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Biomechanical Impact of Pathogenic MYBPC3 Truncation Variant Revealed by Dynamically Tuning In Vitro Afterload.

ABSTRACT: Engineered cardiac microtissues were fabricated using pluripotent stem cells with a hypertrophic cardiomyopathy associated c. 2827 C>T; p.R943x truncation variant in myosin binding protein C (MYBPC3^+/-). Microtissues were mounted on iron-incorporated cantilevers, allowing manipulations of cantilever stiffness using magnets, enabling examination of how in vitro afterload affects contractility. MYPBC3^+/- microtissues developed augmented force, work, and power when cultured with increased in vitro afterload when compared with isogenic controls in which the MYBPC3 mutation had been corrected (MYPBC3^+/+(ed)), but weaker contractility when cultured with lower in vitro afterload. After initial tissue maturation, MYPBC3^+/- CMTs exhibited increased force, work, and power in response to both acute and sustained increases of in vitro afterload. Together, these studies demonstrate that extrinsic biomechanical challenges potentiate genetically-driven intrinsic increases in contractility that may contribute to clinical disease progression in patients with HCM due to hypercontractile MYBPC3 variants.

SUBMITTER: Ramachandran A

PROVIDER: S-EPMC10480352 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Biomechanical Impact of Pathogenic MYBPC3 Truncation Variant Revealed by Dynamically Tuning In Vitro Afterload.

Ramachandran Abhinay A Livingston Carissa E CE Vite Alexia A Corbin Elise A EA Bennett Alexander I AI Turner Kevin T KT Lee Benjamin W BW Lam Chi Keung CK Wu Joseph C JC Margulies Kenneth B KB

Journal of cardiovascular translational research 20230306 4

Engineered cardiac microtissues were fabricated using pluripotent stem cells with a hypertrophic cardiomyopathy associated c. 2827 C>T; p.R943x truncation variant in myosin binding protein C (MYBPC3<sup>+/-</sup>). Microtissues were mounted on iron-incorporated cantilevers, allowing manipulations of cantilever stiffness using magnets, enabling examination of how in vitro afterload affects contractility. MYPBC3<sup>+/-</sup> microtissues developed augmented force, work, and power when cultured wi ...[more]

PMID: 36877449

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Project description:RationaleTruncation mutations in the MYBPC3 gene, encoding for cardiac myosin-binding protein C (MyBP-C), are the leading cause of hypertrophic cardiomyopathy (HCM). Whole heart, fiber and molecular studies demonstrate that MyBP-C is a potent modulator of cardiac contractility, but how these mutations contribute to HCM is unresolved.ObjectivesTo readdress whether MYBPC3 truncation mutations result in loss of MyBP-C content and/or the expression of truncated MyBP-C from the mutant allele and determine how these mutations effect myofilament sliding in human myocardium.Methods and resultsSeptal wall tissue samples were obtained from HCM patients undergoing myectomy (n = 18) and donor controls (n = 8). The HCM samples contained 40% less MyBP-C and reduced levels of MyBP-C phosphorylation, when compared to the donor control samples using quantitative mass spectrometry. These differences occurred in the absence of changes in the stoichiometry of other myofilament proteins or production of truncated MyBP-C from the mutant MYBPC3 allele. The functional impact of MYBPC3 truncation mutations on myofilament sliding was determined using a total internal reflection microscopy (TIRFM) single particle assay. Myosin-thick filaments containing their native complement of MyBP-C, and actin-thin filaments decorated with the troponin/tropomyosin calcium regulatory proteins, were isolated from a subgroup of the HCM (n = 4) and donor (n = 5) heart samples. The maximal sliding velocity of native thin filaments was enhanced within the C-zones of the native thick filaments isolated from the HCM samples, when compared to velocity within the C-zones of thick filaments isolated from the donor samples. Analytical modeling demonstrated that the 40% reduction in MyBP-C content was sufficient to enhance the myofilament sliding velocity, as observed in the TIRFM assay.ConclusionsHCM-causing MYBPC3 truncation mutations result in a loss of MyBP-C content that enhances maximal myofilament sliding velocities, only where MyBP-C is localized within the C-zone. These findings support therapeutic rationale for restoring normal levels of MyBP-C and/or dampening maximal contractile velocities for the treatment of human HCM.

Project description:Afterload plays important roles during heart development and disease progression, however, studying these effects in a laboratory setting is challenging. Current techniques lack the ability to precisely and reversibly alter afterload over time. Here, we describe a magnetics-based approach for achieving this control and present results from experiments in which this device was employed to sequentially increase afterload applied to rat engineered heart tissues (rEHTs) over a 7-day period. The contractile properties of rEHTs grown on control posts marginally increased over the observation period. The average post deflection, fractional shortening, and twitch velocities measured for afterload-affected tissues initially followed this same trend, but fell below control tissue values at high magnitudes of afterload. However, the average force, force production rate, and force relaxation rate for these rEHTs were consistently up to 3-fold higher than in control tissues. Transcript levels of hypertrophic or fibrotic markers and cell size remained unaffected by afterload, suggesting that the increased force output was not accompanied by pathological remodeling. Accordingly, the increased force output was fully reversed to control levels during a stepwise decrease in afterload over 4 hours. Afterload application did not affect systolic or diastolic tissue lengths, indicating that the afterload system was likely not a source of changes in preload strain. In summary, the afterload system developed herein is capable of fine-tuning EHT afterload while simultaneously allowing optical force measurements. Using this system, we found that small daily alterations in afterload can enhance the contractile properties of rEHTs, while larger increases can have temporary undesirable effects. Overall, these findings demonstrate the significant role that afterload plays in cardiac force regulation. Future studies with this system may allow for novel insights into the mechanisms that underlie afterload-induced adaptations in cardiac force development.

Project description:MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and subsequent recovery period. Here we monitored the transcriptome changes by microarray analysis of the whole blood of eight highly trained athletes after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and then mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. We have identified four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation. Each subject performed a treadmill test with an incremental step protocol until exhaustion. Blood samples were taken from the antecubital vein using indwelling catheter before (T1), immediately after ramp test to exaustion (T2) and after 30 min of recovery (T3). Two weeks later, athletes participated in the main exercise, consisting of running at 80% VO2 peak for 30 min on a treadmill. Blood samples were taken from the antecubital vein using indwelling catheter before (T4), immediately after running at 80% VO2 peak for 30 min on a treadmill (T5), after 30 min (T6) and 60 min (T7) of recovery.

Dataset Information

Biomechanical Impact of Pathogenic MYBPC3 Truncation Variant Revealed by Dynamically Tuning In Vitro Afterload.

Publications

Biomechanical Impact of Pathogenic MYBPC3 Truncation Variant Revealed by Dynamically Tuning In Vitro Afterload.

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