Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology

Fernández, Marbely C; Kopton, Ramona A.; Simón-Chica, Ana; Madl, Josef; Hilgendorf, Ingo; Zgierski-Johnston, Callum M.; Schneider-Warme, Franziska

doi:10.1007/978-1-0716-0830-2_17

Cited by 9 publications

(6 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All sharp electrode recordings with maximum diastolic potentials below −40 mV that showed stable APs were included for further analysis. As an alternative, to sharp electrode recordings, we performed whole-cell patch-clamp recordings on aCMs following a previously described protocol ( 52 ) but using a modified extracellular solution containing 4.5 mM KCl. After functional experiments, aCM were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at RT.…”

Section: Methodsmentioning

confidence: 99%

WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

et al. 2022

Self Cite

View full text Add to dashboard Cite

The electric excitability of muscle, heart, and brain tissue relies on the precise interplay of Na + - and K + -selective ion channels. The involved ion fluxes are controlled in optogenetic studies using light-gated channelrhodopsins (ChRs). While non-selective cation-conducting ChRs are well established for excitation, K + -selective ChRs (KCRs) for efficient inhibition have only recently come into reach. Here, we report the molecular analysis of recently discovered KCRs from the stramenopile Hyphochytrium catenoides and identification of a novel type of hydrophobic K + selectivity filter. Next, we demonstrate that the KCR signature motif is conserved in related stramenopile ChRs. Among them, WiChR from Wobblia lunata features a so far unmatched preference for K + over Na + , stable photocurrents under continuous illumination, and a prolonged open-state lifetime. Showing high expression levels in cardiac myocytes and neurons, WiChR allows single- and two-photon inhibition at low irradiance and reduced tissue heating. Therefore, we recommend WiChR as the long-awaited efficient and versatile optogenetic inhibitor.

show abstract

Section: Methodsmentioning

confidence: 99%

WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…MΦ TR were isolated from murine hearts as previously described. (Fernandez, Kopton et al 2021, Simon-Chica, Fernández et al 2022) In short, mice were euthanised by cervical dislocation, hearts were swiftly excised and gently washed with 20 mL of cold phosphate buffered saline (PBS, Sigma Aldrich (in mM: KH 2 PO 4 136, NaCl 58, Na 2 HPO 4 -7H 2 O 268)). Thereafter, tissue was minced into small pieces and enzymatically digested for 50 min under gentle agitation with a mixture of 450 U/mL collagenase I, 125 U/mL collagenase XI, 60 U/mL DNase I, and 60 U/mL hyaluronidase (all Sigma-Aldrich, Munich, Germany) in 1 mL PBS at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…MΦ TR were isolated from murine hearts as previously described. (Fernandez, Kopton et al 2021, Simon-Chica, Fernández et al 2022 MΦ BM were in-vitro-differentiated from bone marrow, harvested from murine femurs and tibiae. Epiphyses were removed and a 23G needle was inserted into one end.…”

Section: Mφ Isolationmentioning

confidence: 99%

Piezo1 stretch-activated channel activity differs between bone marrow-derived and cardiac tissue-resident macrophages

Simon-Chica,

Klesen,

Emig

et al. 2023

Preprint

View full text Add to dashboard Cite

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment recently emerged as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical for cellular activity in particular in a rhythmically contracting organ such as the heart. MΦ,in-vitro-differentiated from bone marrow (MΦBM), form a popular cell model for research. This study explores the activity of stretch-activated ion channels (SAC) in murine MΦBMand compares it to SAC activity in cardiac tissue-resident MΦ (MΦTR). Our main findings are: i) MΦBMand MΦTRhave stretch-induced currents, indicating expression of functional SAC at their plasma membrane; ii) the current profiles in MΦBMand in MΦTRshow characteristics of cation non-selective SAC; iii) unlike in MΦBM, Piezo1 ion channel activity at the plasma membrane of MΦTRis not detectable, neither by assessing electrophysiological activity using the patch clamp technique, nor by measuring cytosolic calcium concentration upon perfusion with Yoda1, a Piezo1 channel agonist. In mature scars after ventricular cryoablation, stretch-induced current characteristics of MΦTRare not significantly different compared to non-injured control tissue, even though scars are expected to contain a mix of pre-existing and circulation-recruited MΦ. This suggests that MΦ invading injured cardiac tissue either phenoconvert their mechanosensitivity from MΦBMto MΦTR, or that thein vitrodifferentiation protocols used to obtain MΦBMgenerate cells that differ from MΦ recruited from the circulation during tissue repairin vivo. Further investigations will explore SAC identity in lineage-traced MΦ in scar tissue, and compare mechanosensitivity of circulating monocytes with that of MΦBM.Key pointsMΦBMand MΦTRhave stretch-induced currents, indicating expression of functional SAC at their plasma membrane;The current profiles in MΦBMand in MΦTRshow characteristics of cation non-selective SAC;Unlike in MΦBM, Piezo1 ion channel activity at the plasma membrane of MΦTRis not detectable

show abstract

“…Heterozygous mice (C57BL/6J) transgenic for Cre recombinase fused to two modified estrogen receptor ligand binding domains under the control of the transcription factor 21 promoter (Tcf21-MerCreMer (Acharya et al ., 2011)), or transgenic for Cre recombinase fused to a single mutant estrogen ligand binding domain under the control of the chemokine receptor 1 promoter (Cx3cr1-CreERT (Yona et al ., 2013)) were crossed with homozygous mice transgenic for floxed Cop4 H134R-eYFP (B6; 129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J (Madisen et al ., 2012)). The resulting offspring expressed ChR2-eYFP in either FB (Tcf21-ChR2) or MΦ (Cx3cr1-ChR2) upon tamoxifen induction (Fernández et al ., 2021). Mice were weaned at P28–35 and ear snips were taken for PCR-based genotyping.…”

Section: Methodsmentioning

confidence: 99%

3D structure of fibroblasts and macrophages in the healthy and cryo-ablated heart

Fernández,

Wülfers,

Madl

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

IntroductionCardiac non-myocytes (NM) play important roles in heart development, homeostasis, and lesion repair. To assess the relevance of different NM populations for cardiac (patho)physiology, a quantitative assessment of their abundance and structure in the different heart chambers is an essential prerequisite. We here present an experimental approach to determine the distribution, dimensions, and 3D morphology of fibroblasts (FB) and macrophages (MΦ) in healthy and pathologically remodelled hearts.Methods and resultsWe used Cre-loxPrecombination to selectively target channelrhopsin-2 (ChR2)-eYFP to either FB or MΦ in healthy and cryo-ablated mouse hearts. Hearts were optically cleared using X-CLARITY and membrane-bound eYFP fluorescence was recorded by confocal microscopy. The resulting image stacks were segmented to generate 3D reconstructions of labelled cell populations innear native tissue. In doing so, we show that FB and MΦ have similar surface areas, volumes and morphologies, but that FB occupy larger fractional volumes than MΦ in all chambers of healthy murine hearts. Furthermore, MΦ appear primarily as single cells, whereas FB form extended networks of interconnected cells. In left-ventricular tissue following cryo-ablation, we observed large disordered networks of FB in the scar area with an increased volume occupied by FB both in the scar and remotely. In cryo-ablated ventricles, MΦ form comparatively small, but dense networks in the scar without changing their abundance in remote myocardium.ConclusionsOur study assesses the 3D distribution and structure of fluorescently labelled FB and MΦ in healthy and lesioned murine hearts. Based on 3D reconstructions of FB and MΦ networks, we quantified the surface areas and volumes of individual non-myocytes in the different chambers of the heart and in ventricular scar tissue, thus providing important quantitative data serving as basis for computational modelling of non-myocyte contributions to cardiac structure and physiology.

show abstract

Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology

Cited by 9 publications

References 30 publications

WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

Piezo1 stretch-activated channel activity differs between bone marrow-derived and cardiac tissue-resident macrophages

3D structure of fibroblasts and macrophages in the healthy and cryo-ablated heart

Contact Info

Product

Resources

About