Physiological Biomimetic Culture System for Pig and Human Heart Slices.

RATIONALE: Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. OBJECTIVE: To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. METHODS AND RESULTS: Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to ß-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. CONCLUSIONS: We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3-dimensional heart model.

Myocardial Slices: an Intermediate Complexity Platform for Translational Cardiovascular Research.

Myocardial slices, also known as "cardiac tissue slices" or "organotypic heart slices," are ultrathin (100-400 µm) slices of living adult ventricular myocardium prepared using a high-precision vibratome. They are a model of intermediate complexity as they retain the native multicellularity, architecture, and physiology of the heart, while their thinness ensures adequate oxygen and metabolic substrate diffusion in vitro. Myocardial slices can be produced from a variety of animal models and human biopsies, thus providing a representative human in vitro platform for translational cardiovascular research. In this review, we compare myocardial slices to other in vitro models and highlight some of the unique advantages provided by this platform. Additionally, we discuss the work performed in our laboratory to optimize myocardial slice preparation methodology, which resulted in highly viable myocardial slices from both large and small mammalian hearts with only 2-3% cardiomyocyte damage and preserved structure and function. Applications of myocardial slices span both basic and translational cardiovascular science. Our laboratory has utilized myocardial slices for the investigation of cardiac multicellularity, visualizing 3D collagen distribution and micro/macrovascular networks using tissue clearing protocols and investigating the effects of novel conductive biomaterials on cardiac physiology. Myocardial slices have been widely used for pharmacological testing. Finally, the current challenges and future directions for the technology are discussed.

Validation of the CREST model & Comparison with SCAI Shock Classification for Prediction of Circulatory Death in Resuscitated Out of Hospital Cardiac Arrest.

OBJECTIVE: We validated the CREST model, a 5 variable score for stratifying risk of circulatory etiology death (CED) following out of hospital cardiac arrest (OHCA), and compared its discrimination with the SCAI shock classification. BACKGROUND: CED occurs in approximately a third of patients admitted after resuscitated OHCA. There is an urgent need for improved stratification of the OHCA patient on arrival to a cardiac arrest centre to improve patient selection for invasive interventions. METHODS: The CREST model and SCAI shock classification were applied to a dual-centre registry of 723 patients with cardiac etiology OHCA, both with and without ST-elevation myocardial infarction, between May 2012 to December 2020. The primary endpoint was 30-day CED. RESULTS: Of 509 patients included (62.3 years, 75.4% male), 125 patients had CREST=0 (24.5%), 162 were CREST=1 (31.8%), 140 were CREST=2 (27.5%), 75 were CREST=3 (14.7%), 7 were CREST of 4 (1.4%) and no patients were CREST=5. CED was observed in 91 (17.9%) patients at 30 days [STEMI - 51/289 (17.6%); NSTEMI - 40/220 (18.2%)]. For the total population, and both NSTEMI & STEMI subpopulations, increasing CREST score was associated with increasing CED (all p<0.001). CREST score and SCAI classification had similar discrimination for the total population (AUC=0.72/calibration slope=0.95), NSTEMI cohort (AUC=0.75/calibration slope=0.940) and STEMI cohort (AUC=0.69 and calibration slope=0.925). AUC meta-analyses demonstrated no significant differences between the two classifications. CONCLUSIONS: The CREST model and SCAI shock classification have similar prediction for the development of CED after OHCA.

Impact of the COVID-19 pandemic on in-hospital mortality in cardiovascular disease: a meta-analysis.

AIMS: The COVID-19 pandemic has resulted in excess mortality due to both COVID-19 directly and other conditions, including cardiovascular (CV) disease. We aimed to explore the excess in-hospital mortality, unrelated to COVID-19 infection, across a range of CV diseases. METHODS AND RESULTS: A systematic search was performed for studies investigating in-hospital mortality among patients admitted with CV disease without SARS-CoV-2 infection compared with a period outside the COVID-19 pandemic. Fifteen studies on 27 421 patients with CV disease were included in the analysis. The average in-hospital mortality rate was 10.4% (n = 974) in the COVID-19 group and 5.7% (n = 1026) in the comparator group. Compared with periods outside the COVID-19 pandemic, the pooled risk ratio (RR) demonstrated increased in-hospital mortality by 62% during COVID-19 [95% confidence interval (CI) 1.20-2.20, P = 0.002]. Studies with a decline in admission rate >50% during the COVID-19 pandemic observed the greatest increase in mortality compared with those with <50% reduction [RR 2.74 (95% CI 2.43-3.10) vs. 1.21 (95% CI 1.07-1.37), P < 0.001]. The observed increased mortality was consistent across different CV conditions (P = 0.74 for interaction). CONCLUSIONS: In-hospital mortality among patients admitted with CV diseases was increased relative to periods outside the pandemic, independent of co-infection with COVID-19. This effect was larger in studies with the biggest decline in admission rates, suggesting a sicker cohort of patients in this period. However, studies were generally poorly conducted, and there is a need for further well-designed studies to establish the full extent of mortality not directly related to COVID-19 infection.

Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.

Although past decades have witnessed significant reductions in mortality of heart failure together with advances in our understanding of its cellular, molecular, and whole-heart features, a lot of basic cardiac research still fails to translate into clinical practice. In this review we examine myocardial slices, a novel model in the translational arena. Myocardial slices are living ultra-thin sections of heart tissue. Slices maintain the myocardium's native function (contractility, electrophysiology) and structure (multicellularity, extracellular matrix) and can be prepared from animal and human tissue. The discussion begins with the history and current advances in the model, the different interlaboratory methods of preparation and their potential impact on results. We then contextualize slices' advantages and limitations by comparing it with other cardiac models. Recently, sophisticated methods have enabled slices to be cultured chronically in vitro while preserving the functional and structural phenotype. This is more timely now than ever where chronic physiologically relevant in vitro platforms for assessment of therapeutic strategies are urgently needed. We interrogate the technological developments that have permitted this, their limitations, and future directions. Finally, we look into the general obstacles faced by the translational field, and how implementation of research systems utilizing slices could help in resolving these.

Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.

Adult cardiac tissue undergoes a rapid process of dedifferentiation when cultured outside the body. The in vivo environment, particularly constant electromechanical stimulation, is fundamental to the regulation of cardiac structure and function. We investigated the role of electromechanical stimulation in preventing culture-induced dedifferentiation of adult cardiac tissue using rat, rabbit and human heart failure myocardial slices. Here we report that the application of a preload equivalent to sarcomere length (SL) = 2.2 µm is optimal for the maintenance of rat myocardial slice structural, functional and transcriptional properties at 24 h. Gene sets associated with the preservation of structure and function are activated, while gene sets involved in dedifferentiation are suppressed. The maximum contractility of human heart failure myocardial slices at 24 h is also optimally maintained at SL = 2.2 µm. Rabbit myocardial slices cultured at SL = 2.2 µm remain stable for 5 days. This approach substantially prolongs the culture of adult cardiac tissue in vitro.

Heterocellularity and Cellular Cross-Talk in the Cardiovascular System.

Cellular specialization and interactions with other cell types are the essence of complex multicellular life. The orchestrated function of different cell populations in the heart, in combination with a complex network of intercellular circuits of communication, is essential to maintain a healthy heart and its disruption gives rise to pathological conditions. Over the past few years, the development of new biological research tools has facilitated more accurate identification of the cardiac cell populations and their specific roles. This review aims to provide an overview on the significance and contributions of the various cellular components: cardiomyocytes, fibroblasts, endothelial cells, vascular smooth muscle cells, pericytes, and inflammatory cells. It also aims to describe their role in cardiac development, physiology and pathology with a particular focus on the importance of heterocellularity and cellular interaction between these different cell types.

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 transforming growth factor-ß (20 ng/ml) or angiotensin II (200 µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at Days 3 and 7 (P < 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 targets.

Preparation of viable adult ventricular myocardial slices from large and small mammals.

This protocol describes the preparation of highly viable adult ventricular myocardial slices from the hearts of small and large mammals, including rodents, pigs, dogs and humans. Adult ventricular myocardial slices are 100- to 400-µm-thick slices of living myocardium that retain the native multicellularity, architecture and physiology of the heart. This protocol provides a list of the equipment and reagents required alongside a detailed description of the methodology for heart explantation, tissue preparation, slicing with a vibratome and handling of myocardial slices. Supplementary videos are included to visually demonstrate these steps. A number of critical steps are addressed that must be followed in order to prepare highly viable myocardial slices. These include identification of myocardial fiber direction and fiber alignment within the tissue block, careful temperature control, use of an excitation-contraction uncoupler, optimal vibratome settings and correct handling of myocardial slices. Many aspects of cardiac structure and function can be studied using myocardial slices in vitro. Typical results obtained with hearts from a small mammal (rat) and a large mammal (human) with heart failure are shown, demonstrating myocardial slice viability, maximum contractility, Ca2+ handling and structure. This protocol can be completed in ∼4 h.

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

Several 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.