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.
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
Cell-cell and cell-substrate interactions alter the contractile behaviour of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cells are often cultured as a mono-layer on rigid tissue culture plastic (TCP) or glass, in contrast to the flexible extracellular matrix of myocardium. We examine the contractile behaviour of hiPSC-CMs on a range of substrates including standard TCP to determine the influence on the time course and synchronicity of cell contraction. HiPSC-CM (Ncardia Cor.4U or CDI iCell 2 ) were seeded on day À2 on either fibronectin-coated TCP or fibronectin-coated recombinant collagen-like polypeptide-based hydrogel (Fujifilm, Tilburg NL) with an estimated stiffness of 10kPa. Spontaneous contractility was monitored using video recordings made on days 0, 1, 3, 5 and 7 (D0-7). To assess the contractile behaviour, video frames (100fps) were subdivided into a 30x30 grid, and each grid square analysed using the MUSCLEMOTION algorithm (Sala et al., 2018). The % of single peaked-transients and the 10 th -90 th percentile difference (IP90) of contraction duration at 50% amplitude (CD50-IP90) and contraction start time (TStart-IP90) values were analysed. Values are reported as means5SD, and groups were compared to each other. Contraction synchronicity (IP90 TStart) and duration (IP90 CD50) in cultures on TCP and hydrogel were not significantly different. However, the percentage of grid squares with single-peaked transients was less in the TCP group (80.150.7% on D1 decreasing to 60.953.9% on D7) than on hydrogel (98.650.1% on D1 decreasing to 84.4513.1% on D7). The time course associated with cultures on TCP was unphysiological and indicates that the substrate is not satisfactory for hiPSC-CMs. In contrast, a single-peaked twitch contraction observed routinely on the collagen hydrogel is consistent with the normal contraction-relaxation cycle in individual cardiomyocytes. These results underline the importance of a suitable substrate for hiPSC-CM culture.
Reduced contractility, caused either by dysfunction or loss of cardiomyocytes, is the leading cause of systolic heart failure (HF). We have demonstrated that deoxy-ATP (dATP) improves systolic cardiac contractility by activating myosin without affecting the diastolic function, suggesting a promising candidate to mitigate cardiac dysfunction. Endogenously, Ribonucleotide Reductase (RNR), a rate-limiting enzyme for de novo synthesis of deoxynucleotides, produces dATP. The goal is to overexpress RNR in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to create cells with elevated dATP levels for use as a novel therapeutic strategy to improve contractility and regeneration in hearts following myocardial infarct. We used CRISPR-Cas9 gene editing to integrate an RNR expression cassette at the AAVS1 locus in hiPSCs WTC11 cell line (#GM25256, Coriell Institute). A double mutant form (RNR-DM) was generated by site-directed mutagenesis to limit proteolytic degradation of RNR in post-mitotic CMs. RNR subunits-R1 and R2-DMintervened by a self-cleavage peptide P2A were expressed under a strong constitutively expressed CAG promoter. The integration of RNR cassette at the AAVS1 locus in hiRNR clones were confirmed by PCR genotyping, sequencing, transgene transcript, and protein expression. The stability and function of RNR-DM transgene were assessed in hiRNR-CMs, differentiated from the hiRNR clone. hiRNR-CMs expressed elevated levels of R1 and R2 subunits as compared to control WTC11-CMs (N=3, where N is an independent run of cardiac differentiation) by immunoblotting. This resulted in an elevation of dATP/ATP ratio (3.451.9 fold) (N=2) for hiRNR-CMs vs. WTC11-CMs, as detected by Mass Spectrometry. Cardiomyocyte shortening measurements by video-based IonOptix were increased (2.0350.7 fold) in hiRNR CMs as compared to WTC11-CMs (N=2). Currently, we are characterizing the hiRNR-CMs, including DNA stability, apoptosis, cell division, calcium cycling, hypertrophy, and structural maturation for future studies of transplantation into models of HF.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.