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

Stein, Jeroen M.; Arslan, Ulgu; Franken, Marnix; Greef, Jessica C. de; Harding, Siân E.; Mohammadi, Neda; Orlova, Valeria V.; Bellin, Milena; Mummery, Christine L.; Meer, Berend J. van

doi:10.1002/cpz1.462

Cited by 9 publications

(8 citation statements)

References 31 publications

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“…MTs were compared in the absence (MToC, Video S1 , Figures 1 B and 3 A) or presence (VMToC, Figure 3 B) of an external vascular network. Sarcomere morphologies appeared similar in the MToC ( Figure 3 C) and VMToC ( Figure 3 D), which was confirmed by similar sarcomere lengths ( Figure 3 E) and alignment indices ( Figure 3 F) assessed using the SOTATool ( Stein et al., 2022 ). This indicated that there was no additional effect on sarcomere organization in the presence of external vascular network.…”

Section: Resultssupporting

confidence: 63%

Vascularized hiPSC-derived 3D cardiac microtissue on chip

et al. 2023

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Section: Resultssupporting

confidence: 63%

Vascularized hiPSC-derived 3D cardiac microtissue on chip

et al. 2023

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“…Moreover, we observed that ACTA1 R256H/+ hPSC-CMs appear to have sarcomeric arrays that were less aligned with the longitudinal cell axis compared to WT. We quantified the alignment using SarcOmere Texture Analysis algorithm (SotaTool) which mathematically scores sarcomere alignment on a scale between 0 to 1, with higher alignment corresponding to a higher score (42). Consistent with our observations, WT cells had a significantly higher alignment score than ACTA1 R256H/+ hPSC-CMs ( Figure 5I ).…”

Section: Resultsmentioning

confidence: 99%

“…Immunofluorescence staining and measurement of sarcomere alignment. Immunostaining was performed similar to prior (38), but SotaTool was used to calculate sarcomere alignment (42). Details can be found in the supplemental methods.…”

Section: Methodsmentioning

confidence: 99%

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility

Garg,

Jansen,

Zhang

et al. 2024

Preprint

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Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1's high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only ~20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 mutation, R256H. We previously identified this mutation in multiple family members with dilated cardiomyopathy (DCM), who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent functional effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using Cryo-EM, and we see alterations in actin's structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1R256H/+human induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric disorganization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy.

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“…Instead, the 4F+ Asns KD group exhibited significantly fewer cardiomyocytes staining for cell cycle markers Ki67, pH3, and Aurora kinase B (Figure 5N through 5P). At the same time, we adopted the software SotaTool 35 to compare the sarcomeric organization of cardiomyocytes between the 4F+ LacZ KD and 4F+ Asns KD groups. Each heart section was imaged at different representative regions with cardiomyocytes sectioned through the longitudinal axis, yielding 80 output results each (Figure S11B).…”

Section: Resultsmentioning

confidence: 99%

Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation

Zhu,

Ackers-Johnson,

Shanmugam

et al. 2024

Circulation

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BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte–targeted delivery of reprogramming factors ( Oct4 , Sox2 , Klf4 , and Myc ) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase ( Asns ) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4 , Sox2 , Klf4 , and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4 , Sox2 , Klf4 , and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns /mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

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

Cited by 9 publications

References 31 publications

Vascularized hiPSC-derived 3D cardiac microtissue on chip

Vascularized hiPSC-derived 3D cardiac microtissue on chip

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility

Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation

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