Ca2+/calmodulin kinase II-dependent regulation of βIV-spectrin modulates cardiac fibroblast gene expression, proliferation, and contractility

Abstract

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 β_IV-spectrin as an important step in CF activation and cardiac fibrosis. Furthermore, loss of β_IV-spectrin was found to depend on Ca²⁺/calmodulin-dependent kinase II (CaMKII). Therefore, we sought to determine the mechanism for CaMKII-dependent regulation of β_IV-spectrin and CF activity. Computational screening and MS revealed a critical serine residue (S2250 in mouse and S2254 in human) in β_IV-spectrin phosphorylated by CaMKII. Disruption of β_IV-spectrin/CaMKII interaction or alanine substitution of β_IV-spectrin Ser2250 (β_IV-S2254A) prevented CaMKII-induced degradation, whereas a phosphomimetic construct (β_IV-spectrin with glutamic acid substitution at serine 2254 [β_IV-S2254E]) showed accelerated degradation in the absence of CaMKII. To assess the physiological significance of this phosphorylation event, we expressed exogenous β_IV-S2254A and β_IV-S2254E constructs in β_IV-spectrin-deficient CFs, which have increased proliferation and fibrotic gene expression compared with WT CFs. β_IV-S2254A but not β_IV-S2254E normalized CF proliferation, gene expression, and contractility. Pathophysiological targeting of β_IV-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 β_IV-spectrin partially reversed these trends, β_IV-S2254A completely negated increased CF proliferation and signal transducer and activation of transcription 3 translocation. Moreover, we observed β_IV-spectrin phosphorylation and associated loss in total protein within human heart tissue following heart failure. Together, these data illustrate a considerable role for the β_IV-spectrin/CaMKII interaction in activating profibrotic signaling.

Keywords: STAT3; calmodulin kinase II; cardiac fibroblast; fibrosis; β(IV)-spectrin.