Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart
Madhu V. Singh, … , Peter J. Mohler, Mark E. Anderson
Madhu V. Singh, … , Peter J. Mohler, Mark E. Anderson
Published March 9, 2009
Citation Information: J Clin Invest. 2009;119(4):986-996. https://doi.org/10.1172/JCI35814.
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Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart

Abstract

Myocardial Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-κB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-κB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb–/– mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.

Authors

Madhu V. Singh, Ann Kapoun, Linda Higgins, William Kutschke, Joshua M. Thurman, Rong Zhang, Minati Singh, Jinying Yang, Xiaoqun Guan, John S. Lowe, Robert M. Weiss, Kathy Zimmermann, Fiona E. Yull, Timothy S. Blackwell, Peter J. Mohler, Mark E. Anderson

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Figure 5

CaMKII regulates LPS-stimulated Cfb expression through the NF-κB pathway.

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CaMKII regulates LPS-stimulated Cfb expression through the NF-κB pathway...
(A) Cultured neonatal cardiomyocytes were transfected with the NF-κB–luciferase reporter gene plasmid and treated with either PBS or LPS for 6 hours. Luciferase activity was measured and normalized to activity from a cotransfected Renilla luciferase plasmid. The data represent the mean ± SEM (n = 3). (B) EMSA showing NF-κB induction in cardiomyocytes upon LPS treatment. Cardiomyocytes were treated for 45 minutes with LPS and nuclear extracts were prepared. The specific NF-κB band is marked by the arrow. The lanes contain no nuclear extract (lane 1), untreated cells (lanes 2 and 3), LPS-treated cells (lanes 4 and 5), LPS-treated cells and 50- and 100-fold WT unlabeled competitor oligonucleotides (oligo) (lanes 6 and 7), and LPS-treated cells and 50- and 100-fold mutated unlabeled oligonucleotides (mut) (lanes 8 and 9). (C) A dominant-negative form of IκB (IκB-DN) attenuates LPS-induced Cfb mRNA expression. Control or dominant-negative IκB–expressing lentivirus particles were infected in cultured cardiomyocytes, followed by LPS induction for 12 hours. RNA was isolated and qRT-PCR performed. (D) In vivo CaMKII inhibition affects LPS-induced NF-κB activation. Luciferase activity of HLL or HLL crossed with CaMKIIN (HLL x CaMKIIN) hearts was measured 6 hours after intraperitoneal injection of LPS (2 μg/g body weight). Luciferase activity was normalized to the total protein concentration; at least 6 animals in each group were used in these experiments.