;-null hearts exhibited a misorganized myocardium as detected by histological and electron microscopic studies and an impaired diastolic function, as suggested by echocardiography and a delay in switching off the slow skeletal troponin I. Loss of mXin&bgr; resulted in the failure of forming mature intercalated discs and the mislocalization of mXin&agr; and N-cadherin. The mXin&bgr;-null hearts showed upregulation of active Stat3 (signal transducer and activator of transcription 3) and downregulation of the activities of Rac1, insulin-like growth factor 1 receptor, protein kinase B, and extracellular signal-regulated kinases 1 and 2. Conclusions: These findings identify not only an essential role of mXin&bgr; in the intercalated disc maturation but also mechanisms of mXin&bgr; modulating N-cadherin-mediated adhesion signaling and its crosstalk signaling for postnatal heart growth and animal survival.

Quantitative phosphoproteomic study of pressure-overloaded mouse heart reveals dynamin-related protein 1 as a modulator of cardiac hypertrophy

Pressure-overload stress to the heart causes pathological cardiac hypertrophy, which increases the risk of cardiac morbidity and mortality. However, the detailed signaling pathways induced by pressure overload remain unclear. Here we used phosphoproteomics to delineate signaling pathways in the myocardium responding to acute pressure overload and chronic hypertrophy in mice. Myocardial samples at 4 time points (10, 30, 60 min and 2 weeks) after transverse aortic banding (TAB) in mice underwent quantitative phosphoproteomics assay. Temporal phosphoproteomics profiles showed 360 phosphorylation sites with significant regulation after TAB. Multiple mechanical stress sensors were activated after acute pressure overload. Gene ontology analysis revealed differential phosphorylation between hearts with acute pressure overload and chronic hypertrophy. Most interestingly, analysis of the cardiac hypertrophy pathway revealed phosphorylation of the mitochondrial fission protein dynamin-related protein 1 (DRP1) by prohypertrophic kinases. Phosphorylation of DRP1 S622 was confirmed in TAB-treated mouse hearts and phenylephrine (PE)-treated rat neonatal cardiomyocytes. TAB-treated mouse hearts showed phosphorylation-mediated mitochondrial translocation of DRP1. Inhibition of DRP1 with the small-molecule inhibitor mdivi-1 reduced the TAB-induced hypertrophic responses. Mdivi-1 also prevented PE-induced hypertrophic growth and oxygen consumption in rat neonatal cardiomyocytes. We reveal the signaling responses of the heart to pressure stress in vivo and in vitro. DRP1 may be important in the development of cardiac hypertrophy.

Xin proteins and intercalated disc maturation, signaling and diseases

Intercalated discs (ICDs) are cardiac-specific structures responsible for mechanical and electrical communication among adjacent cardiomyocytes and are implicated in signal transduction. The striated muscle-specific Xin repeat-containing proteins localize to ICDs and play critical roles in ICD formation and cardiac function. Knocking down the Xin gene in chicken embryos collapses the wall of developing heart chambers and leads to abnormal cardiac morphogenesis. In mammals, a pair of paralogous genes, Xinalpha and Xinbeta exist. Ablation of the mouse Xinalpha (mXinalpha) does not affect heart development. Instead, mXinalpha-deficient mice show adult late-onset cardiac hypertrophy and cardiomyopathy with conduction defects. The mXinalpha-deficient hearts up-regulate mouse Xinbeta (mXinbeta, suggesting a partial compensatory role of mXinbeta. Complete loss of mXinbeta however, leads to failure of forming ICD, mis-localization of mXinalpha, and early postnatal lethality. In this review, we will briefly discuss recent advances in the anatomy and function of ICDs. We will then review what we know about Xin repeat-containing proteins and how this protein family promotes ICD maturation and stability for normal cardiac function.

The Xin repeat-containing protein, mXinβ, initiates the maturation of the intercalated discs during postnatal heart development.

The intercalated disc (ICD) is a unique structure to the heart and plays vital roles in communication and signaling among cardiomyocytes. ICDs are formed and matured during postnatal development through a profound redistribution of the intercellular junctions, as well as recruitment and assembly of more than 200 proteins at the termini of cardiomyocytes. The molecular mechanism underlying this process is not completely understood. The mouse orthologs (mXinα and mXinβ) of human cardiomyopathy-associated (CMYA)/Xin actin-binding repeat-containing protein (XIRP) genes (CMYA1/XIRP1 and CMYA3/XIRP2, respectively) encode proteins localized to ICDs. Ablation of mXinα results in adult late-onset cardiomyopathy with conduction defects and up-regulation of mXinβ. ICD structural defects are found in adult but not juvenile mXinα-null hearts. On the other hand, loss of mXinβ leads to ICD defects at postnatal day 16.5, a developmental stage when the heart is forming ICDs, suggesting mXinβ is required for ICD formation. Using quantitative Western blot, we showed in this study that mXinβ but not mXinα was uniquely up-regulated during the redistribution of intercellular junction from the lateral membrane of cardiomyocytes to their termini. In the absence of mXinβ, the intercellular junctions failed to be restricted to the termini of the cells, and the onset of such defect correlated with the peak expression of mXinβ. Immunofluorescence staining and subcellular fractionation showed that mXinβ preferentially associated with the forming ICDs, further suggesting that mXinβ functioned locally to promote ICD maturation. In contrast, the spatiotemporal expression profile of mXinα and the lack of more severe ICD defects in mXinα-/-;mXinβ-/- double knockout hearts than in mXinβ-/- hearts suggested that mXinα was not essential for the postnatal formation of ICDs. A two-step model for the development of ICD is proposed where mXinβ is essential for the redistribution of intercellular junction components from the lateral puncta to the cell termini.

Emergence of Xin Demarcates a Key Innovation in Heart Evolution

The mouse Xin repeat-containing proteins (mXinα and mXinβ) localize to the intercalated disc in the heart. mXinα is able to bundle actin filaments and to interact with β-catenin, suggesting a role in linking the actin cytoskeleton to N-cadherin/β-catenin adhesion. mXinα-null mouse hearts display progressively ultrastructural alterations at the intercalated discs, and develop cardiac hypertrophy and cardiomyopathy with conduction defects. The up-regulation of mXinβ in mXinα-deficient mice suggests a partial compensation for the loss of mXinα. To elucidate the evolutionary relationship between these proteins and to identify the origin of Xin, a phylogenetic analysis was done with 40 vertebrate Xins. Our results show that the ancestral Xin originated prior to the emergence of lamprey and subsequently underwent gene duplication early in the vertebrate lineage. A subsequent teleost-specific genome duplication resulted in most teleosts encoding at least three genes. All Xins contain a highly conserved β-catenin-binding domain within the Xin repeat region. Similar to mouse Xins, chicken, frog and zebrafish Xins also co-localized with β-catenin to structures that appear to be the intercalated disc. A putative DNA-binding domain in the N-terminus of all Xins is strongly conserved, whereas the previously characterized Mena/VASP-binding domain is a derived trait found only in Xinαs from placental mammals. In the C-terminus, Xinαs and Xinβs are more divergent relative to each other but each isoform from mammals shows a high degree of within-isoform sequence identity. This suggests different but conserved functions for mammalian Xinα and Xinβ. Interestingly, the origin of Xin ca. 550 million years ago coincides with the genesis of heart chambers with complete endothelial and myocardial layers. We postulate that the emergence of the Xin paralogs and their functional differentiation may have played a key role in the evolutionary development of the heart.

XIRP2, an Actin-Binding Protein Essential for Inner Ear Hair-Cell Stereocilia

Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN) are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.

New insights into the roles of Xin repeat-containing proteins in cardiac development, function, and disease.

Since the discovery of Xin repeat-containing proteins in 1996, the importance of Xin proteins in muscle development, function, regeneration, and disease has been continuously implicated. Most Xin proteins are localized to myotendinous junctions of the skeletal muscle and also to intercalated discs (ICDs) of the heart. The Xin gene is only found in vertebrates, which are characterized by a true chambered heart. This suggests that the evolutionary origin of the Xin gene may have played a key role in vertebrate origins. Diverse vertebrates including mammals possess two paralogous genes, Xinα (or Xirp1) and Xinβ (or Xirp2), and this review focuses on the role of their encoded proteins in cardiac muscles. Complete loss of mouse Xinβ (mXinβ) results in the failure of forming ICD, severe growth retardation, and early postnatal lethality. Deletion of mouse Xinα (mXinα) leads to late-onset cardiomyopathy with conduction defects. Molecular studies have identified three classes of mXinα-interacting proteins: catenins, actin regulators/modulators, and ion-channel subunits. Thus, mXinα acts as a scaffolding protein modulating the N-cadherin-mediated adhesion and ion-channel surface expression. Xin expression is significantly upregulated in early stages of stressed hearts, whereas Xin expression is downregulated in failing hearts from various human cardiomyopathies. Thus, mutations in these Xin loci may lead to diverse cardiomyopathies and heart failure.