(Sub-)Cellular Basis of Cardiomyocyte-Interstitium Electro-Mechanical Crosstalk in vitro
Basic research in P1, P2, and P3 explores (sub-)cellular principles of cardiomyocyte/interstitium crosstalk in vitro, focussing on nano- and micro-scale mechanisms of electrical and mechanical interactions of cardiomyocytes (CM), non-myocytes (NM), and extracellular matrix (ECM).
More specifically, P1 (Kohl/Loewe) will explore ‘first principles’ of NM–CM electrical interaction by systematically categorising NM-effects (and their mechanisms) that alter cardiac AP shape and conduction. Specific targets include the nature of passive cable properties, and active (excitation-based continuous or saltatory conduction) signal propagation, as well as the means of altering speed and reach of trans-scar conduction (CM repeater-station theory). This provides a quantitative conceptual basis for analytical interpretation of data in more integrated systems (tissue to organ), aided by provision of shared computational models. P2 (Rog-Zielinska/Lehnart) will complement this in the mechano-structural interaction domain, providing novel insight into the mechanisms of dynamic fibroblast (FB)-mediated ECM nano-organisation, not just in the peri-CM space (which one usually considers when studying interstitial fibrosis), but also within CM extracellular nano-environments, incl. intrastitial fibrosis within transverse-axial tubular system (TATS). This will aid understanding of CM mechanical integration into the deformable cardiac skeleton (ECM), in steady-state, and during progredient or reverse remodelling, using cells from hearts with interstitial remodelling caused by aldosterone/ salt diet (from P6) or trans-aortic constriction (TAC; from P7), and after the removal of respective triggers of remodelling. P3 (Schneider-Warme/Streckfuss-Bömeke) will complement these investigations that focus on how NM affect CM by studying how interstitial cell structure, function, and signalling are shaped by the biophysical activity of CM. To dissociate electrical and mechanical effects from other CM inputs to NM (e.g. biochemical signals), the project will use advanced optogenetic techniques to impose AP-like transmembrane voltage swings onto electrically non-excitable NM, and it will exploit stretchable culture substrates to mimic effects of the cardiac contractile cycle on NM – identifying the response dynamics of NM to stimuli that in native tissue would be generated by CM. Projects will use hiPSC-derived CM and NM (developed in-house, and provided in meaningful, quality-controlled quantities via HELMA) as sources or targets of interstitial interaction. While generally limited by incomplete maturity, hiPSC-derived CM have essential properties of their adult human counterparts (such as ‘long and plateaued’ action potential, crucial for P1). We will enhance maturation (such as by growing cells on structured surfaces that guide cell alignment and aid contractile filament integration in P1), explore induction of TATS structures (P2), and utilise novel long-term maturation protocols (P3). Gene-edited and maintained in culture for extended periods of time, or cryo-preserved, hiPSC-derived cells will serve as a driver of CM-NM crosstalk studies (P1-P4).
Thus, while P1 explores NM-to-CM electrical, and P2 NM-to-CM mechano-structural signalling, P3 complements this by studying CM-to-NM electro-mechanical communication. This will drive insight into roles of the interstitium as a myocardial interaction hub, informing tissue structure and function (see in vitro level), and serve as a basis for assessing and interpreting electro-mechanical behaviour in situ and in vivo. In return, insight into structural and biophysical interaction patterns of interstitial components and CM, obtained in P4-P8, can be tested in simplified in vitro models, to dissect essential parameters not identifiable in more complex systems, aided by quantitative analysis from computational modelling and AI-based tools.
Interstitial Effects on Cardiac Electro-Mechanical Function in situ
Building on the above, P4, P5, and P6 investigate interstitial cell plasticity and its roles for the integration of cardiac electro-mechanical function in native tissue, to identify mechanisms underlying effects of interstitial remodelling on heart function during ageing and in pathological remodelling with focal and diffuse fibrosis.
P4 (Grune/Lenz) will dissect the interrelation of ageing, immune cells (IC), and focal post-myocardial infarction (MI) replacement fibrosis, focussing on contributions by resident (e.g. macrophages [MΦ]) and early-invading IC (neutrophils, monocytes), and assess how protein-mediated signalling of IC is linked to interstitial plasticity and electrical function of the heart. Their work will dissect effects of interstitial remodelling driven by ageing from interstitial remodelling driven by pathology. In addition, identified IC protein signatures and signalling pathways (incl. extracellular vesicles) that are relevant for electrical function, will be validated in human samples. P5 (Zgierski-Johnston/Huisken) will conduct ex vivo whole heart structure−function mapping and computational modelling, to quantitatively explore dynamic, heart rate-dependent effects of TAC-induced diffuse interstitial remodelling on AP conduction in native tissue. This will provide novel mechanistic insight into contributions of interstitial NM and ECM to electrical conduction, as a basis for improving arrhythmia risk assessment from non-invasive imaging data. P6 (Peyronnet/Lother) will explore effects of mechano-sensitive ion channels in NM on cardiac interstitial remodelling, with a view of targeting these channels in a cell-type-specific manner, to prevent fibrosis-associated arrhythmogenesis. This will take mechanical cues ‘full circle’, from remodelling-induced changes in tissue mechanics, to mechano-sensitive NM signalling of relevance for interstitial plasticity, and ECM formation/maintenance.
While P4 explores focal remodelling, P5 and P6 investigatediffuse interstitial remodelling, linked primarily either to systolic or diastolic dysfunction (respectively), thereby covering a broad range of cardiac interstitial changes. All three projects use young adult mice as a reference to compare and contrast features of disease-related interstitial plasticity to age-matched healthy hearts. In addition, P4 studies age-associated changes in interstitial dynamics, to contextualise findings in younger animals, and to add a translationally relevant dimension, in addition to the use of human tissue in P4 and P6. Using native myocardial tissue for in situ structure-function mapping, projects P4–P6 (see in situ level) therefore link (sub-)cellular insight from in vitro research in P1–P3 to in vivo studies in P7 and P8, including the provision of tissue for in vitro research, and the sharing of potential targets for control of interstitial remodelling in vivo.
Steering Interstitial Integration of Cardiac Electro-Mechanical Function in vivo
Topping-out our translational efforts, P7 and P8 develop new approaches for steering interstitial contributions to cardiac function in vivo, controlling collagen cross-linking to prevent and/or reverse interstitial remodelling, and targeting therapeutic interventions selectively to ECM in the inflamed interstitium – as a means of altering interstitial effects on myocardial behaviour exactly where it matters.
P7 (Lindner/Hilgendorf) will study the relevance of cardiac NM for enzymatic collagen cross-linking in replacement and interstitial fibrosis, to establish how collagen-specific post-translational modifications affect cardiac function in vivo, and to assess how this can be used to enhance reversibility of diffuse (post-TAC/TAC release) and focal (post-MI) fibrosis in preclinical models. This will define the role of cardiac NM in regulating collagen quantity and quality, and determine how changes in cross-linking influence passive tissue mechanics and cardiac electro-mechanical function, while also assessing whether identified mechanisms may be amenable targets for intervention. P8 (Briquez/Kessler) in turn will apply a novel approach for targeting fusion protein drugs to the cardiac ECM. Building on expertise with wound healing, this aims to allow systemic application of compounds, while still achieving their selective enrichment in the inflamed ECM, so that treatments may be directed non-invasively to areas of active interstitial remodelling. The ADAMTS-7 (protease)/SVEP1 (target) axis will serve as the initial proof of concept, used for modulating interstitial remodelling, illustrating the novel paradigm of ECM-mediated interventions that steer intercellular NM communication and CM function.
Both P7 and P8 focus on the ECM as unique hallmark of the interstitium and as a target for intervention. P7 pioneers exploration of enzymatic collagen cross-linking, and integrates insight from models for the induction (and termination) of diffuse and focal remodelling in (matching models in P4, P5 and P8). P8 purses a unique and novel strategy for delivering therapeutically relevant interventions specifically to those parts of the cardiac interstitium that may be in need of treatment. The underlying ‘self-homing’ intervention furthermore allows systemic application of low-dose compounds that get enriched in the remodelling target tissue. The exact kind of message that may need to be delivered – whether targeting predominantly CM-NM electrical (e.g. P1, P3, P4, P5), structural (e.g. P2, P5), or mechanical crosstalk (P3, P6), the ECM (P7, P8), or any combination thereof – is one of the key questions at the heart of research across FOR 6051 projects. The answer to this question may vary for different types of interstitial remodelling (focal/diffuse, driven by systolic or diastolic dysfunction, progredient or reverse) – which explains the selection of disease models included by the network.