Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics

Pitoulis, Fotios G.; Hasan, Waseem; Papadaki, Mary; Clavere, Nicolas G.; Perbellini, Filippo; Harding, Siân E.; Kirk, Jonathan A.; Boateng, Samuel Y.; Tombe, Pieter P. de; Terracciano, Cesare M.

doi:10.1016/j.yjmcc.2020.03.007

Cited by 17 publications

(22 citation statements)

References 20 publications

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“…Like other cardiac preparations, the Frank–Starling relationship can be observed in LMS (Fig. 2 ) [ 17 , 36 ]. Reported contractility parameters (isometric force measurements) indicate that freshly prepared viable myocardial slices should generate a force of ~ 15 mN/mm 2 [ 43 , 44 ].…”

Section: Contractile Functionmentioning

confidence: 72%

“…The second was the slicing of tissue blocks in the epicardium-tangential plane, which ensures optimal alignment of cardiomyocytes with the slicing blade and results in substantially reduced cell damage [ 31 , 47 ]. LMS are now prepared from almost all of the animal species including mice [ 20 , 39 , 40 , 52], rats [ 4 – 7 , 11 , 29 , 35 , 39 49, 50, 53], guinea pigs [ 6 , 7 , 48], rabbits [48], dogs [ 8 , 38 , 39 ] and pigs [ 36 ]). LMS can also be prepared from human specimens [ 3 , 9 , 29 , 41 , 43 , 44 ] providing great potential for translational studies.…”

Section: Lms Preparationmentioning

confidence: 99%

“…Biochemical assessment can be used to assess the genomic and proteomic signatures of LMS. Several of these assays can be robustly reproduced, with no need for further characterization or optimization [ 36 , 43 , 44 ]. However, it is important to understand how LMS features are characterized and how these data may be integrated with and utilized for other applications and to address the questions of several research fields.…”

Section: Interrogation Of Lms Physiology—function Structure and Biocmentioning

confidence: 99%

“…Recent findings from Pitoulis et al . have demonstrated transmural differences in contractile function, where LMS from the epicardium and mid-myocardium had a higher active and passive tension than those generated from the endocardium [ 36 ]. This aspect is often overlooked when using isolated cardiomyocytes, and something that should be considered during experimental design.…”

Section: Contractile Functionmentioning

confidence: 99%

“…Previous publications have shown that informative data such as cardiomyocyte morphology, t-tubules or mitochondria density and regularity, and stromal cell numbers can be acquired [ 1 , 32 , 43 ]. Heterogeneity in cardiomyocyte morphology and particularly to their aspect ratio (length:width) has recently been demonstrated in LMS generated from the epicardium compared to midmyocardium and endocardium [ 36 ]. Subcellular targets such as connexins (density and distribution) can be identified and subsequently correlated with conduction velocity parameters [ 43 ].…”

Section: Lms Structure: Visualizing Cells Ecm and Biomoleculesmentioning

confidence: 99%

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A practical guide for investigating cardiac physiology using living myocardial slices

Watson

Dendorfer²,

Thum

et al. 2020

Basic Res Cardiol

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Ex vivo multicellular preparations are essential tools to study tissue physiology. Among them, the recent methodological and technological developments in living myocardial slices (LMS) are attracting increasing interest by the cardiac research field. Despite this, this research model remains poorly perceived and utilized by most research laboratories. Here, we provide a practical guide on how to use LMS to interrogate multiple aspects of cardiac function, structure and biochemistry. We discuss issues that should be considered to conduct successful experiments, including experimental design, sample preparation, data collection and analysis. We describe how laboratory setups can be adapted to accommodate and interrogate this multicellular research model. These adaptations can often be achieved at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software, which is essential to broaden the utilization of this method. We will also highlight how current measurements can be improved to further enhance data quality and reliability to ensure inter-laboratory reproducibility. Finally, we summarize the most promising biomedical applications and envision how living myocardial slices can lead to further breakthroughs.

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Section: Contractile Functionmentioning

confidence: 72%

Section: Lms Preparationmentioning

confidence: 99%

Section: Interrogation Of Lms Physiology—function Structure and Biocmentioning

confidence: 99%

Section: Contractile Functionmentioning

confidence: 99%

Section: Lms Structure: Visualizing Cells Ecm and Biomoleculesmentioning

confidence: 99%

See 3 more Smart Citations

A practical guide for investigating cardiac physiology using living myocardial slices

Watson

Dendorfer²,

Thum

et al. 2020

Basic Res Cardiol

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Mechanosensitive molecular mechanisms of myocardial fibrosis in living myocardial slices

et al. 2022

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Aims Altered mechanical load in response to injury is a main driver of myocardial interstitial fibrosis. No current in vitro model can precisely modulate mechanical load in a multicellular environment while maintaining physiological behaviour. Living myocardial slices (LMS) are a 300 μm-thick cardiac preparation with preserved physiological structure and function. Here we apply varying degrees of mechanical preload to rat and human LMS to evaluate early cellular, molecular, and functionality changes related to myocardial fibrosis. Methods and resultsLeft ventricular LMS were obtained from Sprague Dawley rat hearts and human cardiac samples from healthy and failing (dilated cardiomyopathy) hearts. LMS were mounted on custom stretchers and two degrees of diastolic load were applied: physiological sarcomere length (SL) (SL = 2.2 μm) and overload (SL = 2.4 μm). LMS were maintained for 48 h under electrical stimulation in circulating, oxygenated media at 37°C. In overloaded conditions, LMS displayed an increase in nucleus translocation of Yes-associated protein (YAP) and an up-regulation of mechanotransduction markers without loss in cell viability. Expression of fibrotic and inflammatory markers, as well as Collagen I deposition were also observed. Functionally, overloaded LMS displayed lower contractility (7.48 ± 3.07 mN mm À2 at 2.2 SL vs. 3.53 ± 1.80 mN mm À2 at 2.4 SL). The addition of the profibrotic protein interleukin-11 (IL-11) showed similar results to the application of overload with enhanced fibrosis (8% more of collagen surface coverage) and reduced LMS contractility at physiological load. Conversely, treatment with the Transforming growth factor β receptor (TGF-βR) blocker SB-431542, showed down-regulation of genes associated with mechanical stress, prevention of fibrotic response and improvement in cardiac function despite overload (from 2.40 ± 0.8 mN mm À2 to 4.60 ± 1.08 mN mm À2 ). Conclusions The LMS have a consistent fibrotic remodelling response to pathological load, which can be modulated by a TGF-βR blocker. The LMS platform allows the study of mechanosensitive molecular mechanisms of myocardial fibrosis and can lead to the development of novel therapeutic strategies.

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Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes

Wojtkiewicz

Luecke

Castro

et al. 2022

Journal of Molecular and Cellular Cardiology

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The heart is composed of multiple cell types, each with a specific function. Cell-type-specific approaches are necessary for defining the intricate molecular mechanisms underlying cardiac development, homeostasis, and pathology. While single-cell RNA-seq studies are beginning to define the chamber-specific cellular composition of the heart, our views of the proteome are more limited because most proteomics studies have utilized homogenized human cardiac tissue. To promote future cell-type specific analyses of the human heart, we describe the first method for cardiomyocyte isolation from cryopreserved human cardiac tissue followed by flow cytometry for purity assessment. We also describe a facile method for preparing isolated cardiomyocytes and whole cardiac tissue homogenate for bottom-up proteomic analyses. Prior experience in dissociating cardiac tissue or proteomics is not required to execute these methods. We compare different sample preparation workflows and analysis methods to demonstrate how these can impact the depth of proteome coverage achieved. We expect this how-to guide will serve as a starting point for investigators interested in general and cell-type-specific views of the cardiac proteome.

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Intact myocardial preparations reveal intrinsic transmural heterogeneity in cardiac mechanics

Cited by 17 publications

References 20 publications

A practical guide for investigating cardiac physiology using living myocardial slices

A practical guide for investigating cardiac physiology using living myocardial slices

Mechanosensitive molecular mechanisms of myocardial fibrosis in living myocardial slices

Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes

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