a-cardiac-microphysiological-system-for-studying-ca2-propagation-via

A Cardiac Microphysiological System for Studying Ca<sup>2+</sup> Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

University of Milano-Bicocca

Istituto Italiano di Tecnologia

Brugada syndrome (BrS) is an inherited arrhythmogenic disease that may lead to sudden cardiac death in young adults with structurally normal hearts. No pharmacological therapy is available for BrS patients. This situation highlights the urgent need to overcome current difficulties by developing novel groundbreaking curative strategies. BrS has been associated with mutations in 18 different genes of which loss-of-function (LoF)  mutations constitute the second most common cause. Here we tested the hypothesis that BrS associated with mutations in the  gene encoding the L-type calcium channel (LTCC) pore-forming unit (Caα1.2) is functionally reverted by administration of a mimetic peptide (MP), which through binding to the LTCC chaperone beta subunit (Caβ2) restores the physiological life cycle of aberrant LTCCs. Two novel Caα1.2 mutations associated with BrS were identified in young individuals. Transient transfection in heterologous and cardiac cells showed LoF phenotypes with reduced Ca current (I). In HEK293 cells overexpressing the two novel Caα1.2 mutations, Western blot analysis and cell surface biotinylation assays revealed reduced Caα1.2 protein levels at the plasma membrane for both mutants. Nano-BRET, Nano-Luciferase assays, and confocal microscopy analyses showed (i) reduced affinity of Caα1.2 for its Caβ2 chaperone, (ii) shortened Caα1.2 half-life in the membrane, and (iii) impaired subcellular localization. Treatment of Caα1.2 mutant-transfected cells with a cell permeant MP restored channel trafficking and physiologic channel half-life, thereby resulting in I similar to wild type. These results represent the first step towards the development of a gene-specific treatment for BrS due to defective trafficking of mutant LTCC.

Peptide-Based Targeting of the L-Type Calcium Channel Corrects the Loss-of-Function Phenotype of Two Novel Mutations of the  Gene Associated With Brugada Syndrome.

Cardiovascular diseases are the leading cause of mortality worldwide. Such a widespread diffusion makes the conditions affecting the heart and blood vessels a primary medical and economic burden. It, therefore, becomes mandatory to identify effective treatments that can alleviate this global problem. Among the different solutions brought to the attention of the medical-scientific community, therapeutic angiogenesis is one of the most promising. However, this approach, which aims to treat cardiovascular diseases by generating new blood vessels in ischemic tissues, has so far led to inadequate results due to several issues. In this perspective, we will discuss cutting-edge approaches and future perspectives to alleviate the potentially lethal impact of cardiovascular diseases. We will focus on the consolidated role of resident endothelial progenitor cells, particularly endothelial colony forming cells, as suitable candidates for cell-based therapy demonstrating the importance of targeting intracellular Ca signaling to boost their regenerative outcome. Moreover, we will elucidate the advantages of physical stimuli over traditional approaches. In particular, we will critically discuss recent results obtained by using optical stimulation, as a novel strategy to drive endothelial colony forming cells fate and its potential in the treatment of cardiovascular diseases.

Towards Novel Geneless Approaches for Therapeutic Angiogenesis.

The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC-CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal-like hiPSC-CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC-CMs maturity, spanning from long-term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.

Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation.

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a "superinhibition".

Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Therapeutic neovascularization represents a promising strategy to rescue the vascular network and restore organ function in cardiovascular disorders (CVDs), including acute myocardial infarction, heart failure, peripheral artery disease, and brain stroke. Endothelial colony forming cells (ECFCs), which are mobilized in circulation upon an ischemic insult, are commonly regarded as the most suitable cellular tool to achieve therapeutic neovascularization. ECFCs can be genetically or pharmacologically manipulated to enhance their vasoreparative potential by boosting specific pro-angiogenic signalling pathways. However, optical stimulation represents the most reliable approach to control cellular activity because of its high selectivity and unprecedented spatio-temporal resolution. Herein, we discuss a novel strategy to drive ECFC angiogenic activity in ischemic tissues by combining geneless optical excitation with photosensitive organic semiconductors. We describe how photoexcitation of the conducting polymer poly(3-hexylthiophene-2,5-diyl), also known as P3HT, stimulates extracellular Ca entry through Transient Receptor Potential Vanilloid 1 (TRPV1) channels upon the production of hydrogen peroxide (HO) in the cleft between the nanomaterial and the cell membrane. HO-induced TRPV1-dependent Ca entry stimulates ECFC proliferation and tube formation, thereby providing the proof-of-concept that photoexcitation of organic semiconductors may offer a reliable strategy to stimulate ECFCs-dependent neovascularization in CVDs.

Optical excitation of organic semiconductors as a highly selective strategy to induce vascular regeneration and tissue repair.

Cardiac mesenchymal stromal cells (C-MSC) were recently shown to differentiate into adipocytes and myofibroblasts to promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM). A calcium (Ca) signaling dysfunction, mainly demonstrated in mouse models, is recognized as a mechanism impacting arrhythmic risk in ACM cardiomyocytes. Whether similar mechanisms influence ACM C-MSC fate is still unknown. Thus, we aim to ascertain whether intracellular Ca oscillations and the Ca toolkit are altered in human C-MSC obtained from ACM patients, and to assess their link with C-MSC-specific ACM phenotypes.

Ca dysregulation in cardiac stromal cells sustains fibro-adipose remodeling in Arrhythmogenic Cardiomyopathy and can be modulated by flecainide.

The heterozygous phospholamban (PLN) mutation R14del (PLN R14del ) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. "Superinhibition" of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to (1) to evaluate Ca dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; (2) to test whether they are causally connected.

Early consequences of the phospholamban mutation PLN-R14del in a transgenic mouse model.

Geneless optomodulation of cardiomyocytes membrane potential by sarcolemma-targeted azobenzene photoswitches.

Electrophysiological effects of estrogen on hiPSC-derived cardiomyocytes of a patient affected by estrogen-sensitive Long-QT Syndrome Type 2.

The use of light to control the activity of living cells is a promising approach in cardiac research due to its unparalleled spatio-temporal selectivity and minimal invasiveness. Ziapin2, a newly synthesized azobenzene compound, has recently been reported as an efficient tool for light-driven modulation of excitation-contraction coupling (ECC) in human-induced pluripotent stem cells-derived cardiomyocytes. However, the exact biophysical mechanism of this process remains incompletely understood.

Role of stretch-activated channels in light-generated action potentials mediated by an intramembrane molecular photoswitch.

Arrhythmogenic Cardiomyopathy (ACM) is a life-threatening, genetically determined disease primarily caused by mutations in desmosomal genes, such as PKP2. Currently, there is no etiological therapy for ACM due to its complex and not fully elucidated pathogenesis. Various cardiac cell types affected by the genetic mutation, such as cardiomyocytes (CM) and cardiac mesenchymal stromal cells (cMSC), individually contribute to the ACM phenotype, driving functional abnormalities and fibro-fatty substitution, respectively. However, the relative importance of the CM and cMSC alterations, as well as their reciprocal influence in disease progression remain poorly understood. We hypothesised that ACM-dependent phenotypes are driven not only by alterations in individual cell types but also by the reciprocal interactions between CM and cMSC, which may further impact disease pathogenesis. We utilized a patient-specific, multicellular cardiac system composed of either control or PKP2-mutated CM and cMSC to assess the mutation's role in fibro-fatty phenotype by immunofluorescence, and contractile behaviour of co-cultures using cell motion detection software. Additionally, we investigated reciprocal interactions both in silico and via multi-targeted proteomics. We demonstrated that ACM CM can promote fibro-adipose differentiation of cMSC. Conversely, ACM cMSC contribute to increasing the rate of abnormal contractile events with likely arrhythmic significance. Furthermore, we showed that an ACM-causative mutation alters the CM-cMSC interaction pattern. We identified the CM-sourced DLK1 as a novel regulator of fibro-adipose remodelling in ACM. Our study challenges the paradigm of exclusive cell-specific mechanisms in ACM. A deeper understanding of the cell-cell influence is crucial for identifying novel therapeutic targets for ACM, and this concept is exploitable for other cardiomyopathies.

Cardiomyocyte and stromal cell cross-talk influences the pathogenesis of arrhythmogenic cardiomyopathy: a multi-level analysis uncovers DLK1-NOTCH pathway role in fibro-adipose remodelling.

We introduce a family of membrane-targeted azobenzenes (MTs) with a push-pull character as a new tool for cell stimulation. These molecules are water soluble and spontaneously partition in the cell membrane. Upon light irradiation, they isomerize from trans to cis, changing the local charge distribution and thus stimulating the cell response. Specifically, MTs photoisomerization induces clear and reproducible depolarization. The most promising species, MTP2, was extensively studied. Time-resolved spectroscopy techniques provide insights into the excited state evolution and a complete understanding of its isomerization reaction. Molecular Dynamics simulations reveal the spontaneous and stable partitioning of the compound into the cellular membrane, without significant alterations to the bilayer thickness. MTP2 was tested in different cell types, including HEK293T cells, primary neurons, and cardiomyocytes, and a steady depolarization is always recorded. The observed membrane potential modulation in in-vitro models is attributed to the variation in membrane surface charge, resulting from the light-driven modulation of the MT dipole moment within the cell membrane. Additionally, a developed mathematical model successfully captures the temporal evolution of the membrane potential upon photostimulation. Despite being insufficient for triggering action potentials, the rapid light-induced depolarization holds potential applications, particularly in cardiac electrophysiology. Low-intensity optical stimulation with these modulators could influence cardiac electrical activity, demonstrating potential efficacy in destabilizing and terminating cardiac arrhythmias. We anticipate the MTs approach to find applications in neuroscience, biomedicine, and biophotonics, providing a tool for modulating cell physiology without genetic interventions.

Article	Year
Peptide-Based Targeting of the L-Type Calcium Channel Corrects the Loss-of-Function Phenotype of Two Novel Mutations of the Gene Associated With Brugada Syndrome. Frontiers in physiology\| PubMed ID: 33488405	2020
Towards Novel Geneless Approaches for Therapeutic Angiogenesis. Frontiers in physiology\| PubMed ID: 33551844	2020
Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation. Biology\| PubMed ID: 34439963	2021
Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. International journal of molecular sciences\| PubMed ID: 34948294	2021
Optical excitation of organic semiconductors as a highly selective strategy to induce vascular regeneration and tissue repair. Vascular pharmacology\| PubMed ID: 35589009	2022
Ca dysregulation in cardiac stromal cells sustains fibro-adipose remodeling in Arrhythmogenic Cardiomyopathy and can be modulated by flecainide. Journal of translational medicine\| PubMed ID: 36371290	2022
Early consequences of the phospholamban mutation PLN-R14del in a transgenic mouse model. Acta physiologica (Oxford, England)\| PubMed ID: 38214033	2024
Geneless optomodulation of cardiomyocytes membrane potential by sarcolemma-targeted azobenzene photoswitches. Vascular pharmacology\| PubMed ID: 38985611	2024
Electrophysiological effects of estrogen on hiPSC-derived cardiomyocytes of a patient affected by estrogen-sensitive Long-QT Syndrome Type 2. Vascular pharmacology\| PubMed ID: 38985641	2024
Role of stretch-activated channels in light-generated action potentials mediated by an intramembrane molecular photoswitch. Journal of translational medicine\| PubMed ID: 39605022	2024
Cardiomyocyte and stromal cell cross-talk influences the pathogenesis of arrhythmogenic cardiomyopathy: a multi-level analysis uncovers DLK1-NOTCH pathway role in fibro-adipose remodelling. Cell death discovery\| PubMed ID: 39609399	2024
Membrane-targeted push-pull azobenzenes for the optical modulation of membrane potential. Light, science & applications\| PubMed ID: 39741143	2025

Francesco Lodola

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