Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes
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Evaluation Summary:
This paper will be of interest to scientists in the field of regenerative medicine. The authors compare effects of persistent lentiviral expression of various mitogens in cardiomyocytes in vitro. Technically experiments are of a very high standard, but the data are somewhat difficult to translate to the in vivo situation. The statistical analyses would have to be robust and sufficient for the conclusions to be supported by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in CMs as a potential strategy for regenerative heart repair.
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Reviewer #3 (Public Review):
The authors sought to directly compare manipulations of different signaling pathways for their ability to induce cell cycle activation and proliferation in cardiomyocytes from various species and maturation levels. The manipulation consisted of peristent lentiviral expression of beta catenin, cyclin D2, rat Erbb2, human Erbb2, and Yap8SA.
A major strength of this study is that it shows that most of above expressions appeared to induce negative feed-back responses at the post-transcriptional level to limit protein overexpression, illustrating how difficult it is to manipulate cardiomyocyte proliferation. By contrast, human Erbb2 did induce prominent proliferative effects in both rat and human cardiomyocytes. However, this finding has been shown before. The novelty here is limited to interspecies differences …
Reviewer #3 (Public Review):
The authors sought to directly compare manipulations of different signaling pathways for their ability to induce cell cycle activation and proliferation in cardiomyocytes from various species and maturation levels. The manipulation consisted of peristent lentiviral expression of beta catenin, cyclin D2, rat Erbb2, human Erbb2, and Yap8SA.
A major strength of this study is that it shows that most of above expressions appeared to induce negative feed-back responses at the post-transcriptional level to limit protein overexpression, illustrating how difficult it is to manipulate cardiomyocyte proliferation. By contrast, human Erbb2 did induce prominent proliferative effects in both rat and human cardiomyocytes. However, this finding has been shown before. The novelty here is limited to interspecies differences of the effects of Erbb2 overexpression. Multiple studies in oncology have shown that Erbb2 overexpression increases cell proliferation and is sufficient to induce cancer growth. It has also been shown that transient overexpression of Erbb2 in vivo in the heart results in dedifferentiation and proliferation of cardiomyocytes. The observation that Erbb2 overexpression induces cardiomyocyte dedifferentiation alongside mitosis is not unexpected; in general, stimuli that induce cardiomyocyte proliferation also induce cardiomyocyte dedifferentiation and sarcomere disassembly.
In this study, in a 3D model of rat neonatal cardiobundles Erbb2 overexpression also led to formation of a necrotic core. It also led to loss of sarcomeres and contractile force and tissue stiffening. These effects appeared to be mediated by mTOR-independent, Erk-dependent mechanisms. Although experiments in this study are of a high technical level, and results interesting, the likely impact of this work is minor. Indeed, the overall picture of Erbb2-induced pathologic hypertrophy is likely related to the applied methodologies, i.e., a persistent as opposed to temporally controlled Erbb2 overexpression and the use of an avascular 3D model lacking the cellular complexity of the intact heart.
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Reviewer #2 (Public Review):
This manuscript by Nicholas Strash et al. compares the effects of several potential mitogens on cell cycle of the two most used in vitro models of cardiomyocytes (CMs): neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell (hiPSC)-derived CMs. In addition, they use a 3D model of NRVMs as a model that represents more mature, non-proliferating CMs. The work is interesting for researchers working in the field of cardiac regeneration and provides the first direct comparison of several potential mitogens. The inclusion of several in vitro models to account for potential species differences strengthens the data. The results support previously published findings and the main conclusions are supported by the data presented.
The authors used a 3D model, cardiobundles made from NRVMs, as a …
Reviewer #2 (Public Review):
This manuscript by Nicholas Strash et al. compares the effects of several potential mitogens on cell cycle of the two most used in vitro models of cardiomyocytes (CMs): neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell (hiPSC)-derived CMs. In addition, they use a 3D model of NRVMs as a model that represents more mature, non-proliferating CMs. The work is interesting for researchers working in the field of cardiac regeneration and provides the first direct comparison of several potential mitogens. The inclusion of several in vitro models to account for potential species differences strengthens the data. The results support previously published findings and the main conclusions are supported by the data presented.
The authors used a 3D model, cardiobundles made from NRVMs, as a more mature CM model. However, these cardiobundles still had a considerable number of CMs in active cell cycle in basal conditions. Whether this reflects true proliferation or the postnatal multinucleation process of rat cardiomyocytes, is unclear. Furthermore, post-mitotic human CMs were not studied. These can be obtained from hiPSC-CMs by prolonged culture or using metabolic stimuli as shown by Mills et al. 2017 (PNAS).
The authors demonstrate that the known mitogenic pathway for CMs, Erbb2-mediated signalling, promotes cell cycle activation in 2D cultures or NRVMS and hiPSC-CMs as well as in 3D cardiobundles. Although cell cycle activity was clearly induced, no actual proof of cytokinesis has been presented. For the cardiobundle work, it remains unclear if the increase in cross-sectional size of cardiobundles induced by Erbb2 signalling is due to increased number of CMs or increased size of CMs. Both the physiological ligand of Erbb3, Neuregulin-1, and the downstream ERK pathway are known to induce CM hypertrophy (see for example Zurek et al. 2020 Circulation; Bueno and Molkentin 2002 Circ Res).
The data analysis and statistics raise some concerns, which require clarification. First, the N numbers are really big and according to the Table 1 it is unclear if they all indeed represent independent samples. For example, one field in a monolayer (Table 1, definition of n in Figures 1J, 1P, 4C, 4E, 4G) should not be considered to represent n=1, if several images were analysed from the same sample and/or if several technical replicates (samples prepared from the same cell isolation or differentiation and treated similarly) were analysed. Only samples from separate differentiations or cell isolations should be considered as representatives of n and the results from technical replicates should be averaged to form the n=1 data. Second, the selection of statistical tests is a concern. It is unclear if the data were analysed for equal variances before selecting the test (parametric vs. non-parametric). It is also unclear why the authors carried out multiple t tests instead of using ANOVA or its variations, which are generally considered more suitable for multiple comparisons.
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Reviewer #1 (Public Review):
This manuscript titled "Human Erbb2-induced Erk Activity Robustly Stimulates Cycling and Functional Remodeling of Rat and Human Cardiomyocytes" directly compared a number of previously identified candidate mitogenic genes in different cardiomyocytes and different maturity status and investigated the pathway involved. The authors found that the human Erbb2 triggers the strongest proliferative effect in both human-induced Pluripotent Stem Cells and Neonatal Rat Ventricular Myocyte, and was associated with the Erk pathway. The authors then proved this association by demonstrating that inhibition with Mek inhibitor and Erk inhibitor attenuates the human Erbb2-induced response. In addition, the authors found that Yap8SA failed to trigger proliferation in the cardiomyocyte tested due to negative feedback loop. …
Reviewer #1 (Public Review):
This manuscript titled "Human Erbb2-induced Erk Activity Robustly Stimulates Cycling and Functional Remodeling of Rat and Human Cardiomyocytes" directly compared a number of previously identified candidate mitogenic genes in different cardiomyocytes and different maturity status and investigated the pathway involved. The authors found that the human Erbb2 triggers the strongest proliferative effect in both human-induced Pluripotent Stem Cells and Neonatal Rat Ventricular Myocyte, and was associated with the Erk pathway. The authors then proved this association by demonstrating that inhibition with Mek inhibitor and Erk inhibitor attenuates the human Erbb2-induced response. In addition, the authors found that Yap8SA failed to trigger proliferation in the cardiomyocyte tested due to negative feedback loop. Thus, this study provides helpful information regarding the relative effectiveness of a number of candidate genes.
Strengths:
— This study investigates five candidate genes in different species and different maturation status of cardiomyocyte. In each setting, all genes are studied. Therefore, direct comparison regarding their effectiveness can be made.
— Furthermore, this study demonstrated the mechanism on how the differing responses arose, providing in-depth information.
Weakness:
— Although this study showed induced proliferation of cardiomyocyte following candidate genes expression, the authors did not present sufficient proof that the function would improve. Cardiomyocyte harbor differing functions and parameters that represents it should ideally be investigated.
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Evaluation Summary:
This paper will be of interest to scientists in the field of regenerative medicine. The authors compare effects of persistent lentiviral expression of various mitogens in cardiomyocytes in vitro. Technically experiments are of a very high standard, but the data are somewhat difficult to translate to the in vivo situation. The statistical analyses would have to be robust and sufficient for the conclusions to be supported by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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