bioRxiv Science⌕ Search

bioRxiv · 10.1101/2024.04.05.588306

Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T

Abstract

Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric contraction. While patient studies have defined the genetic landscape of DCM, genetics are not currently used in patient care, and patients receive similar treatments regardless of the underlying mutation. It has been suggested that a precision medicine approach based on the molecular mechanism of the underlying mutation could improve outcomes; however, realizing this approach has been challenging due to difficulties linking genotype and phenotype and then leveraging this information to identify therapeutic approaches. Here, we used multiscale experimental and computational approaches to test whether knowledge of molecular mechanism could be harnessed to connect genotype, phenotype, and drug response for a DCM mutation in troponin T, deletion of K210. Previously, we showed that at the molecular scale, the mutation reduces thin filament activation. Here, we used computational modeling of this molecular defect to predict that the mutant will reduce cellular and tissue contractility, and we validated this prediction in human cardiomyocytes and engineered heart tissues. We then used our knowledge of molecular mechanism to computationally model the effects of a small molecule that can activate the thin filament. We demonstrate experimentally that the modeling correctly predicts that the small molecule can partially rescue systolic dysfunction at the expense of diastolic function. Taken together, our results demonstrate how molecular mechanism can be harnessed to connect genotype and phenotype and inspire strategies to optimize mechanism-based therapeutics for DCM. Significance statementDilated cardiomyopathy (DCM), a leading cause of heart failure, is characterized by the inability of the heart to perfuse the body at normal filling pressures. There are multiple causes of DCM, including point mutations in sarcomeric proteins, but most patients receive similar courses of treatment, regardless of the underlying cause of the DCM. Many patients remain unserved by current therapies, and there is a need for new approaches. Here, we use multiscale experimental and computational approaches to demonstrate how knowledge of molecular mechanism can be harnessed to accurately predict the effects of a patient-specific mutation and responses to presumptive therapeutics. Our approach lays the foundation for a precision medicine approach to DCM.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Greenberg, L., Stump, W. T., Lin, Z. J., Bredemeyer, A. L., Blackwell, T., Hao, X., Greenberg, A. E., Garcia, B., Lavine, K., Greenberg, M. J.. 2024-04-09. Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T. https://doi.org/10.1101/2024.04.05.588306

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

Expression-System Biases Compromise the Functional Assessment of KCNH2 Variants and Clinical Risk Assessment

Background: Functional characterization of KCNH2 variants is essential for assessing pathogenicity in long QT syndrome type 2. Because hERG channels assemble as tetramers, the relative expression of wild-type (WT) and variant subunits critically influences channel function and pathogenicity assessment. However, commonly used experimental systems may not reproduce the balanced allelic expression observed in heterozygous patients. Methods: Allele-specific KCNH2 expression was analyzed in two independent RNA-sequencing datasets from human left ventricular tissue. Using the dominant-negative T74R hERG variant as a model, we calibrated the relationship between WT-to-variant expression ratios and hERG current amplitude. Using automated patch-clamp electrophysiology, we compared 3 dual-expression strategies: internal ribosome entry site (IRES), P2A-linked tandem constructs, and dual cytomegalovirus promoter (dCMV) constructs. Co-electroporation of WT and variant plasmids in different ratios was the reference. Results: Allele-specific expression analysis supported balanced KCNH2 expression in human left ventricular tissue. At a 1:1 WT-to-T74R expression ratio, hERG current amplitude was reduced to 31.0% of WT, consistent with a dominant-negative mechanism in which incorporation of 2 or more T74R subunits renders the channel nonfunctional. All 3 dual-expression strategies produced substantial expression imbalances, with the first cassette consistently favored. IRES constructs showed the greatest asymmetry. P2A constructs partially improved expression balance but introduced an additional N-terminal proline in the second cassette altering T74R-associated trafficking and channel biophysics. The dCMV system also failed to achieve balanced expression, including after stable integration. Co-electroporation of separate WT and variant plasmids provided the most reliable means of controlling their relative expression. Conclusions: Commonly used dual-expression constructs do not reliably reproduce balanced WT and variant hERG expression, potentially distorting assessments of dominant-negative effects and variant pathogenicity. Co-electroporation of separate plasmids provides a robust approach for high-throughput functional phenotyping under heterozygous conditions, with implications for the interpretation of KCNH2 variants and potentially other genes encoding multimeric ion channels.

biophysics↗

Learning from Hi-C reveals nuclear dynamical reorganization in budding yeast across the cell cycle

Hi-C contact maps provide population-averaged measurements of chromosomal contacts. Converting these measurements, however, into three-dimensional structures and associated dynamics remains an underdetermined problem. Existing approaches broadly fall into two classes. Data-driven reconstruction methods that infer either a consensus structure or an ensemble of conformations, but do not inform on chromosome dynamics. Physics-based models, which generate dynamical trajectories, but require prior assumptions about nuclear geometry, chromatin mechanics, tethering, or molecular interactions. Here, we bridge these approaches using the Full-Inversion Chromatin model, a data-driven whole-genome polymer framework that learns an effective force field directly from Hi-C contact probabilities, integrated with Serpentine, a tool to quantify contact-map discrepancies while accounting for differences in sequencing coverage and sparsity. This generates structural ensembles of chromosomes and effective chromatin dynamics. We apply this method at 5-kb resolution to Saccharomyces cerevisiae in G1, mid-S, S/G2, G2/M, anaphase, and quiescence. Without explicitly imposing the spindle pole body, centromere or telomere tethering, predefined chromatin states, or nuclear-envelope interactions, the inferred ensembles recover these major features of yeast nuclear organization. Importantly, although dynamical information is not used during model training, the resulting polymer trajectories reproduce the subdiffusive scaling and chromosomal variations of independently measured chromatin motion. The model further predicts heterogeneous mobility along chromosomes and a distinctive telomere-dependent dynamical organization. Thus, population-averaged Hi-C contacts contain sufficient information to infer not only three-dimensional genome organization, but also effective, locus-dependent chromatin dynamics.

biophysics↗

One high-order ion-coupling mechanism generates multiple SLC1A stoichiometries

Human excitatory amino acid transporter 2 (hEAAT2) is a glial transporter that clears glutamate and aspartate from the extracellular space to terminate neurotransmission. When this essential role is disrupted, excess extracellular glutamate causes excitotoxicity and neurological disease. hEAAT2 transports amino acids into cells against their concentration gradients by coupling uptake to the co-transport of Na+ and H+, and counter-transport of K+. Substrate transport also activates a thermodynamically uncoupled Cl- conductance, whose significance is unknown. Here, to dissect the molecular mechanisms underlying coupled and uncoupled ion transport by EAATs, we leverage the tractable Drosophila melanogaster homologue dEAAT2, which transports taurine as well as glutamate and aspartate. By functionally characterising dEAAT2, we confirmed it has features of a canonical EAAT transport cycle, including an uncoupled Cl- conductance. However, it functions an ion-independent electroneutral exchanger, in stark contrast to its ion-coupled electrogenic human counterpart. Cryo-electron microscopy (cryo-EM) and computational simulations confirmed the inability of dEAAT2 to transport cations, but just two point mutations conferred Na+, H+, and K+ dependence, reconstituting the complex ion coupling profile of human transporters in the Drosophila exchanger. We also showed that dEAAT2 transports taurine in a complex with Cl-, bestowing a unique Cl--dependence on taurine transport, independent of uncoupled Cl- flux. Our findings show that ancestral exchangers in the EAAT family preserve the machinery to achieve complex ion-coupled transport, revealing the molecular basis of high-order ion coupling in hEAATs.

biophysics↗