<i>In vivo</i> analysis of cardiomyocyte proliferation during trabeculation

Uribe, Verónica; Ramadass, Radhan; Dogra, Deepika; Rasouli, S. Javad; Gunawan, Felix; Nakajima, Hiroyuki; Chiba, Ayano; Reischauer, Sven; Mochizuki, Naoki; Stainier, Didier Y. R.

doi:10.1242/dev.164194

Cited by 49 publications

(54 citation statements)

References 74 publications

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“…Not only would this maintain cortical cardiomyocyte number, the disassembly of sarcomeres (known to occur prior to proliferation) might also liberate the cell for delamination. Our findings suggest that such events could have been missed in studies where time-lapse imaging was acquired only in a single plane, potentially underestimating the contribution of dividing cortical cardiomyocytes to pioneer trabecular cardiomyocytes [5,6,30,37]. This insight was only possible due to the high spatial and temporal resolution of our optically-gated imaging system.…”

Section: Discussionmentioning

confidence: 91%

“…The ability to image an embryo at a cellular and subcellular level is crucial for understanding the dynamic processes and interactions underpinning development [1][2][3][4][5][6]. The ideal imaging system would acquire high resolution 3D images of specific organs and structures continuously over relevant time-periods, typically hours or days, without causing tissue damage or interfering with the anatomical or physiological state of the organism.…”

Section: Introductionmentioning

confidence: 99%

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Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart

et al. 2019

Preprint

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Three-dimensional fluorescence time-lapse imaging of structural, cellular and subcellular processes in the beating heart is an increasingly achievable goal using the latest imaging and computational techniques. However, previous approaches have had significant limitations. Temporarily arresting the heart using drugs disrupts the heart's physiological state, and the use of ultra-high frame-rates for fluorescence image acquisition causes phototoxic cell damage. Real-time triggered imaging, synchronized to a specific phase in the cardiac-cycle, can computationally "freeze" the heart to acquire the minimal number of fluorescence images required for 3D time-lapse imaging. However, until now no solution has been able to maintain phase-lock to the same point in the cardiac cycle for more than about one hour. Our new hybrid optical gating system maintains phase-lock for up to 24 h, acquiring synchronized 3D+time video stacks of the unperturbed heart in vivo. This approach has enabled us to observe detailed developmental, structural, cellular and subcellular processes, including live cell division and cell fate tracking, in the embryonic zebrafish heart using transgenic fish lines expressing cell-specific fluorophores. We show that our approach not only provides high spatial and temporal resolution 3D-imaging, but also avoids phototoxic injury, where alternative approaches induce measurable harm. This provides superb cellular and subcellular imaging of the heart while it is beating in its normal physiological state, and opens up new and exciting opportunities for further study in the heart and other moving cellular and subcellular structures in vivo.

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Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart

et al. 2019

Preprint

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“…This completion of cytokinesis is precluded by the disassembly of the contractile apparatus. 67,68 With CMs requiring disassembly of their contractile apparatus to undergo mitosis the process of cell division has to be strictly regulated. The molecular events that mediate CM mitotic exit are poorly understood and in order to elucidate the underlying mechanisms resulting in activation of the endogenous cardiac regeneration process, a method for identifying CMs with true proliferative potential is needed.…”

Section: Discussionmentioning

confidence: 99%

Isolation of cardiomyocytes undergoing mitosis with complete cytokinesis

Milliron

Weiland

Kort

et al. 2019

Preprint

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Rationale-Adult human cardiomyocytes (CMs) do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result polyploidization and multinucleation occur. In order to get a deeper understanding of the mechanisms surrounding division of CMs there is a crucial need for a technique to isolate CMs that complete cell division/cytokinesis.Objective-Markers of cell cycle progression based on DNA content cannot distinguish between mitotic CMs that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MB) targeting specific mRNAs we aimed to identify truly proliferative CMs derived from hiPSCs.Methods and Results-Fluorescence activated cell-sorting combined with molecular beacons was performed to sort CM populations enriched for mitotic cells. Expressions of cell-cycle specific genes were confirmed by means of RT-qPCR, single-cell RNA sequencing (scRNA-seq). We further characterized the sorted groups by proliferation assays and time-lapse microscopy which confirmed the proliferative advantage of MB-positive cell populations relative to MB-negative and G2/M populations. Gene expression analysis revealed that the MB-positive CM subpopulation exhibited patterns consistent with the biological processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs (CDC20 + SPG20 + ) enabled the enrichment of cytokinetic events. Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20 -SPG20 + while polyploid CMs that replicated DNA but failed to complete karyokinesis were found to be CDC20 -SPG20 -.Conclusions-This study demonstrates a novel alternative to existing DNA content-based approaches for sorting CMs with true mitotic potential that can be used to study in detail the unique dynamics of CM nuclei during mitosis. Together with high-throughput scRNA-seq, our technique for sorting live CMs undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue.

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“…At the level of the cardiomyocytes, chamber outgrowth occurs via an increase in cell size and coordinated changes in cell shape and directionality (Lin et al, 2012). This is augmented by a localized resumption of cell proliferation and the continued addition of newly differentiated tissue to the ends of the tube (the heart poles) (Kelly et al, 2014;Uribe et al, 2018). Directed hypertrophy determines the overall shape of a chamber while cell division is more evident during ballooning stages in mammalian hearts that undergo rapid increases in size (Günthel et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Defective heart chamber growth and myofibrillogenesis after knockout ofadprhl1gene function by targeted disruption of the ancestral catalytic active site

Smith

Towers

Demetriou

et al. 2020

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1 Cardiac adprhl1 CRISPR knockout Smith et al. Mohun Highlights.Comparison of adprhl1 morpholinos. Knockdown of the two Xenopus cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of ventricle outgrowth. CRISPR/Cas9 targeted gene mutation of adprhl1 with multiple gRNAs reveals exonic locations that encode critical amino acids for Adprhl1 function.Repair of DSBs at exon 6 yields small in-frame deletions that cause specific ventricle myofibril assembly defects.The deletions disturb a conserved di-arginine containing peptide loop at the centre of the ancestral substrate binding cleft/ADP-ribosylhydrolase site of this pseudoenzyme.Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion. 2 Cardiac adprhl1 CRISPR knockout Smith et al. Mohun Abstract.ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.

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In vivo analysis of cardiomyocyte proliferation during trabeculation

Cited by 49 publications

References 74 publications

Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart

Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart

Isolation of cardiomyocytes undergoing mitosis with complete cytokinesis

Defective heart chamber growth and myofibrillogenesis after knockout ofadprhl1gene function by targeted disruption of the ancestral catalytic active site

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