Automatic detection of adult cardiomyocyte for high throughput measurements of calcium and contractility

Cao, Lu; Manders, Emmy M.; Helmes, Michiel

doi:10.1371/journal.pone.0256713

“…We made a user‐friendly application in Python called SotaTool, which aims to enhance the performance of SOTA by implementing various background subtraction methods, adding an alignment index and including automated segmentation. In contrast to other approaches, the method does not require regions of interest (ROIs) to be pre‐defined or selected, or images of fluorescent sarcomeres to be aligned manually (Cao, Manders, & Helmes, 2021; Pasqualin et al., 2016; Peterson, Kalda, & Vendelin, 2013). While current algorithms typically rely on manual user input per image to indicate the directionality of the sarcomeres, SotaTool detects the optimal angle automatically, reducing analysis time and selection bias.…”

Section: Introductionmentioning

confidence: 99%

Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro

Stein

¹

,

Arslan

²

,

Franken

³

et al. 2022

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Sarcomeres are the structural units of the contractile apparatus in cardiac and skeletal muscle cells. Changes in sarcomere characteristics are indicative of changes in the sarcomeric proteins and function during development and disease. Assessment of sarcomere length, alignment, and organization provides insight into disease and drug responses in striated muscle cells and models, ranging from cardiomyocytes and skeletal muscle cells derived from human pluripotent stem cells to adult muscle cells isolated from animals or humans. However, quantification of sarcomere length is typically time consuming and prone to user‐specific selection bias. Automated analysis pipelines exist but these often require either specialized software or programming experience. In addition, these pipelines are often designed for only one type of cell model in vitro. Here, we present an easy‐to‐implement protocol and software tool for automated sarcomere length and organization quantification in a variety of striated muscle in vitro models: Two dimensional (2D) cardiomyocytes, three dimensional (3D) cardiac microtissues, isolated adult cardiomyocytes, and 3D tissue engineered skeletal muscles. Based on an existing mathematical algorithm, this image analysis software (SotaTool) automatically detects the direction in which the sarcomere organization is highest over the entire image and outputs the length and organization of sarcomeres. We also analyzed videos of live cells during contraction, thereby allowing measurement of contraction parameters like fractional shortening, contraction time, relaxation time, and beating frequency. In this protocol, we give a step‐by‐step guide on how to prepare, image, and automatically quantify sarcomere and contraction characteristics in different types of in vitro models and we provide basic validation and discussion of the limitations of the software tool. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Staining and analyzing static hiPSC‐CMs with SotaTool Alternate Protocol: Sample preparation, acquisition, and quantification of fractional shortening in live reporter hiPSC lines Support Protocol 1: Finding the image resolution Support Protocol 2: Advanced analysis settings Support Protocol 3: Finding sarcomere length in non‐aligned cells

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“…Fig. 2A ) 47 . Treatment with nutlin-3 significantly decreased fractional shortening (i.e., percent of contraction) of the muscle fibers in response to electrical stimulation, in a nutlin-3 dose-dependent manner ( Fig.…”

Section: Resultsmentioning

confidence: 97%

“…The order that the different treatments were measured was randomized and the researcher who performed the measurements was blinded to the treatments. All contractility measurements were performed at 25°C using a MultiCell High Throughput System (IonOptix, Westwood, MA) (45). Fibers were electronically stimulated using custom pacer with a pulse duration of 5.0 ms duration, 10 V and 1 Hz.…”

Section: Fdb Contractility Studiesmentioning

confidence: 99%

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in Lamin A/C-deficient mice

Kirby

¹

,

Zahr

²

,

Fong

³

et al. 2022

Preprint

1

0

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Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

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“…Muscle fibers were then assessed for contractile function using a novel high-throughput system for assessing intact myofiber function (Supplementary Fig. 2A ) [ 47 ]. Treatment with nutlin-3 significantly decreased fractional shortening (i.e., percent of contraction) of the muscle fibers in response to electrical stimulation, in a nutlin-3 dose-dependent manner (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The order that the different treatments were measured was randomized and the researcher who performed the measurements was blinded to the treatments. All contractility measurements were performed at 25 °C using a MultiCell High Throughput System (IonOptix, Westwood, MA) [ 47 ]. Fibers were electronically stimulated using custom pacer with a pulse duration of 5.0 ms duration, 10 V and 1 Hz.…”

Section: Methodsmentioning

confidence: 99%

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in lamin A/C-deficient mice

Kirby,

Zahr,

Fong

et al. 2024

Cell Death Discov.

2

0

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Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

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Automatic detection of adult cardiomyocyte for high throughput measurements of calcium and contractility

Cited by 4 publications

References 24 publications

Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro

Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in Lamin A/C-deficient mice

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in lamin A/C-deficient mice

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