Dataset Information

CaMKII phosphorylates βIV-spectrin in heart

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 βIV-spectrin as an important step in CF activation and cardiac fibrosis. Further, 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 mass spectrometry revealed a critical serine residue (S2250 in mouse, S2254 in human) in βIV-spectrin phosphorylated by CaMKII. Disruption of βIV-spectrin/CaMKII interaction or ablation of βIV-spectrin Ser2250 through alanine substitution (βIV-S2250A) prevented CaMKII-induced degradation, while a phospho-mimetic construct (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 to wild-type (WT). βIV-S2254A but not βIV-S2254E normalized proliferation and gene expression. Pathophysiologic targeting of βIV-spectrin phosphorylation and subsequent degradation was identified in CFs activated with the profibrotic ligand angiotensin II (AngII), resulting in increased proliferation and STAT3 nuclear accumulation. While therapeutic delivery of exogenous WT βIV-spectrin partially reversed these trends, βIV-S2254A completely negated increased CF proliferation and STAT3 translocation. Moreover, we observed βIV-spectrin phosphorylation and associated loss in total protein within human heart failure (HF) tissue. Together, these data illustrates a considerable role for the βIV-spectrin/CaMKII interaction in activating profibrotic signaling.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Chlorocebus Sabaeus Mus Musculus (mouse)

DISEASE(S): Endomyocardial Fibrosis

SUBMITTER: Ling Li

LAB HEAD: Thomas Hund

PROVIDER: PXD025776 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

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

Nassal Drew M DM Patel Nehal J NJ Unudurthi Sathya D SD Shaheen Rebecca R Yu Jane J Mohler Peter J PJ Hund Thomas J TJ

The Journal of biological chemistry 20210618 1

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 ...[more]

PMID: 34153319

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Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.

Project description:Background: Organ fibrosis due to excessive production of extracellular matrix (ECM) by resident fibroblasts is estimated to contribute to >45% of deaths in the Western world, including those due to cardiovascular diseases such as heart failure (HF). Here, we screened for small molecule inhibitors with a common ability to suppress activation of fibroblasts across organ systems. Methods: High content imaging of cultured cardiac, pulmonary and renal fibroblasts was employed to identify non-toxic compounds that blocked induction of markers of activation in response to the profibrotic stimulus, TGF-β. SW033291, which inhibits the eicosanoid-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), was chosen for follow-up studies with cultured adult rat ventricular fibroblasts (ARVFs) and human cardiac fibroblasts (CFs), and for evaluation in mouse models of cardiac fibrosis and diastolic dysfunction. Additional mechanistic studies were performed with CFs treated with exogenous eicosanoids. Results: Nine compounds, including SW033291, shared a common ability to suppress TGF-β-mediated activation of cardiac, pulmonary and renal fibroblasts. SW033291 dose-dependently inhibited TGF-β-induced expression of activation markers (e.g. α-smooth muscle actin and periostin) in ARVFs and normal human CFs, and reduced contractile capacity of the cells. Remarkably, the 15-PGDH inhibitor also reversed constitutive activation of fibroblasts obtained from explanted hearts from patients with HF. SW033291 blocked cardiac fibrosis induced by angiotensin II (Ang II) infusion and ameliorated diastolic dysfunction in an alternative model of systemic hypertension driven by combined uninephrectomy (UNX) and deoxycorticosterone acetate (DOCA) administration. Mechanistically, SW033291-mediated stimulation of ERK1/2 mitogen-activated protein kinase signaling was required for SW033291 to block CF activation. Of the 12 exogenous eicosanoids that were tested, only 12-hydroxyeicosatetraenoic acid (12[S]-HETE), which signals through the G protein-coupled receptor (GPCR), GPR31, recapitulated the suppressive effects of SW033291 on CF activation. Conclusions: Inhibition of degradation of eicosanoids, arachidonic acid-derived fatty acids that signal through GPCRs, is a potential therapeutic strategy for suppression of pathological organ fibrosis. In the heart, we propose that 15-PGDH inhibition triggers CF-derived autocrine/paracrine signaling by eicosanoids, including 12(S)-HETE, to stimulate ERK1/2 and block conversion of fibroblasts into activated cells that secrete excessive amounts of ECM and contribute of HF pathogenesis.

Dataset Information

CaMKII phosphorylates βIV-spectrin in heart

Publications

Ca<sup>2+</sup>/calmodulin kinase II-dependent regulation of β<sub>IV</sub>-spectrin modulates cardiac fibroblast gene expression, proliferation, and contractility.

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