preparation-of-human-myocardial-tissue-for-long-term-cultivation

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

University Hospital, LMU Munich

German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA)

In vitro models incorporating the complexity and function of adult human tissues are highly desired for translational research. Whilst vital slices of human myocardium approach these demands, their rapid degeneration in tissue culture precludes long-term experimentation. Here, we report preservation of structure and performance of human myocardium under conditions of physiological preload, compliance, and continuous excitation. In biomimetic culture, tissue slices prepared from explanted failing human hearts attain a stable state of contractility that can be monitored for up to 4 months or 2000000 beats in vitro. Cultured myocardium undergoes particular alterations in biomechanics, structure, and mRNA expression. The suitability of the model for drug safety evaluation is exemplified by repeated assessment of refractory period that permits sensitive analysis of repolarization impairment induced by the multimodal hERG-inhibitor pentamidine. Biomimetic tissue culture will provide new opportunities to study drug targets, gene functions, and cellular plasticity in adult human myocardium.

Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.

The original version of this Article incorrectly acknowledged Elisabeth Reiser and Rene Schramm as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article.

Publisher Correction: Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.

The normally positive cardiac force-frequency relationship (FFR) becomes flat or negative in chronic heart failure (HF). Here we explored if remodeling of the cardiomyocyte transverse tubular system (t-system) is associated with alterations in FFR and contractile kinetics in failing human myocardium. Left-ventricular myocardial slices from 13 failing human hearts were mounted into a biomimetic culture setup. Maximum twitch force (F), 90% contraction duration (CD), time to peak force (TTP) and time to relaxation (TTR) were determined at 37°C and 0.2-2 Hz pacing frequency. F /F.  and F /F.  served as measures of FFR, intracellular cardiomyocyte t-tubule distance (ΔTT) as measure of t-system remodeling. Protein levels of SERCA2, NCX1, and PLB were quantified by immunoblotting. F /F.  (  = 0.82) and F /F.  (  = 0.5) correlated negatively with ΔTT, i.e., samples with severe t-system loss exhibited a negative FFR and reduced myocardial wall tension at high pacing rates. PLB levels also predicted F /F. , but to a lesser degree (  = 0.49), whereas NCX1 was not correlated (  = 0.02). CD correlated positively with ΔTT (  = 0.39) and negatively with SERCA2/PLB (  = 0.42), indicating that both the t-system and SERCA activity are important for contraction kinetics. Surprisingly, ΔTT was not associated with TTP (  = 0) but rather with TTR (  = 0.5). This became even more pronounced when interaction with NCX1 expression was added to the model (  = 0.79), suggesting that t-system loss impairs myocardial relaxation especially when NCX1 expression is low. The degree of t-system remodeling predicts FFR inversion and contraction slowing in failing human myocardium. Moreover, together with NCX, the t-system may be important for myocardial relaxation.

The Degree of t-System Remodeling Predicts Negative Force-Frequency Relationship and Prolonged Relaxation Time in Failing Human Myocardium.

Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection.

SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.

Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies. All of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task. In particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and co-morbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models cannot provide a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on a organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and improved current animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction and refinement (3R) as a guiding concept.

Animal models and animal-free innovations for cardiovascular research: current status and routes to be explored. Consensus document of the ESC working group on myocardial function and the ESC Working Group on Cellular Biology of the Heart.

Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host-graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.

Article	Year
Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro. Nature communications\| PubMed ID: 30631059	2019
Publisher Correction: Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro. Nature communications\| PubMed ID: 30692546	2019
The Degree of t-System Remodeling Predicts Negative Force-Frequency Relationship and Prolonged Relaxation Time in Failing Human Myocardium. Frontiers in physiology\| PubMed ID: 32231589	2020
SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes. Cardiovascular research\| PubMed ID: 32966582	2020
Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome. Circulation\| PubMed ID: 34694888	2021
Animal models and animal-free innovations for cardiovascular research: current status and routes to be explored. Consensus document of the ESC working group on myocardial function and the ES... Cardiovascular research\| PubMed ID: 34999816	2022
Migratory and anti-fibrotic programmes define the regenerative potential of human cardiac progenitors. Nature cell biology\| PubMed ID: 35550611	2022

Andreas Dendorfer

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