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

Harvard University

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
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

Chiara Florindi

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