Original articleMyD88 mediated inflammatory signaling leads to CaMKII oxidation, cardiac hypertrophy and death after myocardial infarction
Highlights
► MyD88−/− mice have improved post-MI survival. ► MI induces TLR/MyD88 dependent inflammatory gene expression. ► MyD88−/− mice have reduced cardiac hypertrophy and fibrosis. ► MyD88−/− mice have reduced post-MI oxidation-dependent CaMKII activation.
Introduction
Myocardial infarction (MI) is marked by inflammatory gene expression [1], [2], [3], [4], [5] and is associated with increased reactive oxygen species (ROS) production, pathological myocardial hypertrophy, heart failure and increased mortality [6]. The multifunctional Ca2+/calmodulin dependent protein kinase II (CaMKII) has recently emerged as a MI and ROS-activated signaling molecule that regulates expression of inflammatory genes and affects adverse outcomes after MI [5], [7]. Cardiac-specific transgenic overexpression of CaMKII results in cardiac hypertrophy, heart failure and premature death [8], [9]. CaMKII becomes constitutively active by threonine-287 ‘autophosphorylation’ (phospho-CaMKII) and by oxidation of methionines 281/282 (ox-CaMKII) and constitutively active CaMKII appears to contribute to increased mortality in mice after MI [7], [10]. In contrast, inhibition of CaMKII activity can attenuate many of these MI-related adverse effects [7], [11]. We found that CaMKII modulates post-MI expression of genes involved in inflammation such as complement factor B (Cfb) [5]. Mice deficient in Cfb gene were protected from cardiac hypertrophy after an MI and had improved survival. Cfb and other proinflammatory genes, such as TNF-α are dependent on the NF-κB transcription factor and are induced through activation of toll-like receptors (TLRs), suggesting that TLR signaling may activate CaMKII and contribute to diverse adverse consequences to MI [5], [6].
TLRs are integral components of the innate immune system that recognize pathogens and sterile injury and elaborate the inflammatory response, including expression of cytokines, chemokines and complement pathways [12], [13]. TLR-4 deficient mice have reduced myocardial injury after ischemia-reperfusion [14], [15] and had reduced hypertrophy in pressure overload and MI models [16], [17]. Adenoviral infection with a dominant negative form of myeloid differentiation protein 88 (MyD88) was protective against hypertrophy, apoptosis and fibrosis in a rat model of aortic banding, [18] whereas in an ischemia-reperfusion model, MyD88 inhibition suppressed NF-κB induction and myocyte apoptosis [19]. Inhibition of NF-κB by cardiomyocyte-specific expression of a dominant-negative IκB mutant reduced cardiac hypertrophy in a myotrophin transgene mediated hypertrophy/heart failure model [20]. These studies suggest that following injury or stress, activation of the NF-κB pathway through TLR is pathologically important to the myocardium. Most TLRs function through an adapter MyD88 that has no known enzymatic activity, but is required for downstream signaling and activation of the NF-κB transcription factors [13]. TLR-3 does not require MyD88 for its signaling [21] whereas TLR-4, the predominant TLR isoform in myocardium [4], utilizes MyD88-dependent and MyD88-independent pathways for downstream signaling to NF-κB [22]. Loss of MyD88 severely compromises many TLR mediated inflammatory responses [23], [24]. MyD88 signaling is important in pathological responses to aortic banding and dilated cardiomyopathy [18], [19], [25], but little is known about the potential role of MyD88 in inflammatory responses after MI. Furthermore, potential molecular mechanisms underlying TLR/MyD88 signaling in post-MI hearts are not understood. Here we show a role of MyD88 in TLR-mediated inflammation and hypertrophy, while MyD88−/− mice are protected from MI-mediated increases in CaMKII oxidation and phosphorylation, myocardial hypertrophy, inflammation, cell death, fibrosis and mortality compared to wild type littermate controls.
Section snippets
Animals
C57BL6/J mice were obtained from Jackson Laboratories. MyD88−/− mice were developed by Shizuo Akira's group [23] in Japan and were kindly provided to us by Dr. M. Nedim Ince, University of Iowa. HLL mice were a generous gift from Drs. Timothy Blackwell and Fiona Yull, Vanderbilt University [5], [26]. All experiments with animals were done in accordance with the regulations put forth by the Institutional Animal Care and Use Committee of University of Iowa.
MI surgery and echocardiography
MI surgery and pre- and post-MI
MyD88 activates NF-κB and proinflammatory gene expression in post-MI hearts
NF-κB activation is a major determinant of inflammation after infection and injury [29]. MI causes increased NF-κB-mediated expression of pro-inflammatory genes. We used a transgenic NF-κB-luciferase reporter mouse [5], [26] to test activation of the NF-κB pathway in post-MI hearts. Post-MI induction of NF-κB was determined 1 and 7 days after MI (Fig. 1A). We observed an increase in the NF-κB activity in hearts at day 1 after MI that persisted through 7 days post-MI. Luciferase activity at these
CaMKII in post-MI hypertrophic and inflammatory signaling
MI causes profound changes in cardiac gene expression, including induction of pro-hypertrophic and pro-inflammatory genes. MI and heart failure are associated with increased CaMKII activity, ROS production, cardiac hypertrophy and inflammation [6]. We have previously shown that CaMKII regulates TLR regulated inflammatory gene expression in LPS treated cardiomyocytes and in post-MI hearts [5]. Inhibition of CaMKII [11] or ablation of its target gene complement factor B (Cfb) [5] improves post-MI
Conclusions
The TLR/MyD88 pathway is important for adverse responses to MI and contributes to increasing myocardial ox-CaMKII. These data suggest that MyD88 contributes to clinically important pro-inflammatory and pro-oxidant responses after MI.
Disclosures
Madhu V. Singh and Mark E. Anderson are named inventors on a patent application claiming to treat inflammatory heart disease by CaMKII inhibition.
Source of funding
This study was funded by the NIH grants R01 HL 079031, R01 HL 096652, and R01 HL 070250 to MEA, and NIH RR026293 to RMW. This work was also supported by the University of Iowa Research Foundation and, in part, by the Fondation Leducq Award to the Alliance for Calmodulin Kinase Signaling in Heart Disease.
Acknowledgments
We thank Kathy Zimmerman for expert technical help on mouse echocardiographic data acquisition and analyses and Jinying Yang for animal help. University of Iowa Central Microscopy Core Facility provided technical support and Virus Vector Core Facility provided Rac1DN adenovirus. We also thank Shari Lifson (Columbia University) for help with image analyses.
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