Dataset Information

Mechanical forces regulate cardiomyocyte myofilament maturation via the VCL-SSH1-CFL axis

ABSTRACT: Mechanical forces regulate cell behavior and tissue morphogenesis. In particular, cardiac tissues require mechanical stimuli generated by the heartbeat for differentiation and maturation, but the molecular mechanisms underlying these processes remain unclear. Here, we first show that mechanical forces acting via the mechanosensitive factor Vinculin (VCL) are essential for cardiomyocyte myofilament maturation and that cardiac contractility regulates the localization and activation of Vinculin. To further analyze the role of Vinculin in myofilament maturation, we examined its interactome in contracting cardiomyocytes and found many cytoskeletal factors including actinins. We also identified Slingshot protein phosphatase 1 (SSH1), which we show is recruited by Vinculin to regulate F-actin rearrangement and myofilament maturation through its association with the actin depolymerizing factor Cofilin (CFL). Together, our results reveal that mechanical forces generated by cardiac contractility regulate cardiomyocyte maturation through the VCL-SSH1-CFL axis, providing mechanistic insight into how mechanical forces are transmitted intracellularly to regulate myofilament maturation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture, Cardiac Muscle Cell

SUBMITTER: Johannes Graumann

LAB HEAD: Didier Y.R. Stainier

PROVIDER: PXD014624 | Pride | 2019-09-06

REPOSITORIES: Pride

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Publications

Mechanical Forces Regulate Cardiomyocyte Myofilament Maturation via the VCL-SSH1-CFL Axis.

Fukuda Ryuichi R Gunawan Felix F Ramadass Radhan R Beisaw Arica A Konzer Anne A Mullapudi Sri Teja ST Gentile Alessandra A Maischein Hans-Martin HM Graumann Johannes J Stainier Didier Y R DYR

Developmental cell 20190905 1

Mechanical forces regulate cell behavior and tissue morphogenesis. During cardiac development, mechanical stimuli from the heartbeat are required for cardiomyocyte maturation, but the underlying molecular mechanisms remain unclear. Here, we first show that the forces of the contracting heart regulate the localization and activation of the cytoskeletal protein vinculin (VCL), which we find to be essential for myofilament maturation. To further analyze the role of VCL in this process, we examined ...[more]

PMID: 31495694

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Project description:Background The axon guidance cue Slit2 recently has been found to regulate calcium homeostasis and molecular signaling in various stress events in different organs. However, whether Slit2 plays a role in cardiac ischemia-reperfusion (IR) injury has not been reported. Here, we aimed to investigate the role of Slit2 and the underlying mechanisms in cardiac IR injury. Methods Langendorff-perfused isolated hearts from Slit2-overexpressing (Slit2-Tg) mice and their background strain C57BL/6J mice were subjected to 20 min of global ischemia followed by 40 min of reperfusion. Left ventricular function of isolated hearts was monitored. Infarct size of post-IR hearts was determined by staining with 2,3,5-triphenyltetrazolium chloride (TTC) and histological changes of cardiac tissues and cells were determined with hematoxylin-eosin (HE) staining and transmission electron microscopy. Transcriptomic analysis was used to predict the biological processes and signaling pathways affected by Slit2 overexpression in the post-IR myocardium. Pro-Q staining and Western blotting was used to assess the phosphorylation levels of cardiac myofilaments and expression levels of myofilament-associated protein kinase and phosphatases. Results Slit2 overexpression increased post-IR left ventricular developed pressure (LVDP) by 35% and reduced infarct size by 53%, along with decreased myofibrillar disruption, mitochondrial swelling, and mitochondrial cristae dissolution. Slit2 overexpression significantly changed post-IR gene expression profiles. Functional products of these genes include regulation of cation transmembrane transport, cation homeostasis, collagen fibril organization, and regulation of heart rate. And post-IR myocardial KEGG pathways upregulated by Slit2 overexpression include ECM-receptor interaction, PI3K-Akt signaling pathway, and adrenergic signaling. Slit2 overexpression impacted myofilament phosphorylation together with myofilament-associated protein kinase C (PKC) isoforms and protein phosphatases (PPs). IR in C57BL/6J hearts upregulated phosphorylation of cardiac troponin-I (cTnI), which was suppressed by Slit2 overexpression. Myofilament‐associated PKCε, PKCδ, and PP2A were significantly increased post‐IR in C57BL/6J hearts, but in Slit2‐Tg hearts, myofilament‐associated PKCε and PP2A were increased and PKCδ was suppressed. Conclusions Our results demonstrate that Slit2 overexpression protects cardiac function and reduces IR injury, which is associated with Slit2‐induced gene profile shifts. The suppression of MyBP‐C and troponin‐I phosphorylation, and myofilament‐associated PKCδ levels induced by Slit2 overexpression could contribute to the cardioprotection of Slit2 in post-IR myocardium.

Project description:Engineered cardiac tissues (ECTs) are platforms to investigate cardiomyocyte maturation and functional integration, to evaluate the feasibility of generating implantable tissues for cardiac repair and regeneration, and may be useful models for pharmacology and toxicology bioassays. These ECTs rapidly mature in vitro to acquire the features of functional cardiac muscle and respond to mechanical load with increased proliferation and maturation. ECTs can be generated from various immature cardiac cell sources and little is known regarding the broad changes in regulatory transcript expression that occur in these in vitro tissues during normal maturation and in response to mechanical or pharmacologic interventions. We tested the hypothesis that global ECT gene expression patterns are sensitive to mechanical loading conditions and tyrosine kinase inhibitors, similar to the maturing myocardium. We generated 3D ECTs from day 14.5 rat embryo ventricular cells, as previously published, and then treated constructs after 5 days in culture for 48 hours with mechanical stretch (5%, 0.5 Hz) and/or the p38MAPK (p38 mitogen-activated protein kinase) selective inhibitor BIRB796. RNA was isolated from 3 sets of experiments and assayed using a standard Agilent rat 4x44k V3 microarray and Pathway Analysis software for transcript expression fold changes and changes in regulatory molecules and networks. At the threshold of a 1.5 fold change in expression, mechanical stretch altered 1,559 transcripts, versus 1,411 for BIRB796, and 1,846 for stretch plus BIRB796. As anticipated, top pathways altered in response to these stimuli include Cellular Development, Cellular Growth and Proliferation; Tissue Development; Cell Death, Cell Signaling, and Small Molecule Biochemistry as well as numerous other pathways. Changes in transcript expression were confirmed by quantitative-PCR for selected regulatory molecules. Thus, ECTs display a broad spectrum of altered gene expression in response to mechanical load and/or tyrosine kinase inhibition, reflecting the complex regulation of proliferation, differentiation, and architectural alignment that occurs during ECT maturation and adaptation. This approach can now be used to test the role of individual molecules and pathways on the regulation of ECT maturation and remodeling. 7 and 4 biological replicates with four groups (control, mechanical stretch, BIRB and mechanical stretch with BIRB)

Dataset Information

Mechanical forces regulate cardiomyocyte myofilament maturation via the VCL-SSH1-CFL axis

Publications

Mechanical Forces Regulate Cardiomyocyte Myofilament Maturation via the VCL-SSH1-CFL Axis.

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