Loss of microRNA-128 promotes cardiomyocyte proliferation and heart regeneration

The goal of replenishing the cardiomyocyte (CM) population using regenerative therapies following myocardial infarction (MI) is hampered by the limited regeneration capacity of adult CMs, partially due to their withdrawal from the cell cycle. Here, we show that microRNA-128 (miR-128) is upregulated in CMs during the postnatal switch from proliferation to terminal differentiation. In neonatal mice, cardiac-specific overexpression of miR-128 impairs CM proliferation and cardiac function, while miR-128 deletion extends proliferation of postnatal CMs by enhancing expression of the chromatin modifier SUZ12, which suppresses p27 (cyclin-dependent kinase inhibitor) expression and activates the positive cell cycle regulators Cyclin E and CDK2. Furthermore, deletion of miR-128 promotes cell cycle re-entry of adult CMs, thereby reducing the levels of fibrosis, and attenuating cardiac dysfunction in response to MI. These results suggest that miR-128 serves as a critical regulator of endogenous CM proliferation, and might be a novel therapeutic target for heart repair.

The major concerns are as follows.
The explanation why the hearts of miR-128-expressing mice in Fig. 2C and 2D are hypertrophic is unclear. Should the authors' interpretation be that miR-128 blocks cardiomyocyte proliferation and thus induces compensatory cardiomyocyte hypertrophy, why should this also result in massive organ hypertrophy? The authors state that (p. 6): "Interestingly, miR-128OE mice displayed pathologically dilated cardiomyopathy that was consistent with focal replacement fibrosis, CM hypertrophy, and severe heart failure compared with Ctrl mice at the same adult stage (data not published)." This information is not consistent with what shown in Fig. 2. Given the relevance of these findings, these should be presented in detail in the manuscript.
The author state that, upon treatment with anti-miR-128, cardiomyocyte became "dedifferentiated" based on immunstaining for cardiac troponin T. It is unclear how the authors can define a "differentiated" or "undifferentiated" state based on the images shown in Suppl. Fig. 3. What are the cells with elongated filaments they define as "dedifferentiated"?? What are the majority of cells in these cultures, which do not stain with the anti-TnT antibody?
Suppl. Fig. 3E and 3G. Staining for phospho-H3 is not convincing. The number of positive cells is astonishingly high (more than 15%) for a marker that identifies cells that only transiently travel through the G2M phases of the cell cycle. The pictures show high magnification of a single cell, which is totally anecdotal and not significant. Fig. 3G-H: For a heart with such a marked hyperplasty as that shown in Fig. 3C, one would expect a remarkably high proliferation activity, while it appears that the number of cycling cells is only 2fold than that of controls. This is in sharp disagreement with the picture shown in Fig. 3G (by the way, in this picture the actual cell reactivity to Ki67 is covered by the arrow marks). In the same picture the representative image is rather ambiguous. Is this a cardiomyocyte nucleus? What is the red halo around the nucleus?? Again, it is unclear how quantification of "disassembled" cardiomyocytes was performed, and certainly this value should not be expressed as "# per section" -out of how many analyzed cells?
The authors wish to identify targets for miR-128 action and reach the conclusion that Suz12 as one of these. Why this one only? There are a number of already described targets for this miRNA and many more are predicted. This information needs to be reported and a more systematic analyses has to be conducted. It is hard to believe that the authors by chance picked up the only gene that mediates the effect of the miRNA based on notoriously fallacious prediction analysis! Figure 6F. Quantification of the number of cardiac myocytes cannot be based on the assessment of those that are isolated, especially when the difference is so relatively small between KO and controls Other minor issues Fig 1 panels A-C show the normal heart development at birth. This is textbook information, there is no need to show it A previous report indicates that miR-128 is elevated in both cardiomyocytes and noncardiomyocytes close to the regenerating zone during newt cardiac regeneration (Witman et al. Dev Biol 2013). Since apical regeneration in mice is believed to mimic fish and amphibian heart regeneration, this information appears relevant and needs to be compared with the findings reported in the manuscript. This study reports that miR-128 is induced in the postnatal heart and diminishes the regenerative activity of the heart. These key findings are based on loss and gain of function studies in vivo demonstrating an important effect of this miRNA on cardiomyocyte dedifferentiation/proliferation. Using adult mice, the deletion of miR-128 in cardiomyocytes increased the regenerative window and improved healing after myocardial infarctions. The authors additionally provide evidence that miR-128 is targeting SUZ12, and that this regulates the expression of the cell cycle inhibitor p27. The study is novel and well performed. I only have a few specific comments. Specific concerns 1. Figure 2H: the EF appears very high (90%) and is reduced to about 80 %, which would be normal in adult mice. Are these measurements correct or related to the measurement at P1? Are there are confounding effects on anesthesia/technical limitation of echo at these very early stages? 2. Figure 3F: please provide cell numbers for CM (a reduction of size does not necessarily indicate proliferation). 3. Cardiomyocyte proliferation should be documented in the therapeutic study by using Aurora Kinase staining. 4. The authors only investigated and named SUZ12 as target for miR-128. It is well known that miRNAs do not work by inhibiting one target. Although this reviewer understands that it is beyond the scope of the present study to address the full mechanism, the data showing regulation of miR-128 target should be provided as suppl. Figure. It would be essential to know how many miR-128 targets are regulated and how these might influence cell cycle progression/dedifferentiation. 5. miR-128 is highly expressed in neurons. Since innervation was shown to drive cardiac regeneration, the authors might wish to speculate about a putative function of miR-128 in this process. The manuscript by Huang et al., describes their work to show how miR-128 suppresses cardiomyocyte cell cycle activity in cardiac homeostasis and disease. miR-128 is expressed at low levels in neonatal hearts (P1) and elevated at P7 and P28. In a mouse model with cardiomocyte(CM)-specific overexpression of miR-128, they find that heart size is enlarged at P1 with increased CM size and CM proliferation. Deletion of miR-128 from Nkx2-5Cre lineage during cardiogenesis did not affect heart function at P7, although CM size was decreased and CM cell cycle activity was increased at P7 and P14. Using a bioinformatics approach, authors search for predicted target genes underexpressed in adult heart relative to neonatal heart and identify Suz12 as a potential direct miR-128 target. The direct regulation of Suz12 mRNA by miR-128 is shown in in vitro assays, and knockdown of Suz12 in Nkx-25Cre;miR-128 mutants rescues the proliferation phenotype. The authors then go on to test if miR-128 may interfere with cardiac regeneration after injury, using the neonatal apical resection model. In cardiomyocyte miR-128 overexpression mice, neonatal hearts do not regenerate to the same extent as in control mice, and cardiomyocyte proliferation is decreased. Furthermore, it is shown that after myocardial infarction in adult mice, loss of miR-128 enhances cardiac function and suppresses tissue fibrosis. Comments − Previous work has shown a direct regulation of Suz12 by miR-128 (Peruzzi et al., Neuro Oncol 2013), it is suggested to cite this paper. Moreover, work from the Pu lab has demonstrated the relevance of the PRC2 complex for cardiomyocyte development (He et al., Circ Res 2012). Since this study also uses nkx2-5Cre to interefere with PRC2 complex function, it is recommended authors discuss this paper. − For neonatal hearts and isolated CM, the authors propose that miR-128 suppresses Suz12 leading to decreased p27, cyclinE and Cdk1 in neonatal hearts and isolated CM. However, to evaluate cell cycle activity in miR-128 iKO heart after MI, the authors decide to use a different panel of genes compared to previous experiments in the manuscript. Can the authors show p27, CyclinE and Cdk2 levels in mutants versus controls? What happens to Suz12 levels in this experiment? − What is the baseline expression level of miR-128 in the heart and which cell types is it expressed in? − The authors claim that miR-128 regulates neonatal cell cycle withdrawal. However, for their overexpression and loss of function models, miR-128 levels are manipulated from embryonic stages onwards. Is miR-128 also expressed during development, and can the authors show at which timepoint during development miR-128 levels are changed in their overexpression and loss of function models? An alternative explanation to the observation that CM size is reduced and cell cycle activity is enhanced in the loss of function model is that embryonic CM do not mature in the absence of miR-128 and thus retain an embryonic or fetal proliferative phenotype. Previous studies using nkx2-5Cre to ablate the PRC2 complex subunit Ezh2 indicate requirements of this complex for CM development. As such the current study does not formally prove that miR-128 in heart directly regulates neonatal cell cycle withdrawal, as this observation may be secondary to embryonic onset defects. Can the authors comment on this? − Authors report that miR-128 mice display dilated cardiomyopathy consistent with focal replacement fibrosis, CM hypertrophy and severe heart failure in adults. Can data be provided to substantiate these claims? − EdU incorporation is quantified by counting the number of EdU+ CM per section. Because the authors also report a change in cell size between conditions, these quantifications should be corrected for the total number of CM per section. − Authors use a datamining approach to predict miR-128 target genes that are underexpressed in adult heart versus neonatal heart. Which dataset was used to establish differential expression? − Resolution of immuno images is not great -for instance for figure 1c, Figure 2i, Figure 6b: it can not be appreciated whether nuclei are CM nuclei or adjacent fibroblasts; for 6b: are we to believe that nearly all EdU+ cells are CM? − The exact genotype and treatment of controls is often missing, making it impossible to evaluate if the right contros were used. Please specify the genotype, and treatment (for instance with/without tamoxifen) of control and mutant for each experiment, for instance in figure legends. − The rescue experiment using Siz12 siRNA in miR-128 mutant hearts is not well described, please provide details. − The authors should discuss the recent paper by Zeng et al. showing that miR-128 inhibition during IR induced injury blocks cardiomyocyte apoptosis through the regulation of PPARG. What happens to apoptosis and PPARG in the studies presented in the current manuscript?
To Reviewer #1: We appreciate the reviewer's comments, which were very helpful in improving the manuscript.
The manuscript by Huang and collaborators reports the role of miR-128 in cardiomyocyte proliferation during fetal life and myocardial regeneration after cardiac damage. Through the use of a remarkable number of genetically modified mice to overexpress or conditionally block this miRNA, the authors wish to prove that its major effect is to repress cardiomyocyte proliferation. Yet, the manuscript leaves the reader both unconvinced of the effect and disappointed by the major conclusions, since most of the experiments are performed superficially and lack specific controls, thus rendering the major conclusions inconsistent.
The major concerns are as follows.

Reviewer comment 1: The explanation why the hearts of miR-128-expressing mice in Fig. 2C and 2D are hypertrophic is unclear. Should the authors' interpretation be that miR-128 blocks cardiomyocyte proliferation and thus induces compensatory cardiomyocyte hypertrophy, why should this also result in massive organ hypertrophy?
Author's Reply: Transgenic mice with cardiomyocyte (CM) specific miR-128 overexpression (miR-128 OE ) displayed cardiac hypertrophy and cardiac dysfunction, which coincides with a suppressed CM cell cycle. Our RNA-seq data supported this, revealing that the differentially expressed genes in miR-128 OE hearts belong to pathways involved in DNA replication, cell cycle, hypertrophic cardiomyopathy, and dilated cardiomyopathy (Supplementary Fig. 3). Therefore, we proposed that the suppressed CM cell cycle induced by miR-128 overexpression might be associated with the compensatory CM hypertrophy. Although the mechanism of cardiac hypertrophy is not fully understood, our ongoing studies include investigation into the mechanisms involved in miR-128 induced cardiac hypertrophy.
The discussion section has been modified accordingly on page 17-18: 'Cell hypertrophy is defined as cell enlargement due to an increase in protein and RNA content without DNA replication. KEGG pathway analysis revealed that the differentially expressed genes in miR-128 OE hearts belong to pathways involved in DNA replication, cell cycle, hypertrophic cardiomyopathy, and dilated cardiomyopathy. Transgenic mice overexpressing miR-128 displayed premature cell cycle exit, cardiac hypertrophy, and cardiac dysfunction. Further analysis is underway to explore the association of miR-128 activation in pathogenesis of congenital heart disease involving abnormalities of myocardial growth.' Reviewer comment 2: Fig. 2G-H (Nat Genet. 2015;47(7): 776-83. J Vis Exp. 2016;111). To analyze heart function by echocardiography, mouse pups (P1) were restrained by hand in the absence of anesthesia drugs. Gentle pressure was then used to apply an ultrasound probe to the pup's chest, eliminating the common anesthesia/technical limitations. To sustain this finding, we have added additional echocardiography parameters (left ventricular diastolic diameter-LVDd and left ventricular systolic diameter-LVDs) in Figs. 2G and H. EF was calculated as: EF%= [(LVDd) 3 -(LVDs) 3 /(LVDd) 3 ×100]. FS was determined as: FS%= [(LVDd-LVDs)/LVDd×100].
Reviewer comment 3: The authors state that (p. 6): "Interestingly, miR-128 OE mice displayed pathologically dilated cardiomyopathy that was consistent with focal replacement fibrosis, CM hypertrophy, and severe heart failure compared with Ctrl mice at the same adult stage (data not published)." This information is not consistent with what shown in Fig. 2. Given the relevance of these findings, these should be presented in detail in the manuscript.
Author's Reply: As suggested, additional details of our phenotype observations in adult miR-128 OE mice have been provided (Supplementary Figs. 2D-F, Supplementary Fig. 3).
Accordingly, this statement has been modified in the results section on page 7: 'To study the role of miR-128 in heart development, miR-128 OE mice were mated in the absence of Dox (Supplementary Fig. 2D). Assessment of miR-128 level by qPCR confirmed its marked overexpression by embryonic day 10.5 (E10.5) in the hearts of miR-128 OE mice (Supplementary Fig. 2E). These miR-128 OE mutant mice displayed enlarged heart chambers, myocardial fibrosis, CM hypertrophy, and impaired left ventricular systolic heart function at P28 (Supplementary Figs. 2F-H). Moreover, KEGG pathway analysis showed that oxidative phosphorylation, metabolism, hypertrophic cardiomyopathy, and dilated cardiomyopathy pathways were enriched in miR-128 OE hearts. Concomitantly, cell cycle and DNA replication pathways were suppressed in miR-128 OE hearts (Supplementary Fig. 3). Taken together, these data indicate that CM-specific overexpression of miR-128 induces early CM cell cycle exit, compensatory pathological growth of CM (hypertrophy), and impaired cardiac homeostasis.' Reviewer comment 4: The author state that, upon treatment with anti-miR-128, cardiomyocyte became dedifferentiated" based on immunostaining for cardiac troponin T. It is unclear how the authors can define a "differentiated" or "undifferentiated" state based on the images shown in Suppl. Fig. 3. What are the cells with elongated filaments they define as "dedifferentiated"?? What are the majority of cells in these cultures, which do not stain with the anti-TnT antibody?
Author's Reply: We appreciate this comment. Accordingly, new images with higher resolution were presented in Supplementary Fig. 4 (previously Supplementary Fig. 3). Dedifferentiation can be characterized by partially disorganized sarcomere structure with cell proliferation markers and progenitor markers (Ref. 4,8). Our results showed that inhibition of miR-128 resulted in dedifferentiation of CM (including disassembly and reduction of sarcomere structure) (Supplementary Fig. 4C), and increased expression of proliferation marker such as pH3, Aurora B, and EdU (Supplementary Figs. 4F-H). GATA4 recently was used to identify the dedifferentiated CM (Ref. 8). In Supplementary Fig. 4I. an increased amount of GATA4 expression was observed in the anti-miR-128 treated CM. Some cTnT-negative cells were also observed in Supplementary Fig. 4 due to the limitation of CM isolation techniques. The majority of these cells were mesenchymal cells (such as fibroblast cells). Despite of the presence of cTnT-negative cells, we only focus on the dedifferentiated cTnT + CMs, which were characterized by sarcomere disassembly.
Reviewer comment 5: Suppl. Fig. 3E and 3G. Staining for phospho-H3 is not convincing. The number of positive cells is astonishingly high (more than 15%) for a marker that identifies cells that only transiently travel through the G2M phases of the cell cycle. The pictures show high magnification of a single cell, which is totally anecdotal and not significant.
Author's Reply: We appreciate this comment. New data has been presented in Supplementary  Fig. 4 (previously Supplementary Fig. 3). To investigate the effect of anti-miR-128 treatment on CM proliferation, analysis methods included phospho-H3 (pH3) staining (Supplementary Fig.  4F), Aurora B staining (Supplementary Fig. 4G), and EdU incorporation assay ( Supplementary  Fig. 4H). In addition, the number of EdU, pH3, and Aurora B positive CM was re-analyzed with respect topercentage of cells rather than the number per section. Fig. 3G-H: For a heart with such a marked hyperplastic as that shown in Fig. 3C, one would expect a remarkably high proliferation activity, while it appears that the number of cycling cells is only 2 fold than that of controls. This is in sharp disagreement with the picture shown in Fig. 3G (by the way, in this picture the actual cell reactivity to Ki67 is covered by the arrow marks). In the same picture the representative image is rather ambiguous. Is this a cardiomyocyte nucleus? What is the red halo around the nucleus?? Again, it is unclear how quantification of "disassembled" cardiomyocytes was performed, and certainly this value should not be expressed as"# per section"-out of how many analyzed cells?

Reviewer comment 6:
Author's Reply: In the previous version of Fig. 3G, yellow arrow marks were used to indicate the sarcomere disassembled CMs rather than the cell proliferation activity. As suggested, we avoided confusion by presenting new higher resolution images in Fig. 3G. Yellow arrows in the new images are indicative of Ki67 + CM with sarcomere disassembly. In Figs. 3G-5 and G-6, the CM dedifferentiation phenotype was characterized by disorganized sarcomere structure as identified by cTnT immunofluorescence staining (red color around the nucleus). As suggested, all quantification data is now expressed as percentage (%) of cells. Finally, the number of CMs we analyzed was added to the figure legend accordingly.

Reviewer comment 7:
The authors wish to identify targets for miR-128 action and reach the conclusion that Suz12 as one of these. Why this one only? There are a number of already described targets for this miRNA and many more are predicted. This information needs to be reported and a more systematic analyses has to be conducted. It is hard to believe that the authors by chance picked up the only gene that mediates the effect of the miRNA based on notoriously fallacious prediction analysis! Author's Reply: As suggested, we have added systematic analyses of the miR-128 target gene screening (Supplementary Fig. 7).
Accordingly, this statement has been modified in the results section on pages 9-10: 'RNA-Seq was performed on control (Ctrl) and miR-128 OE hearts (P7) to identify the putative target genes of miR-128 responsible for cell cycle regulation. By comparing the downregulated mRNAs identified in miR-128 OE hearts relative to Ctrl hearts with all possible predicted candidate miR-128 target genes 19 , we found 87 genes that contained the predicted binding site at the 3'UTR (Supplementary Fig. 7A). Gene Ontology (GO) PANTHER Analysis was then performed to identify the affected cellular biological processes. The leading biological category was 'cellular process' category, with nearly 28.7% of all associated genes (GO: 0009987, Supplementary Fig.  7B). A further subgroup analysis of the 'cellular process' indicated the potential for miR-128 to affect multiple pathways that are related to regulation of the cell cycle, cell communication, and cellular component movement (Supplementary Fig. 7B). Moreover, the analysis of genes downregulated in miR-128 OE showed statistically significant enrichment of genes downregulated after siRNA inhibition of components of polycomb repressive complex 2 (PRC2), Suz12 in particular (Supplementary Fig. 7C). PRC2 is a major epigenetic modifier that affects multiple genes and is crucial for organogenesis. Perturbation of the epigenetic landscape during early cardiac development inhibits CM proliferation, and eventually leads to fatal cardiac malformations 20, 21 . Significantly, Suz12 was identified among the predicted downstream target genes of miR-128. In contrast to neonatal hearts, the protein levels of SUZ12 was lower in the adult heart (where the CM proliferation ability is quite limited) (Figs. 4B-C), paralleling the upregulation of miR-128. These data suggested a potential interaction between miR-128 and Suz12, and was a key factor in generating our hypothesis that miR-128 regulates CM proliferation in part through the PRC2-Suz12 signaling pathway.'