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| Autor: | Drew M, Nassal, Nehal J, Patel, Sathya D, Unudurthi, Rebecca, Shaheen, Jane, Yu, Peter J, Mohler, Thomas J, Hund |
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| Rok vydání: | 2020 |
| Předmět: |
Male
βIVifKO inducible fibroblast-specific βIV-spectrin knockout mouse βIV-spectrin FDR false discovery rate macromolecular substances CF cardiac fibroblast STAT3 qv3J mutant βIV-spectrin allele C-terminal region containing CaMKII interaction motif Mice βIV-S2254E βIV-spectrin with glutamic acid substitution at serine 2254 (phosphomimetic) cDNA complementary DNA NIH National Institutes of Health CHX cycloheximide FBS fetal bovine serum Chlorocebus aethiops Animals Phosphorylation Myofibroblasts Cells Cultured Cell Proliferation calmodulin kinase II DMEM Dulbecco's modified Eagle's medium Myocardium AngII angiotensin II fibrosis qPCR quantitative real-time PCR Spectrin cardiac fibroblast Endomyocardial Fibrosis Myocardial Contraction ECM extracellular matrix Mice Inbred C57BL qv4J mutant βIV-spectrin allele lacking repeats 10 through C terminus CaMKII Ca2+/calmodulin-dependent kinase II Amino Acid Substitution COS Cells STAT3 signal transducer and activation of transcription 3 Female Calcium-Calmodulin-Dependent Protein Kinase Type 2 Research Article DAPI 4′ 6-diamidino-2-phenylindole HA hemagglutinin MS/MS tandem MS/MS |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | Fibrosis is a pronounced feature of heart disease and the result of dysregulated activation of resident cardiac fibroblasts (CFs). Recent work identified stress-induced degradation of the cytoskeletal protein βIV-spectrin as an important step in CF activation and cardiac fibrosis. Furthermore, loss of βIV-spectrin was found to depend on Ca2+/calmodulin-dependent kinase II (CaMKII). Therefore, we sought to determine the mechanism for CaMKII-dependent regulation of βIV-spectrin and CF activity. Computational screening and MS revealed a critical serine residue (S2250 in mouse and S2254 in human) in βIV-spectrin phosphorylated by CaMKII. Disruption of βIV-spectrin/CaMKII interaction or alanine substitution of βIV-spectrin Ser2250 (βIV-S2254A) prevented CaMKII-induced degradation, whereas a phosphomimetic construct (βIV-spectrin with glutamic acid substitution at serine 2254 [βIV-S2254E]) showed accelerated degradation in the absence of CaMKII. To assess the physiological significance of this phosphorylation event, we expressed exogenous βIV-S2254A and βIV-S2254E constructs in βIV-spectrin-deficient CFs, which have increased proliferation and fibrotic gene expression compared with WT CFs. βIV-S2254A but not βIV-S2254E normalized CF proliferation, gene expression, and contractility. Pathophysiological targeting of βIV-spectrin phosphorylation and subsequent degradation was identified in CFs activated with the profibrotic ligand angiotensin II, resulting in increased proliferation and signal transducer and activation of transcription 3 nuclear accumulation. While therapeutic delivery of exogenous WT βIV-spectrin partially reversed these trends, βIV-S2254A completely negated increased CF proliferation and signal transducer and activation of transcription 3 translocation. Moreover, we observed βIV-spectrin phosphorylation and associated loss in total protein within human heart tissue following heart failure. Together, these data illustrate a considerable role for the βIV-spectrin/CaMKII interaction in activating profibrotic signaling. |
| Databáze: | OpenAIRE |
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