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Title

THE STRUCTURAL BASIS OF MYOFILAMENT LENGTH DEPENDENT ACTIVATION

Creator

Hsu, Karen Hsiaoman

Date

2014, 2014-12

Description

Titin-based passive tension is one of the mechanisms that modulate myofilament length dependent activation, the phenomenon that underlies the...
Show moreTitin-based passive tension is one of the mechanisms that modulate myofilament length dependent activation, the phenomenon that underlies the so-called Frank Starling Law of the Heart. The Greaser N2BA-G rat model, with a homozygous autosomal mutation in the titin gene, produce a longer isoform of titin that produce much less passive tension as well as a reduced degree of LDA. This provides a useful tool to test whether titin modulates sarcomere structure with changes in sarcomere length. Here we assessed sarcomere structure using small angle X-ray diffraction of intact, twitching papillary muscle in the diastolic phase of contraction at short and long sarcomere length. Comparison of our results from wild type and titin mutant rats indicated that the equatorial intensity ratio, I11/I10, decreases in both wild type and the titin mutant rats with increases in length indicating that the myosin heads are more associated with the thick filament backbone than the thin filament at long lengths. Analysis of the first myosin layer line shows that there is a radially inward movement of myosin heads in WT myocardium but not in the titin mutant when stretched. Difference electron density maps show the thick filament densities in wild type increases more than in titin mutant rats and that the densities of the thin filament are less in the titin mutant rats. Our data suggest, therefore, that increased calcium sensitivity at longer sarcomere length is not due to a radial extension of the myosin heads at long length. Rather, it is associated with transmission of titin-based passive tension from the thick filaments to the thin filament, resulting in increased ordering of the myofilaments at the hexagonal lattice positions requiring some sort of communicating structure, presumably crossbridges, between the thick and the thin filaments as part of the length transduction mechanism behind LDA. The X-ray diffraction studies showed that there are gross troponin structural cahnges when muscle is stretched indicating that troponin is an important regulator of LDA in the myofilaments. But the precise way in which troponin regulates muscle contraction is only partly understood. In the research reported here, we investigated the effects of cTnI phosphorylation of sites in human cardiac tissue using site-directed mutagenesis. Serine and threonine were replaced by aspartic acid (D) or glutamic acid (E) to mimic the phosphorylated state. Six recombinant human cardiac troponin complexes (hcTn), containing either hcTnI-WT, hcTnI-S5/6D, hcTnI-S23/24D, hcTnI-S42/44E, hcTnI-T143E and hcTnI-S150D were exchanged into skinned human cardiac fibers. Force and ATPase activity was measured as function of [Ca2+] at short and long sarcomere length (SL=2.0 and 2.3um). We found that LDA in the hcTnI-S150D substitution was attenuated, and was unchanged with the other sites. CD, as indexed by tension cost was decreased for hcTnI-S42/44E and hcTnI-S150D, and was unaffected for the other site substitutions. While some of these results were consistent with previous findings form rodent myocardium, others were not, suggesting that one should use caution in extrapolating from results in rodetn cardiac tissue to human cardiac tissue.
Ph.D. in Biological and Chemical Sciences, December 2014
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