The general objective of the experiments carried out in this thesis was to fill major gaps in our understanding of how thin filament control mechanisms translate to regulation of cardiac function. Filling these gaps is essential to understanding and treating acquired and familial cardiac disorders linked to sarcomeric protein mutations. Here we focused on á-tropomyosin (α- TM) as a nodal point in control of the thin filament state. Structural flexibility of α-TM represents a significant, but poorly understood property, in the control of thin filament state and cardiac function. In this study, we specifically addressed the following: (i) the implications of the D137L mutation on the global structural flexibility of α-TM and (ii) the effects of D137L mutation on cardiac function. A highly integrative study employing a range of approaches from recombinantly expressed proteins to ejecting heart of a novel transgenic mouse model was carried out in order to address these objectives.
α-TM is a coiled-coil protein that cooperatively binds along the actin filaments and serves as a nodal point in control of calcium regulated cardiac muscle dynamics. α-TM has a conserved, charged residue (Asp137) located in the hydrophobic core of its coiled-coil structure. This is distinct to the α-TM coiled-coil in that the residue is found at a position typically occupied by a hydrophobic residue. In a previous in vitro study, which substituted this Asp137 residue with a more expected canonical Leu, it was demonstrated that Asp137 destabilizes a local region in the middle of α-TM, inducing a more flexible region that is important for modulating the cooperative activation of the thin filaments. In the first part of this thesis, we extended these earlier findings and demonstrated that the D137L mutation decreased structural flexibility of α-TM, which was a global effect that caused long-range structural rearrangements.
We know next to nothing of the relative significance of α-TM flexibility in sarcomeric control mechanisms in vivo. Therefore in the second part of this study, we investigated implications of α-TM flexibility on in situ cardiac function of a novel transgenic mouse model expressing α-TM-D137L in the heart. To our knowledge, our findings are the first to show that a marked decrease in α-TM's structural flexibility due to substitution of Asp137 with Leu depressed systolic parameters of cardiac contraction and ultimately led to a phenotype similar to dilated cardiomyopathy in α-TM-D137L transgenic mouse heart.
α-TM molecules undergo calcium and myosin dependent regulatory relocations, azimuthally, over the surface of the actin filaments during cardiac muscle contraction and relaxation. Structural flexibility of α-TM is thought to have a key role in these relocations. Our results demonstrated that expression of α-TM-D137L in transgenic mouse hearts depressed calcium dependent activation of thin filaments. However, there was no change in the strongly bound cross-bridge dependent activation in skinned fiber preparations. Therefore we proposed a mechanism that the decreased flexibility of α-TM-D137L impede calcium dependent relocation of α-TM on actin resulting in a delay in time sensitive activation and relaxation processes of cardiac muscle, which eventually lead to systolic dysfunction in transgenic mouse hearts.
Collectively, this work has shed light on a functionally important structural characteristic of α-TM and suggested a possible association between flexibility of α-TM and cardiac disorders. A change in flexibility of α-TM has been previously reported for some cardiomyopathy linked α- TM mutations. While α-TM-D137L mutation is not associated with inherited cardiomyopathies, our findings provide unique insights into our understanding of both how disease-linked α-TM point mutations can significantly alter the dynamic properties of α-TM, as well as, how altering α-TM flexibility can have a significant effect on calcium-dependent thin filament regulation and ultimately on cardiac function.
|Advisor:||Solaro, R. John|
|Commitee:||Solaro, Ross J.|
|School:||University of Illinois at Chicago|
|Department:||Biochemistry and Molecular Genetics|
|School Location:||United States -- Illinois|
|Source:||DAI-B 75/07(E), Dissertation Abstracts International|
|Subjects:||Biochemistry, Physiology, Biophysics|
|Keywords:||Asp137, Cardiac muscles, Cardiovascular disease, D137l, Flexibility, Sarcomere, Thin filaments, Transgenic mouse model, Tropomyosin, Tropomyosin flexibility, Troponin|
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