Myocardial slices as an in vitro platform to study cardiac disease

In vitro models are the pillars of fundamental research and drug discovery, offering reductionist methods to better understand cellular responses in isolation. Often these methods are oversimplified, which makes their relevance to human biology and clinical translation ambiguous. Living myocardial slices (LMSLMSs) are viable thin (200-400μm) cardiac tissue slices, with preserved native multicellularity, architecture, mechanical and electrophysiological responses. Recent development in their culture, by us and others, paved the way for long-term preservation of adult mammalian heart tissue in vitro, without significant changes in its function and structure. This model has been extensively used in healthy tissue; however, to date, there are no established pathological models to study disease progression in vitro. Here we hypothesised that LMSLMSs can be used as an informative in vitro disease model to study temporal and spatial changes in cardiac function/structure in response to local cardiac damage. Before inducing cardiac damage, we further improved and characterised the cultured LMS model by designing robust tissue holders, optimising the oxygenation of the media, and establishing the best slice thickness (300μ) for oxygen diffusion and tissue stability in culture. We found that the LMSLMSs were adequately oxygenated in the inner layers and responded to mechanical stimuli with an increase in their contraction and hyperpolarisation of the mitochondrial membrane. We then developed a cryoinjury model, by applying a cooled probe on the LMSLMSs. We found that injury created a distinct necrotic area, surrounded by a border zone (BZ). The injury resulted in preserved force but electrical instability, with the presence of spontaneous contractions. Microscopic analysis of the BZ showed the presence of high numbers of spontaneous Ca2+ sparks, which could be affected by inhibiting the activation of Ca2+/calmodulin-dependent protein kinase II (CamKII). The inhibitory effect was more pronounced in endocardial LMSLMSs, showing transmural differences of CamKII under pathological conditions. Structural analysis of the BZ also showed an acute increase of the sarcomere length and loss of t-tubule density upon culture, that could also account for the arrhythmogenicity of the injured LMSLMSs. One application of therapeutic interventions on the model, by using extracellular vesicles (EVs), did not show any functional or molecular improvements. This thesis demonstrates the significance of using diseased LMSLMSs to study the way that local injury affects tissue stability, function, and structure. Further work is required to better understand the link between spontaneous Ca2+ and contraction events, as well as finding successful therapeutic interventions.Open Acces

Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) for three dimensional visualization of myocardial tissue

AbstractSeveral pathologic conditions of the heart lead to cardiac structural remodelling. Given the high density and the opaque nature of the myocardium, deep three dimensional (3D) imaging is difficult to achieve and structural analysis of pathological myocardial structure is often limited to two dimensional images and of thin myocardial sections. Efficient methods to obtain optical clearing of the tissue for 3D visualisation are therefore needed. Here we describe a rapid, simple and versatile Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) protocol specifically designed for cardiac tissue. With this method 3D information regarding collagen content, collagen localization and distribution could be easily obtained across a whole 300 µm-thick myocardial slice. FASTClear does not induce structural or microstructural distortion and it can be combined with immunostaining to identify the micro- and macrovascular networks. In summary, we have obtained decolorized myocardial tissue suitable for high resolution 3D imaging, with implications for the study of complex cardiac tissue structure and its changes during pathology.</jats:p

Investigation of cardiac fibroblasts using myocardial slices

Aims: Cardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro , CFs undergo rapid phenotypic changes including increased expression of \u3b1-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices. Methods and Results: Unloading of myocardial slices induced CF proliferation without \u3b1-SMA expression up to 7 days in culture . CFs migrating onto the culture plastic support or cultured on glass expressed \u3b1SMA within 3 days. The cells on the slice remained \u3b1SMA(-) despite TGF-\u3b2 (20ng/mL) or angiotensin II (200\ub5M) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at day 3 and 7 (P\u2009&lt;\u20090.001). Conclusions: Myocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential target

Investigation of cardiac fibroblasts using myocardial slices

Aims: Cardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro , CFs undergo rapid phenotypic changes including increased expression of α-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices. Methods and Results: Unloading of myocardial slices induced CF proliferation without α-SMA expression up to 7 days in culture . CFs migrating onto the culture plastic support or cultured on glass expressed αSMA within 3 days. The cells on the slice remained αSMA(-) despite TGF-β (20ng/mL) or angiotensin II (200µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at day 3 and 7 (P &lt; 0.001). Conclusions: Myocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential target