The classic clinical definition of hypertrophic cardiomyopathy (HCM) as originally described by Teare is deceptively simple, "left ventricular hypertrophy in the absence of any identifiable cause". Longitudinal studies, however, including a seminal study performed by Frank and Braunwald in 1968, clearly described the disorder much as we know it today, a complex, progressive and highly variable cardiomyopathy affecting ~1/500 individuals worldwide. Subsequent genetic linkage studies in the early 1990's identified mutations in virtually all of the protein components of the cardiac sarcomere as the primary molecular cause of HCM. In addition, a substantial proportion of inherited dilated cardiomyopathy (DCM) has also been linked to sarcomeric protein mutations. Despite our deep understanding of the overall function of the sarcomere as the primary driver of cardiac contractility, the ability to use genotype in patient management remains elusive. A persistent challenge in the field from both the biophysical and clinical standpoints is how to rigorously link high-resolution protein dynamics and mechanics to the long-term cardiovascular remodeling process that characterizes these complex disorders. In this review, we will explore the depth of the problem from both the standpoint of a multi-subunit, highly conserved and dynamic "machine" to the resultant clinical and structural human phenotype with an emphasis on new, integrative approaches that can be widely applied to identify both novel disease mechanisms and new therapeutic targets for these primary biophysical disorders of the cardiac sarcomere. Keywordsthin filament; hypertrophic cardiomyopathy; dilated cardiomyopathy; troponin; tropomyosin Current Challenges in our Understanding of Sarcomeric CardiomyopathiesMutations in the genes that encode the protein components of the cardiac thin filament were first linked to clinical cardiomyopathies in 1994 [85]. Mutations in the regulatory thin filament comprise ~ 7-10% of hypertrophic cardiomyopathy (HCM) cases and represent a distinct and complex subset of the disorder with marked mutation-specific phenotypes [11,84]. Despite 25 years of basic research, many questions remain regarding the clinical

The structural analysis of large protein complexes has been greatly enhanced through the application of electron microscopy techniques. One such multiprotein complex, the cardiac thin filament (cTF), has cyclic interactions with thick filament proteins to drive contraction of the heart that has recently been the subject of such studies. As important as these studies are, they provide limited or no information on highly flexible regions that in isolation would be characterized as inherently disordered. One such region is the extended cardiac troponin T (cTnT) linker between the regions of cTnT which have been labeled TNT1 and TNT2. It comprises a hinge region (residues 158–166) and a highly flexible region (residues 167–203). Critically, this region modulates the troponin/tropomyosin complex’s position across the actin filament. Thus, the cTnT linker structure and dynamics are central to the regulation of the function of cardiac muscles, but up to now, it was ill-understood. To establish the cTnT linker structure, we coupled an atomistic computational cTF model with time-resolved fluorescence resonance energy transfer measurements in both ±Ca2+ conditions utilizing fully reconstituted cTFs. We mapped the cTnT linker’s positioning across the actin filament, and by coupling the experimental results to computation, we found mean structures and ranges of motion of this part of the complex. With this new insight, we can now address cTnT linker structural dynamics in both myofilament activation and disease.

Point mutations within sarcomeric proteins have been associated with altered function and cardiomyopathy development. Difficulties remain, however, in establishing the pathogenic potential of individual mutations, often limiting the use of genotype in management of affected families. To directly address this challenge, we utilized our all-atom computational model of the human full cardiac thin filament (CTF) to predict how sequence substitutions in CTF proteins might affect structure and dynamics on an atomistic level.Utilizing molecular dynamics (MD) calculations, we simulated 21 well-defined genetic pathogenic cardiac troponin T and tropomyosin variants to establish a baseline of pathogenic changes induced in computational observables. Computational results were verified via differential scanning calorimetry on a subset of variants to develop an experimental correlation.Calculations were performed on 9 independent variants of unknown significance (VUS) and results were compared to pathogenic variants to identify high resolution pathogenic signatures.Results for VUS were compared to the baseline set to determine induced structural and dynamic changes and potential variant reclassifications were proposed. This unbiased, highresolution computational methodology can provide unique structural and dynamic information that can be incorporated into existing analyses to facilitate classification both for de novo variants and those where established approaches have provided conflicting information.

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