Journal
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
Volume 54, Issue -, Pages 90-97Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.yjmcc.2012.11.012
Keywords
myofilament; regulation; titin; passive stiffness; CaMKII; diastolic function
Categories
Funding
- NIH [HL068821]
- NIH/NCRR [S10 RR028868-01]
- NATIONAL CANCER INSTITUTE [P30CA023074] Funding Source: NIH RePORTER
- NATIONAL CENTER FOR RESEARCH RESOURCES [S10RR028868] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL084579, R01HL062881, T32HL007249] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [P30ES006694] Funding Source: NIH RePORTER
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Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titin's elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKII delta, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titin's spring elements might be targeted by CaMKII delta. It was found that CaMKII delta phosphorylates titin in mouse LV skinned fibers, that the CaMKII delta sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKII delta sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKII delta at several conserved serine residues. Whether phosphorylation of titin by CaMKII delta occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKII delta sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKII delta sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKII delta, both in vitro and in vivo, and that titin's molecular spring region that determines diastolic stiffness is a target of CaMKII delta. (C) 2012 Elsevier Ltd. All rights reserved.
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