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.

During the last decade, optogenetics has emerged as a paradigm-shifting technique to monitor and steer the behavior of specific cell types in excitable tissues, including the heart. Activation of cation-conducting channelrhodopsins (ChR) leads to membrane depolarization, allowing one to effectively trigger action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarization-induced inhibition of AP generation has remained challenging. The green-light activated ChR from Guillardia theta (GtACR1) mediates Cl⁻-driven photocurrents that have been shown to silence AP generation in different types of neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocyte activity. Using single-cell electrophysiological recordings and contraction tracking, as well as intracellular microelectrode recordings and in vivo optical recordings of whole hearts, we find that GtACR1 activation by prolonged illumination arrests cardiac cells in a depolarized state, thus inhibiting re-excitation. In line with this, GtACR1 activation by transient light pulses elicits AP in rabbit isolated cardiomyocytes and in spontaneously beating intact hearts of zebrafish. Our results show that GtACR1 inhibition of AP generation is caused by cell depolarization. While this does not address the need for optogenetic silencing through physiological means (i.e., hyperpolarization), GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarization.

Macrophages (MΦ), known for immunological roles, such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrioventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterize passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM. We combined classic electrophysiological approaches with 3D florescence imaging, RNA-sequencing, pharmacological interventions, and computer simulations. We used Cx₃ $cr1eYFP/+$ mice wherein cardiac MΦ are fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2±0.1 GΩ (all data mean±SEM), capacitance 18.3±0.1 pF, resting membrane potential −39.6±0.3 mV, and several voltage-activated, outward or inwardly rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, and DIDS), flow cytometry, immuno-staining, and RNA-sequencing, we identified Kv1.3, Kv1.5, and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarize resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1. Our results provide a first electrophysiological characterization of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ–CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources.

In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes. Despite high specificity when using the same Cre driver lines in a previous study in combination with a different optogenetic probe, we found patchy off-target ChR2 expression in cryo-sections and extended z-stack imaging through the ventricular wall of hearts cleared using CLARITY. Based on immunohistochemical analysis, single-cell electrophysiological recordings and whole-genome sequencing, we reason that non-specificity is caused on the Cre recombination level. Our study highlights the importance of careful design and validation of the Cre recombination targets for reliable cell class specific expression of optogenetic tools.

The heart is one of the least regenerative organs in humans, and ischemic heart disease is the leading cause of death worldwide. Understanding the cellular and molecular processes that occur during cardiac wound healing is an essential prerequisite to reducing health burden and improving cardiac function after myocardial tissue damage. Here, by integrating single-cell RNA sequencing with high-resolution spatial transcriptomics, we reconstruct the spatiotemporal dynamics of the fibrotic niches after cardiac injury in adult mice. We reveal a complex multicellular network that regulates cardiac repair, including fibroblast proliferation silencing by Trem2 ^high macrophages to prevent excessive fibrosis. We further discovered a rare population of progenitor-like cardiomyocytes after lesion, promoted by myeloid and lymphoid niche signals. Culturing non-regenerative mouse cardiomyocytes or human heart tissue with these niche factors reactivated progenitor gene expression and cell cycle activity. In summary, this spatiotemporal atlas provides valuable insights into the heterocellular interactions that control cardiac repair.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical – in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦ_BM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦ_BM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue‐resident MΦ; MΦ_TR). We show that MΦ_BM and MΦ_TR have stretch‐induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦ_BM and in MΦ_TR show characteristics of cation non‐selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦ_BM using the patch‐clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦ_TR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦ_TR and in MΦ_BM. In MΦ isolated from left‐ventricular scar tissue 28 days after cryoablation, stretch‐induced current characteristics are not significantly different compared to non‐injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre‐existing cardiac and circulation‐recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦ_BM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage‐traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦ_BM. image Key points: Bone marrow‐derived (MΦ_BM) and tissue resident (MΦ_TR) macrophages have stretch‐induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch‐activated current profiles show characteristics of cation non‐selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦ_BM and in MΦ_TR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦ_TR and in MΦ_BM. Piezo1 ion channel activity is detected in the plasma membrane of MΦ_BM but not in MΦ_TR, suggesting that MΦ_BM may not be a good model to study the mechanotransduction of MΦ_TR. Stretch‐induced currents, Piezo1 mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28 days after cryoinjury compared to sham operated mice.