Engineered Cardiac Troponin C Structure-Function Studies: Designing Proteins for Treatment of Cardiomyopathies

Abstract

The functional effects of mutations associated with cardiomyopathies generally suggest that the Ca2+ responsiveness of the myofilament was affected. This functional change appears to be independent of which protein contains the mutation and therefore indicates that the altered Ca2+ sensitivity could be a critical or causative component of disease expression and progression. However, the correlation underlying this functional change with the disease phenotype is still unclear. Thus, in this work a series of single amino acid-substituted cardiac troponin C (cTnC) variants with altered Ca2+ binding affinities were studied to determine how they influence the Ca2+ activation pathway in myofilament contraction and whether this change in Ca2+ binding will result in adaptive changes in intact cardiomyocytes. These variants have not been identified as associated with any cardiomyopathies and therefore may eventually provide clues as to whether altered Ca2+ signaling of myofilament contraction is causal or an adaptive response in diseased hearts. Firstly, we sought structural and mechanistic explanations for the increased/decreased Ca2+ sensitivity of contraction for the cTnC variants using an array of biophysical techniques. The properties of these cTnC variants were characterized by determining their effects on Ca2+ binding ability, cTnC-cTnI interaction and their modulation by PKA phosphorylation in solution, and their structural alterations using molecular dynamic simulations. We found that cTnC variants have different effects on both binding of Ca2+ and cTnI to cTnC, and they also respond differently upon PKA phosphorylation. MD simulations show, for the first time, that cTnC variants could disrupt crucial hydrophobic interactions so that the closed form of cTnC or the Ca2+ binding loop is destabilized. The findings emphasize the importance of the regulatory domain of cTnC's conformation in the regulation of contraction and suggest that mutations in cTnC that alter myofilament Ca2+ sensitivity can do so by modulating Ca2+ and cTnI binding. Secondly, the functional capacity of the Ca2+ desensitizing variants was characterized by expressing them in cardiomyocytes using adenovirus. Additionally, we demonstrate that engineered cTnC variants can correct the disease-induced abnormal Ca2+ binding sensitivity. Our study provides insights for the development of novel therapeutic strategies for the treatment of cardiomyopathies.