Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts

Sander, Veronika; Suñé, Guillermo; Jopling, Chris; Morera, Cristina; Belmonte, Juan Carlos Izpisúa

doi:10.1038/nprot.2013.041

Cited by 71 publications

(57 citation statements)

References 32 publications

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“…Pools of three ventricles from naive control fish ( cmlc2:nlsGFP ) and Tnni3k transgenics ( cmlc2:Tnni3k-2a-nlsGFP , abbreviated as cmlc2:mTn-ni3k in the text) were digested in 5 mg/ml collagenase type II and 5 mg/ml collagenase type IV as described 51 . Following stopping buffer washes, cells were filtered through a 250-μm mesh and fixed with 2% PFA for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Patterson¹,

Barske²,

Handel³

et al. 2017

Nat Genet

275

301

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Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

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Section: Methodsmentioning

confidence: 99%

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Patterson¹,

Barske²,

Handel³

et al. 2017

Nat Genet

275

301

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show abstract

“…Isolation and culture of primary cardiomyocytes was done as published 29 . Hearts were isolated from one year old fish.…”

Section: Methodsmentioning

confidence: 99%

Actin Binding GFP Allows 4D In Vivo Imaging of Myofilament Dynamics in the Zebrafish Heart and the Identification of Erbb2 Signaling as a Remodeling Factor of Myofibril Architecture

Reischauer

Arnaout

Ramadass

et al. 2014

Circulation Research

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Rationale Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of anti-neoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date. Objective Imaging in vivo myocardial contractile filament dynamics and assess potential causes of dilated cardiomyopathy in anti-neoplastic therapies targeting Erbb2. Methods and Results We generated a transgenic zebrafish line expressing an actin-binding GFP in cardiomyocytes, allowing in vivo imaging of myofilaments. Analysis of this line revealed architectural differences in myofibrils of the distinct cardiomyocyte subtypes. We used this model to investigate the effects of Erbb2 signaling on myofibrillar organization, since drugs targeting ERBB2 (HER2/NEU) signaling, a mainstay of breast cancer chemotherapy, cause dilated cardiomyopathy in many patients. High-resolution in vivo imaging revealed that Erbb2 signaling regulates a switch between a dense apical network of filamentous myofibrils and the assembly of basally localized myofibrils in ventricular cardiomyocytes. Conclusions Using this novel line, we compiled a reference for myofibrillar microarchitecture among myocardial subtypes in vivo and at different developmental stages, establishing this model as a tool to analyze in vivo cardiomyocyte contractility and remodeling for a broad range of cardiovascular questions. Further, we applied this model to study Erbb2 signaling in cardiomyopathy. We show a direct link between Erbb2 activity and remodeling of myofibrils, revealing an unexpected mechanism with potentially important implications for prevention and treatment of cardiomyopathy.

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“…1 One of the most widely used clinical CM models involves the use of rodent neonatal ventricular CMs (NRVMs) on tissue culture plates (TCPS). This model is popular because of its versatility 2 and convenience, compared to whole animal heart experiments. 3 Research with this model has enabled morphological, biochemical and electrophysiological characterization of the heart, 4 providing insight into the contraction, ischemia, hypoxia and toxicity of various compounds.…”

Section: Introductionmentioning

confidence: 99%

Biomimetic Microstructure Morphology in Electrospun Fiber Mats is Critical for Maintaining Healthy Cardiomyocyte Phenotype

et al. 2015

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Despite recent advances in biomimetic substrates, there is still only limited understanding of how the extracellular matrix (ECM) functions in the maintenance of cardiomyocyte (CM) phenotype. In this study, we designed electrospun substrates inspired by morphologic features of non-failing and failing human heart ECM, and examined how these substrates regulate phenotypes of adult and neonatal rat ventricular CMs (ARVM and NRVM, respectively). We found that poly(ε-caprolactone) fiber substrates designed to mimic the organized ECM of a non-failing human heart maintained healthy CM phenotype (evidenced by cell morphology, organized actin/myomesin bands and expression of β-MYH7 and SCN5A.1 and SCN5A.2) compared to both failing heart ECM-mimetic substrates and tissue culture plates. Moreover, culture of ARVMs and NRVMs on aligned substrates showed differences in m- and z-line alignment; with ARVMs aligning parallel to the ECM fibers and the NRVMs aligning perpendicular to the fibers. The results provide new insight into cardiac tissue engineering by illustrating the importance models that mimic the cardiac ECM microenvironment in vitro.

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Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts

Cited by 71 publications

References 32 publications

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Actin Binding GFP Allows 4D In Vivo Imaging of Myofilament Dynamics in the Zebrafish Heart and the Identification of Erbb2 Signaling as a Remodeling Factor of Myofibril Architecture

Biomimetic Microstructure Morphology in Electrospun Fiber Mats is Critical for Maintaining Healthy Cardiomyocyte Phenotype

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