Cathleen Cover

Desmoplakin and Talin2 Are Novel mRNA Targets of Fragile X–Related Protein-1 in Cardiac Muscle

Rationale: The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied. Objective: To investigate the molecular mechanisms and targets of the muscle-specific fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish. Methods and Results: Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an up-regulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis. Conclusions: Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.

Desmoplakin and Talin2 Are Novel mRNA Targets of Fragile X–Related Protein-1 in Cardiac Muscle Whitman; Dsp and Tln2 Are mRNA Targets of FXR1 in the Heart

Rationale: The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied. Objective: To investigate the molecular mechanisms and targets of the muscle-specific fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish. Methods and Results: Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an up-regulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis. Conclusions: Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.

Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality.

Significance Modulation of actin filament architecture underlies a plethora of cellular processes including cell shape, division, adhesion, and motility. In heart muscle cells actin-containing thin filaments form highly organized structures with precisely regulated lengths. This precision is required for efficient interaction with myosin-containing filaments and provides the basis for contraction. The mechanism whereby heart muscle cells regulate thin filament assembly and its consequences for cardiac physiology are largely unknown. We discovered that Leiomodin 2 (Lmod2) elongates thin filaments to a proper length. Mice lacking Lmod2 have abnormally short thin filaments, experience severe contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy, and die at age ∼3 wk. Therefore, Lmod2 and proper thin filament lengths are essential for heart function. Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.

Band 3 and its peptides during aging, radiation exposure, and Alzheimer's disease: Alterations and self-recognition

An aging antigen, senescent cell antigen, resides on the 911 amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG autoantibody binding. Band 3 is a ubiquitous membrane transport protein found in the plasma membrane of diverse cell types and tissues, and in nuclear, mitochondrial, and golgi membranes. Band 3 in tissues such as brain performs the same functions as it does in red blood cells forming senescent cell antigen. Oxidation is a mechanism for generating senescent cell antigen. The aging antigenic sites reside on human band 3 map residues 538-554, and 812-830. Carbohydrate moieties are not required for the antigenicity or recognition of senescent cell antigen. Anion transport site were mapped to residues 588-594, 822-839, and 869-883. The aging vulnerable site which triggers the antigenic site and the transport sites of band 3 were mapped using overlapping synthetic peptides along the molecule. Naturally occurring autoantibodies to regions of band 3 comprising both senescent cell antigen and B cells producing these antibodies were demonstrated in the sera of normal, healthy individuals. The presence of these antibodies tend to increase with age. Individuals with autoimmune diseases (rheumatoid arthritis and systemic lupus erythematosus) have increased antibodies to senescent cell antigen peptides. Radiation exposure results in an increase in antibodies to peptides 588-602 which lies in a transport region containing the aging vulnerable site. Band 3 ages as cells and tissues age. Our studies, to date, indicate, that the anion transport ability of band 3 decreases in brains and lymphocytes from old mice. This decreased transport ability precedes obvious structural changes such as band 3 degradation and generation of SCA, and is the earliest change thus far detected in band 3 function. Other changes include a decreased efficiency of anion transport (decreased Vmax) in spite of an increase in number of anion binding sites (increased Km), decreased glucose transport, increased phosphorylation, increased degradation to smaller fragments as detected by quantitative binding of antibodies to band 3 breakdown products and residue 812-830, and binding of physiologic IgG autoantibodies in situ. The latter 3 findings indicate that post-translational changes occur. In Alzheimers Disease (AD), our results indicate that post-translational changes occur in band 3. These include decreased band 3 phosphorylation of a 25-28kD segment, increased degradation of band 3, alterations in band 3 recognized by antibodies, and decreased anion and glucose transport by blood cells. Serum autoantibodies were increased in AD patients compared to controls to band 3 peptide 822-839. This band 3 residue lies in an anion transport/binding region.

Inflammation and drug hepatotoxicity: Aggravation of injury or clean‐up mission?

Inflammatory mediators released by nonparenchymal inflammatory cells in the liver have been implicated in the progression of acetaminophen (APAP) hepatotoxicity. Among hepatic nonparenchymal inflammatory cells, we examined the role of the abundant natural killer (NK) cells and NK cells with T‐cell receptors (NKT cells) in APAP‐induced liver injury.

Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1

Background: Gap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear. Methods: To identify the mechanisms regulating gap junction remodeling in cardiac disease, we sought to identify the functional properties of FXR1 expression, direct targets of FXR1 in human left ventricle dilated cardiomyopathy (DCM) biopsy samples and mouse models of DCM through BioID proximity assay and RNA immunoprecipitation, how FXR1 regulates its targets through RNA stability and luciferase assays, and functional consequences of altering the levels of this important RNA-binding protein through the analysis of cardiac-specific FXR1 knockout mice and mice injected with 3xMyc-FXR1 adeno-associated virus. Results: FXR1 expression is significantly increased in tissue samples from human and mouse models of DCM via Western blot analysis. FXR1 associates with intercalated discs, and integral gap junction proteins Cx43 (connexin 43), Cx45 (connexin 45), and ZO-1 (zonula occludens-1) were identified as novel mRNA targets of FXR1 by using a BioID proximity assay and RNA immunoprecipitation. Our findings show that FXR1 is a multifunctional protein involved in translational regulation and stabilization of its mRNA targets in heart muscle. In addition, introduction of 3xMyc-FXR1 via adeno-associated virus into mice leads to the redistribution of gap junctions and promotes ventricular tachycardia, showing the functional significance of FXR1 upregulation observed in DCM. Conclusions: In DCM, increased FXR1 expression appears to play an important role in disease progression by regulating gap junction remodeling. Together this study provides a novel function of FXR1, namely, that it directly regulates major gap junction components, contributing to proper cell-cell communication in the heart.