Dataset Information

Organelle Optogenetics: Direct Manipulation of Intracellular Ca²⁺ Dynamics by Light.

ABSTRACT: As one of the ubiquitous second messengers, the intracellular Ca²⁺, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca²⁺-dependent signals: influx from the extracellular space and release from the intracellular Ca²⁺ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca²⁺ release from the stores under high spatiotemporal precision, we established a new method termed "organelle optogenetics." That is, one of the light-sensitive cation channels (channelrhodopsin-green receiver, ChRGR), which is Ca²⁺-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGR_ER) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGR_ER co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGR_ER; (3) an increase of fluorometric Ca²⁺ was evoked by the optical stimulation (OS) in the cells expressing ChRGR_ER in a manner independent on the extracellular Ca²⁺ concentration ([Ca²⁺]_o); (4) the ?F/F₀ was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and (5) the store-operated Ca²⁺ entry (SOCE) was induced by the OS in the ChRGR_ER-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca²⁺ from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca²⁺ dynamics under spatiotemporal precision.

SUBMITTER: Asano T

PROVIDER: S-EPMC6107701 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Organelle Optogenetics: Direct Manipulation of Intracellular Ca2+ Dynamics by Light.

Asano Toshifumi T Igarashi Hiroyuki H Ishizuka Toru T Yawo Hiromu H

Frontiers in neuroscience 20180817

As one of the ubiquitous second messengers, the intracellular Ca2+, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca2+-dependent signals: influx from the extracellular space and release from the intracellular Ca2+ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca2+ release from the stores under high spatiotemporal precision, we established ...[more]

PMID: 30174581

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Project description:Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.

Project description:RationaleCa2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown.ObjectiveTo determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR.Methods and resultsModified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after β-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics.ConclusionsThe interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

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Organelle Optogenetics: Direct Manipulation of Intracellular Ca²⁺ Dynamics by Light.

Publications

Organelle Optogenetics: Direct Manipulation of Intracellular Ca<sup>2+</sup> Dynamics by Light.

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