During the filling of the heart or during the contraction of opposing muscle groups, the sarcomere experiences passive stretch that increases its overall length by several hundred nanometers. In the past, it was believed that extracellular structures, mainly collagen, were responsible for the integrity of the sarcomere and provided resistance to prevent over-stretching of the sarcomere. Within the past thirty years, it has become clear that titin, the third filament of the sarcomere, bears the majority of the force during passive stretch f muscle tissue, and is responsible for setting the optimal working length of the sarcomere. Only in 2012, after genetic sequencing of hundreds of individuals, was it shown that mutations in titin are the leading cause of inherited dilated cardiomyopathy. Now that there exists a clear link between titin mutations and disease, attention has turned towards identifying the normal physiological role of titin. Besides organizing the thick filament, the I-band segment of titin deforms to accommodate stretching of the sarcomere. Unstructured regions of titin rich in proline extend like molecular springs. Structured Ig domains, on the other hand, unfold at forces of several piconewtons to reveal cryptic residues that can undergo post-translational modification. The I-band of titin is unusually rich in cryptic cysteine residues, which can react with both oxidative and nitrosylative species when exposed by force. Hence, titin is thought to be an important redox sensor in skeletal and cardiac muscle. I propose to study the effects of post-translational modifications on titin elasticity and folding. These studies have important implications for how myocardial mechanics change after myocardial infarction, or in the setting of diseases such as diabetes, hypertension, and atherosclerosis.
Aim 1 will study how reactive oxygen species alter the stability of titin Ig domain by blocking folding or inducing disulfide formation.
Aim 2 is to determine if reactive nitrogen species react with residues in titin Ig domains alter titin mechanics and how mechanical stability depends on the location of the modification within the Ig fold.
Aim 3 seeks to utilize a novel mouse model containing a genetically encoded tag in titin to measure the extent of titin Ig unfolding in muscle tissue using super-resolution and electron microscopy. This unique combination of single molecule and single myofibril experiments will demonstrate how changes at the molecular level translate into a mechanical phenotype. These studies provide insights into how oxidative insults, such as those present in ischemia/reperfusion tend to affect the cytoskeleton of muscle, altering myocardial mechanics, and initiating signaling pathways that lead to remodeling and further impairment of cardiac function and diastolic performance.
Oxidative stress in the myocardium is a hallmark of myocardial infarction, atherosclerosis, hypertension, diabetes, and aging. The mechanisms by which oxidative stress alters heart elasticity and initiates myocardial remodeling remain unknown. Emerging studies point to titin as a determinant factor in muscle tissue compliance and heart disease. This project seeks to characterize the mechanical properties of titin Ig domains in the presence of reactive oxygen and nitrogen species at both the single molecule and tissue level, thereby changing the way we think about muscle elasticity and diastolic extensibility.
| Eckels, Edward C; Tapia-Rojo, Rafael; Rivas-Pardo, Jamie Andrés et al. (2018) The Work of Titin Protein Folding as a Major Driver in Muscle Contraction. Annu Rev Physiol 80:327-351 |
| Valle-Orero, Jessica; Tapia-Rojo, Rafael; Eckels, Edward C et al. (2017) Proteins Breaking Bad: A Free Energy Perspective. J Phys Chem Lett 8:3642-3647 |
| Haldar, Shubhasis; Tapia-Rojo, Rafael; Eckels, Edward C et al. (2017) Trigger factor chaperone acts as a mechanical foldase. Nat Commun 8:668 |
| Rivas-Pardo, Jaime Andrés; Eckels, Edward C; Popa, Ionel et al. (2016) Work Done by Titin Protein Folding Assists Muscle Contraction. Cell Rep 14:1339-1347 |
