Key pointsr Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall.r Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent.r From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca 2+ ] are present, such as the failing myocardium.r Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction.Abstract Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca 2+ ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca 2+ handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca 2+ ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca 2+ in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca 2+ both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca 2+ ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca 2+ overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca 2+ ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy A. Najafi and M. McConnell contributed equally to this work.

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm–Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.

Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere. Understanding the molecular-level dysfunction elicited by TF mutations is crucial to elucidate cardiac disease mechanisms. The hypertrophic cardiomyopathy-causing cardiac troponin T (cTnT) mutation ⌬160Glu (⌬160E) is located in a putative "hinge" adjacent to an unstructured linker connecting domains TNT1 and TNT2. Currently, no high-resolution structure exists for this region, limiting significantly our ability to understand its role in myofilament activation and the molecular mechanism of mutation-induced dysfunction. Previous regulated in vitro motility data have indicated mutation-induced impairment of weak actomyosin interactions. We hypothesized that cTnT-⌬160E repositions the flexible linker, altering weak actomyosin electrostatic binding and acting as a biophysical trigger for impaired contractility and the observed remodeling. Using time-resolved FRET and an all-atom TF model, here we first defined the WT structure of the cTnT-linker region and then identified ⌬160E mutation-induced positional changes. Our results suggest that the WT linker runs alongside the C terminus of tropomyosin. The ⌬160E-induced structural changes moved the linker closer to the tropomyosin C terminus, an effect that was more pronounced in the presence of myosin subfragment (S1) heads, supporting previous findings. Our in silico model fully supported this result, indicating a mutation-induced decrease in linker flexibility. Our findings provide a framework for understanding basic pathogenic mechanisms that drive severe clinical hypertrophic cardiomyopathy phenotypes and for identifying structural targets for intervention that can be tested in silico and in vitro.

from a pit before abortive CCP termination or endocytic vesicle production. Surprisingly, the binding times of cargo molecules associating to CCPs are much shorter than the overall endocytic process. By measuring tens of thousands of capturing events, we build the distribution of capture times and the times that cargo remains confined to a CCP. An analytical stochastic model is developed and compared to the measured distributions. Due to the dynamic nature of the pit, the model is non-Markovian and it displays long-tail power law statistics. Our findings identify one source of the large heterogeneities observed in CCP maturation and provide a mechanism for the anomalous diffusion of proteins in the plasma membrane.

Inherited mutations in cardiac thin filament proteins create primary alterations in structure. These changes then lead to pathologic remodeling of the heart. We have proposed that two mutations, tropomyosin (Tm) D230N which leads to dilated cardiomyopathy (DCM) and cardiac troponin T (cTnT) R92L, which leads to hypertrophic cardiomyopathy (HCM), directly affect the Tm overlap, a crucial structure regulating myofilament activation. These mutations lead to divergent ventricular remodeling, which we hypothesize is caused by differential effects on the Tm overlap, an inherently dynamic system. In order to investigate this hypothesis, we used time-resolved Forster resonance energy transfer (FRET) to measure discrete distances across the proteins of the overlap, and differential scanning calorimetry (DSC) in order to characterize the baseline structure of this region as well as the effects of mutants. We found that the cTnT R92L mutant increased interprotein distance at the proximal N-terminal end of this region. In contrast, the Tm D230N mutant decreases interprotein distance at the proximal C-terminal end of the overlap. DSC studies showed a significant increase in the association of Tm with actin in the presence of Tm D230N, predicted to increase cooperativity of thin filament activation. These finding are in agreement both with the human population who carry the disease, as well as the mouse lines in our lab, which phenocopy the human disease. Overall, the study further characterized the wild type structure of the Tm overlap, and revealed the differential structural effects of the mutants, thereby, for the first time linking local changes in structure to phenotypic changes and ventricular remodeling leading to HCM and DCM. Further studies will investigate the role of isoform and phosphorylation modifiers, elucidating how these modify human disease in the presence and absence of cardiomyopathy linked mutations.