Transcriptional Reversion of Cardiac Myocyte Fate During Mammalian Cardiac RegenerationNovelty and Significance
Rationale: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level.
Objective: The objectives of our study were to determine whether myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process.
Methods and Results: We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart.
Conclusions: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.
The adult mammalian heart has a limited capacity for self-renewal after injury.1–3 Shortly after birth, mammalian cardiac myocytes (CMs) exit the cell cycle, and subsequent heart growth is achieved primarily by hypertrophy of existing CMs.4 Substantial evidence suggests that even these terminally differentiated adult CMs retain some limited ability for cell division.5,6 However, the innate ability of the adult mammalian heart to repair itself after injury, such as myocardial infarction, is inadequate to replace the loss of functional myocardium.7 In contrast, some vertebrates, such as zebrafish and newts, can fully regenerate their hearts after amputation throughout their adult lives, primarily by proliferation of mature CMs.8,9
Editorial, see p 767
In This Issue, see p 763
Although adult mammalian hearts fail to regenerate after injury, neonatal mice can fully regenerate their heart after resection of the left ventricular apex.10 Genetic fate mapping demonstrated that new CMs in the regenerating apex were derived from preexisting CMs as opposed to a resident stem cell or progenitor population. CMs in the regenerating neonatal mouse heart demonstrate loss of distinct sarcomere structures, and a significant proportion of these cells enter the cell cycle as indicated by phosphorylated histone H3 (pH3) expression and upregulation of aurora B kinase suggestive of cell fate reversion.10 Thus, identifying mechanisms by which myocytes naturally undergo cell cycle activity during regeneration is fundamental for elucidating the molecular roadblocks that prevent regeneration in the adult heart. The idea that CMs undergo partial reversion of cell fate during mouse heart repair has been based primarily on observations at the ultrastructural level.10,11 The transcriptional changes that accompany this phenotypic response to injury remain largely unknown.
Here, we profiled global gene expression patterns over the course of mouse CM differentiation both in vitro (mouse embryonic stem cells differentiated to CMs) and in vivo (CM maturation from neonate to adult) and compared this transcriptional signature of differentiation to a CM explant model, whereby CMs lose the fully differentiated phenotype (mouse adult CMs explanted and cultured over 72 hours) to identify genes and gene networks that changed dynamically during these processes. We then examined global expression changes in the neonatal mouse whole heart ventricle as well as in purified CMs after apical resection and found that heart regeneration is characterized by a transcriptional reversion of the CM differentiation process, including reactivation of cell cycle genes and developmental programs. We interrogated the RNA sequencing (RNAseq) data sets by using a systematic approach to predict and validate upstream regulators and associated pathways that can modulate the cell cycle state of CMs. We identified interleukin 13 (IL13) as a new regulator of CM cell cycle entry and found that STAT6, STAT3, and periostin are critical mediators of IL13 signaling in CMs. As recently described in the zebrafish,12 STAT3 is predicted to regulate differentially expressed gene networks during heart regeneration in our data set, and STAT3 protein expression is increased in the regenerating neonatal mouse heart. Together, our data suggest that cardiac regeneration involves a transcriptional reversion of the differentiation process, and our systematic approach used to interrogate these RNAseq data sets provides significant insight into signaling pathways of both myocyte cell cycle activity and cardiac regeneration.
Animal Care and Usage
All animal studies were performed in accordance with the local animal care and use committee.
Embryonic Stem Cell Differentiation
RNAseq data for the differentiation of mouse embryonic stem cells into CMs through mesodermal and cardiac progenitor intermediates were obtained from Wamstad et al.13 Raw data from both biological replicates at each time point (embryonic stem cell, mesoderm, cardiac progenitor, and CM) were reprocessed according to the methodology described below.
Whole Heart Ventricle Isolation
Crl:CD1(CD-1) neonatal mice (Charles Rivers Laboratories, MA) were euthanized by decapitation at postnatal day 0 (P0), 4 (P4), and 7 (P7) and by isoflurane overdose at 8 to 10 weeks of age. Hearts were excised, washed in ice cold PBS, and snap frozen in liquid nitrogen. Heart atria were dissected and discarded, and ventricles were processed for RNAseq. At least 2 heart ventricles were pooled for each replicate. Two replicates were processed for RNAseq.
Adult Cardiac Myocyte Isolation
Adult mouse CMs were isolated from 8- to 10-weeks-old male CD-1 mice based on a protocol described previously.14
Neonatal Mouse Apical Resection
Apical resection surgeries were performed on postnatal day 1 CD-1 mice as described previously.10 The chests of sham-operated mice were opened, and the hearts were exposed but not resected. Hearts from sham and resected animals were collected at 24 hours and 7 days post surgery, washed in PBS, and snap frozen in liquid nitrogen. The apex of the heart was dissected and processed for RNAseq. Heart apex from 3 animals was pooled for each biological replicate. We consistently observe significantly elevated CM cell cycle entry at 7 days post operation in resected versus sham-operated hearts.
Neonatal Cardiac Myocyte Purification
Neonatal CMs were dissociated from whole mouse hearts (P0 and P4) and also from sham and resected neonatal mouse hearts at 7 days post surgery using the Neonatal Heart Dissociation Kit (Miltenyi Biotec). CMs were purified from the dissociated cell population using the Neonatal Cardiac myocyte Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Three biological replicates were generated per timepoint and experimental treatment for RNAseq. Hearts from 5 to 10 mouse pups were pooled for each biological replicate.
RNA Sequencing and Analysis
Total RNA was extracted from all samples using Trizol (Invitrogen) according to the manufacturer’s instructions. Polyadenylated RNA was isolated from 1 to 10 μg of total RNA using the Dynabeads mRNA purification kit (Invitrogen). Polyadenylated RNA was fragmented, and the first strand was synthesized using the Superscript III reverse transcription kit (Invitrogen). Double-stranded DNA was then synthesized with DNA polymerase I (Invitrogen). End repair, A-tailing, adapter ligation, and size selection were then performed using the SRPI-Works System (Beckman Coulter) followed by minimal amplification and addition of barcodes by PCR. Paired-end 40 base pair read length sequencing was then performed on an Illumina HiSeq 2000. Sequence alignment and assembly was performed as described previously.15,16 For the purified neonatal CM samples, the TrueSeq (Invitrogen) sample preparation protocol was performed because of the low RNA yield. Raw RNA-Seq data files are available through the NCBI Gene Expression Omnibus, GEO accession number GSE64403.
Isolation, Culture, and Treatment of Ventricular Neonatal Rat Cardiac Myocytes
Rat ventricular CMs were isolated from 1-day-old Sprague–Dawley rats (Charles River) as described previously.14 Cultured CMs were exposed to 100 ng/mL of each growth factor or cytokine followed by quantification of 3H-thymidine incorporation. 3H-thymidine or bromodeoxyuridine incorporation, flow cytometry, expression analysis, siRNA knockdown, qPCR, and western blot protocols can be found in the Online Data Supplement.
Data Analysis and Statistics
Data were analyzed by either 1-way analysis of variance or unpaired t test unless otherwise stated. All data are presented as mean±SEM. Statistical analysis was performed with the Prism Graphpad Software.
Determining the Transcriptional Signature of Cardiac Myocyte Differentiation
To elucidate the transcriptional changes that drive the switch from a proliferative mode of growth to cell cycle exit during CM terminal differentiation and maturation, we first established a reference transcriptional signature of this process by identifying a common set of differentially expressed genes during in vitro differentiation and in vivo CM maturation. We analyzed RNAseq data from cells collected at the embryonic stem cell, mesoderm, cardiac progenitor, and CM stages as described previously13 (Figure 1A; FPKM [Fragments Per Kilobase of exon per Million fragments] values for all RNAseq data sets in Online Table IA). We then analyzed gene expression in an in vivo maturation model and compared these data to the in vitro differentiation model. Within the first week of life, and into adulthood, mammalian CMs undergo a maturation process that is characterized by development of a rigid and organized sarcomeric structure, binucleation, increased metabolic demand, and exit from the cell cycle.17 We collected and analyzed mouse heart ventricles from P0, P4, and P7 and from 8- to 10-weeks-old adult mice (Figure 1A). We defined the transcriptional changes that occur during both in vitro and in vivo differentiation as the core gene network and postulated that these genes, at least in part, underpin critical transcriptional determinants of CM differentiation.
As expected, differentially expressed genes during in vitro and in vivo differentiation showed primarily upregulation of sarcomere and mitochondrial-related genes and downregulation of cell cycle genes as determined by analysis of enriched gene ontology terms18,19 (Online Figure IA and IB). Although the embryonic stem cell differentiation model and in vivo CM maturation have been studied previously, the full set of genes dynamically changing that are common to both data sets has not been described. In total, 927 genes were commonly upregulated in both data sets (Figure 1B; Online Table IB), which function in mitochondrial, sarcoplasm, and sarcomere-specific processes, among others (Figure 1C and 1D; Online Table ID). Genes critical for sarcomere assembly, such as Titin Cap and Cardiac troponin I type 3, showed pronounced increases in expression over the course of differentiation, reflecting sarcomere assembly and organization during CM differentiation and maturation.
Genes whose expression decreased in both differentiation data sets function primarily in RNA processing and cell cycle (Figure 1C and 1D; Online Table IC and IE). RNA processing encompasses mechanisms, such as RNA transport (eg, Thoc1, 3, 4, and 5, Ncbp1, and Khsrp), RNA splicing (eg, Xab2, Wbp11, Aqr), and RNA capping (eg, Rnmt, Rngtt, Ncbp1, and Tgs1), among others. Post-transcriptional modification is a critical mechanism that regulates cell fate commitment and differentiation,20 and our data support that RNA processing is necessary during the process of CM differentiation both in vitro and in vivo. Cell cycle exit is a hallmark of mature CMs, and a failure to re-enter the cell cycle is thought to contribute to the lack of cardiac regeneration in adult mammals. We found that cell cycle regulators, such as E2F2, Brca2, and several cell division genes (eg, Cdc6, 7, 23, 26, and 27) were consistently downregulated in both differentiation data sets. Importantly, differentially expressed genes unique to either in vitro or in vivo differentiation did not strongly reflect hallmarks of myocyte differentiation (Online Table IF–II).
Explanted Cardiac Myocytes Display a Transcriptional Reversion of Differentiation
Adult mammalian CMs are terminally differentiated cells that rarely proliferate in vivo; however, adult rodent CMs isolated and cultured ex vivo over several days can revert to a more immature state and re-enter the cell cycle, suggesting that adult CMs retain some plasticity.21 We hypothesized that the transcriptional response of explanted mouse CMs would result in a loss of the differentiated phenotype, providing clues to the critical factors required for maintaining the myocyte differentiated state. Thus, we analyzed gene expression patterns by RNAseq from adult mouse CMs explanted and cultured for 0, 24, 48, and 72 hours (Figure 1A; Online Figure IC) to model the early transcriptional changes that occur during destabilization of the CM state.
To compare changes in gene expression programs in ex vivo cultured CMs to the differentiation process, we performed hierarchical clustering and identified groups of genes with common functions (Figure 2A; Online Table IIA). Strikingly, we found gene clusters that were repressed during differentiation were reactivated during the CM explant time course, specifically those with functions in RNA processing and chromatin modification (Figure 2A, green, brown and light blue; Online Table II A). We next used principle component analysis to gain further insights into the relationship between the differentiation models and ex vivo time course (Figure 2B). The CM explant samples showed a directional reversion along the PC1 axis, represented by a downregulation of sarcomere components and activation of the cell cycle. The shift along the PC2 axis in the in vivo samples likely shifts in the opposite direction of the embryonic stem cell data set as a result of transcriptional changes involved in blood vessel morphogenesis and extracellular matrix processes or perhaps in response to early developmental genes turned on during in vitro differentiation and turned off during in vivo maturation.
The vast majority (2253; 93%) of genes differentially expressed during ex vivo culture showed changes in the opposite direction during the differentiation process (Figure 2C; Online Table IIB and IIC). Genes deactivated during differentiation overlapped those genes that are activated during explant culture and function in RNA processing, cell cycle, transcription, and DNA repair. Moreover, genes activated during differentiation and downregulated during the ex vivo time course have roles in cell metabolism and regulation of heart contraction (Figure 2C and 2D; Online Table IID and IIE). Several sarcomere-related genes highly expressed in the adult heart (ie, Tnnt2, Tcap, Myl3, Tnni3, Tpm1, Myh7, Myh6, Mybpc3, and Tmod1) were downregulated on explantation (Online Figure II).
Neonatal Mouse Cardiac Regeneration Involves a Transcriptional Reversion of Cardiac Myocyte Differentiation
Neonatal mice maintain the capacity to regenerate their hearts after resection of ≤15% of the apex; however, this ability is lost by 7 days after birth.10 During this critical developmental window, CMs develop rigid and highly organized sarcomere structures, and many of these cells become multinucleated.17 On injury, some CMs display apparent loss of clear sarcomere structures at 1 and 7 days post resection (dpr),10 suggesting that CM fate changes during neonatal mouse heart regeneration. However, the process of CM regeneration has not been elucidated at the transcriptional level in mammals. Thus, we used the transcriptional signature derived from the in vitro differentiation, in vivo maturation, and ex vivo culture models to characterize the molecular events that occur in the neonatal mouse heart during regeneration.
We analyzed transcripts by RNAseq from the resected hearts 1 and 7 dpr and compared these data to gene expression profiles measured from hearts of sham-operated animals to determine differential gene expression during regeneration. The biological processes modulated during these 2 time points are strikingly different, with only a small percentage of differentially expressed genes common at both time points (Figure 3A). At 1 dpr, genes with roles in inflammatory response and wound healing processes were primarily upregulated, reflecting an acute infiltration of immune cells. Conversely, at 7 dpr, cell cycle genes were activated and sarcomere and heart contractility-related genes showed lower expression, consistent with a transition of CMs to a less mature state (Figure 3A; Online Figure ID).
We next compared the expression of the 2253 genes inversely expressed during differentiation and ex vivo culture (see Figure 2C) with those genes that showed significant change during regeneration. In total, 22% (14.8% downregulated and 23.1% upregulated) of all differentially expressed genes at 7 dpr significantly overlapped with this core gene set (P=0.002), whereas differentially expressed genes at 1 dpr did not significantly overlap these genes (Figure 3B), suggesting that a transcriptional reversion occurs by 7 dpr in the regenerating neonatal mouse heart. Genes found only differentially expressed during regeneration included genes related to inflammatory response (eg, Cxcl15 and Cxcl13) and adhesion molecules (eg, Fn1 and Cldn18; Online Table III). These may provide important insight into regenerative-specific factors.
We next developed a metric to assess whether differentially expressed genes at each time point relative to sham controls were associated with genes driving a differentiated or undifferentiated state (Figure 3C). Genes that changed during in vitro differentiation, in vivo maturation, and ex vivo explant CM data sets were classified according to how strongly they were expressed at different stages of differentiation (y axes in Figure 3C; see Experimental Procedures). Genes upregulated at 7 dpr showed a significant shift toward a less differentiated state, with the strongest shift occurring toward the P0 neonatal state. In contrast, genes downregulated at 7 dpr displayed the opposite trend, largely shifting toward genes that defined a more differentiated state. Moreover, genes expressed at 7 dpr more closely resembled a less differentiated state than at 1 dpr.
The majority of genes upregulated during ex vivo culturing of CMs that showed higher expression in the regenerating mouse heart have functions in cell cycle, mitosis, and RNA processing, consistent with the increased CM cell cycle activity in the regenerating mouse heart10 (Online Figure IIIA). Genes upregulated during differentiation and downregulated in the explant model that were also significantly downregulated during regeneration included titin-associated genes (eg, Fhl2, Ank1; Online Figure IIIB), calcium-regulating genes (eg, Dmpk, Asph and Hrc; Online Figure IIIC), and transcriptional or translational regulators (eg, Tsc22d3, Zbtb16). Titin and titin-associated genes are critical components of the sarcomere, and it is thought that sarcomeres are physically disassembled during regeneration based on ultrastructure analysis; however, this process has not been characterized at the molecular level. Another CM-specific gene, atrial natriuretic peptide (Nppa), showed significant increased expression in the regenerating heart. Nppa is a secreted protein involved in heart development and is reactivated upon heart injury, such as myocardial infarction,22 suggesting a functional role for this developmental gene during heart injury. Our data suggests a transcriptional reversion consistent with a change in CM fate to an immature state occurs during mammalian heart regeneration.
Neonatal Mouse Cardiac Regeneration Involves a Transcriptional Reversion in Isolated Cardiac Myocytes
Given the potential for cell heterogeneity, we isolated cardiac myocytes (iCMs) from whole hearts and compared these data to our gene expression analysis in ventricular samples. Myocytes were specifically isolated from neonatal hearts (P0 and P4) and adult CMs, resulting in ≈95% purified CMs. We analyzed differentially expressed genes in these samples and used Ingenuity Pathway Analysis to map gene networks that dynamically change over the course of CM maturation from P0 to adult. Consistent with a more proliferative state, many of the top differentially expressed gene networks in P0 isolated CMs relative to those isolated from adult hearts involved cell cycle, cellular assembly and organization, DNA replication, and recombination and repair (Online Figure IV). Importantly, differentially expressed genes in whole ventricular P0 tissue relative to adult heart samples overlapped the majority of genes (51 out of 70) present in the network generated from iCMs data sets (Online Figure IV outlined in pink). We observed an upregulation of representative sarcomere and mitochondrial genes and downregulation of cell cycle genes in P0 relative to adult iCMs, similar to the patterns observed in whole heart tissue as displayed in Figure 1 (Online Figure VA). Principal component analysis of iCMs at P0 and P4 was highly similar to results obtained using ventricular heart tissue. Additionally, hierarchical clustering demonstrated that gene clusters upregulated during differentiation and maturation (ie, oxidation reduction) were downregulated during in vitro explantation and genes downregulated during differentiation and maturation (ie, cell cycle) were activated on explantation (Online Figure VB and VC). These analyses demonstrate that the core transcriptional changes are replicated between ventricular samples and purified CMs, suggesting that the transcriptional changes are largely specific for CMs.
We next isolated CMs at 7 dpr from hearts of mice that had undergone sham or resection at 1 day of age to further test the hypothesis that genes differentially expressed in regenerating myocytes revert to a less differentiated state. Genes upregulated in regenerating iCMs at 7 dpr showed a shift toward a less differentiated state compared with sham iCMs, with the strongest shift occurring toward the P0 neonatal state. Genes downregulated at 7 dpr displayed the opposite trend, specifically shifting toward genes that defined a more differentiated state in the in vitro differentiation data set (Online Figure VD). Of interest, atrial natriuretic peptide was also significantly elevated in iCMs during regeneration, suggesting a strong candidate factor involved in the myocyte response during regeneration. Although some differentially expressed genes (ie, downregulation of sarcomere-related genes) were not robustly reproduced in the iCMs data sets (likely because of the sensitivity of day 7 mouse hearts to the digestion and purification process), our data support the conclusions derived from the sham and resected ventricle tissue that genes turned on in regenerating myocytes shift to a less differentiated state at the transcriptional level.
Identification of Candidate Regulators of Cardiac Myocyte Cell State
We hypothesized that by examining genes differentially expressed in each of our data sets, we could predict factors that regulate CM cell cycle state. Differentially expressed genes from in vitro and in vivo differentiation, ex vivo culture, and 7 dpr regeneration data sets were selected as seed genes to predict upstream regulators using Ingenuity Pathway Analysis (www.ingenuity.com). In total, 150 factors were predicted to be upstream regulators of differentially expressed genes in all 4 of the data sets (Online Table IV). To test whether putative upstream regulators play a role in modulating cell cycle state, we investigated the effect of the exogenous addition of growth factors and cytokines predicted in our analysis on the capacity of cultured neonatal mammalian CMs to undergo cell cycle activity. Of the 15 growth factors and cytokines predicted as upstream regulators (Figure 4A), 2 factors, Neuregulin 1 (Nrg1) and Oncostatin M (OSM), are known to play a significant role in inducing CM cell cycle activity. Nrg1 induces cell division of mature mammalian CMs via ErbB4 receptor signaling and protects against myocardial infarction scar formation in vivo.23 CMs exposed to the cytokine OSM undergo differentiation reversion characterized by loss of sarcomere structure and reexpression of alpha smooth muscle actin.24 In vivo, OSM signaling results in a dilated cardiomyopathy phenotype. These studies demonstrate that our systematic approach successfully identified factors with critical functions in regulating CM cell cycle activity or differentiated state, leading us to examine other candidates from our analysis.
We screened the effect of several candidate cytokines and growth factors on CM DNA synthesis. Exposure of cultured neonatal rat CMs to Nrg1, IL3, IL13, CTGF, and Fgf1 significantly stimulated DNA synthesis as detected by 3H-thymidine incorporation (Figure 4B). Among these, IL13 most consistently stimulated DNA synthesis in a dose-dependent manner (data not shown). Importantly, IL13 was also predicted to be an upstream regulator of differentially expressed gene networks in the iCMs from resected versus sham mice at 7 dpr (Online Figure VE). Immunohistochemistry confirmed that CMs exposed to IL13 had significantly increased DNA synthesis indicated by bromodeoxyuridine incorporation (Figure 4C). Furthermore, IL13-treated CMs showed almost a 2-fold increase in Ki67 expression (Figure 4D) and increased nuclear pH3 expression. The number of neonatal rat CMs appearing to undergo cell cycle entry as determined by absence of clear sarcomere structures and strong pH3 staining in the nucleus (Figure 5A, right panel) was significantly higher in IL13-treated cells compared with control cells (Figure 5A and 5B). Furthermore, CMs with absence of clear sarcomere structures stained positive for aurora b kinase (Figure 5C), a marker of cell cycle entry. Twenty-four hours post IL13 treatment, a significantly higher percentage of total cultured cells stained positive for cardiac troponin T compared with untreated cells (Figure 5D), suggesting that IL13 preferentially induces either proliferation or survival of CMs compared with other cell types (such as fibroblasts). Collectively, these data suggest that IL13 stimulates CM cell cycle entry in vitro. IL13 is a cytokine secreted primarily by activated TH2 immune cells, suggesting that inflammatory factors could play an important role in initiating CM cell cycle activity during heart regeneration via IL13 signaling.
IL13 Signals Through IL4R/IL13Ra1 in Cardiac Myocytes
We next sought to elucidate the IL13 signaling mechanism in CMs. IL13 can transmit signals to target cells through the IL4Ra/IL13Ra1 receptor heterodimer or through IL13Ra2.25 We compared expression of these 3 receptors across whole mouse tissues that are known to respond to IL13, including lung, skeletal muscle, and brain. IL4Ra is expressed at similar levels in all tissues examined but showed highest expression in the lung (Figure 6A), whereas IL13Ra1 demonstrated significantly higher expression in the heart compared with other tissues examined (Figure 6B). We found that both IL4Ra and IL13Ra1 are expressed in cultured neonatal rat CMs, whereas IL13Ra2 transcripts could not be detected (data not shown), suggesting that IL13 signals on CMs via the IL4/IL13Ra1 receptor heterodimer. Knockdown of IL4Ra by siRNA slightly but significantly diminished CM 3H-thymidine incorporation in response to IL13 exposure, whereas knockdown of IL13Ra1, or both IL13Ra1 and IL4Ra, substantially decreased 3H-thymidine incorporation to baseline levels (Figure 6C; Online Figure VI). Thus, IL13Ra1, and to a lesser extent IL4Ra, plays a significant role in mediating the DNA synthesis response to IL13 signaling.
STAT6 and STAT3/Periostin Mediate the IL13 Response in Cardiac Myocytes
A recent study demonstrated that Jak1/STAT3 signaling is critical for myocyte proliferation during zebrafish heart regeneration.12 In response to IL13 stimulation and binding IL4Ra/IL13Ra1, TYK2 activates STAT1 and STAT6, whereas JAK2 signaling is upstream of STAT3 activation.26 Although STAT6 expression remained constant, IL13-treated CMs demonstrated visibly pronounced STAT6 phosphorylation as early as 15 minutes after treatment (Figure 7A). STAT6 phosphorylation decreased from 30 minutes to 24 hours after IL13 exposure. On the other hand, STAT3 phosphorylation was delayed, appearing by 3 hours and increasing at 24 hours post IL13 treatment. We investigated the upstream regulators of differentially expressed gene networks to examine the possibility that STAT6 or STAT3 signaling regulates gene expression in the regenerating mouse heart. STAT3 (activation score, 2.621; P=7.67×10−7) was among the 22 transcription factors predicted to significantly activate differentially expressed gene networks during heart regeneration at 7 dpr (Online Table V). Although STAT3 RNA expression did not change during regeneration, STAT3 protein levels were significantly increased in resected hearts compared with sham-operated hearts at 1 dpr (Figure 7B).
Delivery of recombinant periostin has been shown to induce cell cycle re-entry in differentiated, mononucleotide mammalian CMs in vivo.27 Interestingly, periostin expression has been used as a marker of IL13 activity in vivo,28 and its expression is regulated by STAT3 activation.29 Given its role in heart regeneration, we hypothesized that periostin mediates the CM response downstream of the IL13/STAT3 pathway. IL13 exposure stimulated expression of periostin by 24 hours post treatment in cultured CMs (Figure 7C). In vivo, periostin expression shows a trend toward upregulation during neonatal mouse heart regeneration 7 dpr; however, this difference is not statistically significant (data not shown). Notably, siRNA knockdown of STAT3 in neonatal rat CMs decreased periostin expression (Figure 7C), demonstrating that STAT3 mediates expression of periostin in cultured CMs. Moreover, both periostin and STAT6 knockdown diminished the DNA synthesis phenotype induced by IL13 (Figure 7D). Our data demonstrate that IL13 induces CM-specific expression of several factors involved in the regeneration response, including STAT3 and periostin.
Heart regeneration in lower organisms as well as in the neonatal mouse has been proposed to occur by proliferation of preexisting CMs.10,30 This assumption has been based primarily on observations of loss of clear sarcomeres at the ultrastructural level and expression of cell cycle entry markers, such as pH3; however, the molecular changes that precisely characterize this process have not been studied in detail. We identified a core transcriptional signature of CM differentiation and found that explanted adult CMs cultured over several days demonstrate a transcriptional reversion of the differentiation process. Similarly, our data show that regenerating neonatal mouse heart 7 dpr seem to undergo a transcriptional reversion after apical resection.
Although CMs account for the majority of total myocardial mass (between 60% and 80% depending on the developmental stage),31 numerous additional cell types may also contribute to the transcriptional profiles obtained via RNA profiling in ventricular tissue and some transcriptional changes may be caused by changes in cell type composition. To address this confounding factor, we profiled in vitro systems that specifically model a transient CM state (in vitro differentiation and adult CM explants) to identify expression changes specific to CMs during the differentiation and loss of differentiation processes. This method of profiling multiple model systems increases the likelihood that transcriptional changes observed across all 4 models represent a change in myocyte cell state. To further verify these findings, we profiled purified CMs over the same time course during maturation (P0 to adult) and in the regenerating neonatal mouse heart 7 dpr. These data sets support our conclusions that CM regeneration involves a transcriptional reversion of maturation.
Our work suggests that transcriptional alteration of a specific subset of genes that regulate sarcomere organization, RNA processing, and cell cycle progression is likely critical for remodeling the terminally differentiated program of CMs. Several genes involved in sarcomere organization and function (eg, Smyd1, Fhl2, Ankrd1, Tcap, and Ttn) showed increased and sustained expression during differentiation and were suppressed during myocyte explantation and at 7 dpr during heart regeneration. Consistent with our findings, experimental evidence demonstrates that diminished expression of Smyd1b, a methyltransferase involved in sarcomere organization that we found downregulated during regeneration,32 causes significant disruption of myofibril organization in zebrafish cardiac muscles.33 Additional studies will be required to determine whether modulating the expression of these genes influences the proliferative state of CMs.
Accumulating evidence suggests that cytokines and growth factors trigger regeneration in other cell types, such as skeletal muscle,34 optic nerve,35 and hepatocytes.36,37 Thus, we were particularly interested in identifying novel cytokines and growth factors that could initiate cell cycle entry of CMs. Our systematic approach identified Nrg1 and OSM, 2 factors demonstrated to induce CM proliferation and dedifferentiation both in vitro and in vivo.23,24 The identification of Nrg1 and OSM establishes the validity of our model and suggests that other identified factors may also be physiologically relevant for inducing CM cell cycle activity. We found that IL13, a cytokine produced primarily by activated TH2 cells, can activate CM cell cycle activity in vitro. IL13 acts on nonhematopoietic cell types, such as smooth muscle cells, endothelial cells, and fibroblasts, and can induce proliferation of cultured vascular smooth muscle cells.38 Thus, we reasoned that IL13 could play a role in mediating CM cell cycle activity after an inflammatory response. We demonstrate that the IL4Ra/IL13Ra1 heterodimer mediates CM response to IL13. The reduction in DNA synthesis was most pronounced after IL13Ra1 knockdown, which approached the level of untreated cells. These data suggest that IL13Ra1 may play a key regulatory role in transducing IL13 signal compared with IL4Ra.
We demonstrate that IL13 induces myocyte cell cycle entry, and the response to IL13 is mediated in part by the STAT3/periostin pathway. STAT3 activation is required for CM proliferation in response to injury in zebrafish12 and has also been implicated in mammalian muscle regeneration as a critical mediator of myoblast proliferation.39 Our analysis identified IL13 and STAT3 as regulators of differentially expressed gene networks during mouse heart regeneration and STAT3 protein expression was increased in the regenerating mouse heart; thus, IL13/STAT3 could be an initiating factor in mouse cardiac regeneration. Periostin delivery has been shown to stimulate myocyte proliferation and reduced injury size and fibrosis after experimental myocardial infarction.27 We found periostin expression to be mediated by STAT3 expression in cultured CMs; thus, it is plausible that IL13 initiates STAT3 and periostin induction to facilitate mouse heart regeneration.
To date, STAT6 signaling has not been described in the context of heart regeneration. Our data demonstrate that STAT6 mediates IL13 signaling in CMs in vitro; however, we did not find compelling evidence that STAT6 plays a role in heart regeneration in vivo. In iCMs, IL13 induced rapid STAT6 phosphorylation that quickly diminished, whereas STAT3 phosphorylation was strongly detected 24 hours after IL13 exposure. We postulate that STAT3 activity persists several days after the injury event that initiates regeneration, whereas STAT6 signaling is transient and therefore undetectable in the days after resection. Further experiments will be required to determine whether STAT6 signaling mediates heart regeneration processes. Together, this work demonstrates that our approach to identify the core transcriptional changes that occur during CM maturation and regeneration can be applied to gain a systems level understanding of the molecular factors that coordinate these critical processes.
Our study suggests that cardiac regeneration is not a stochastic loss of the mature cell state, but rather a direct transcriptional reversion of the differentiation process. We observed this trend in the in vitro CM explant model as well as during heart regeneration at 7 dpr. We demonstrate a proof of principal concept by which interrogation of high throughput RNA expression data sets elucidated novel pathways that stimulate in myocyte cell cycle activity, and importantly, this signaling occurs during mammalian heart regeneration. Although post-transcriptional events, such as transcription factor translocation to the nucleus, are also critical mediators, the regenerative response40 our study provides a critical framework for understanding the transcriptional expression changes required for CM repair in response to injury that will be invaluable for ultimately guiding efforts to promote adult mammalian cardiac regeneration.
This study was performed with financial support from the National Institutes of Health (C.C. O’Meara, F32HL117595; J.A. Wamstad, F32HL104913 and K99HL122514; R.T. Lee, R01AG032977 and R01AG040019; L.A. Boyer, U01HL098179), the Harvard Stem Cell Institute (RTL), and the National Science Foundation under the Science and Technology Center Emergent Behaviors of Integrated Cellular Systems (EBICS) Grant No. CBET-0939511 (R.T. Lee and L.A. Boyer). C.C. O’Meara, J.A. Wamstad, R. Gladstone, R.T. Lee, and L.A. Boyer conceived and designed the experiments.C.C. O’Meara, J.A. Wamstad, R. Gladstone, G.M. Fomovsky, and J. Gannon performed the experiments. C.C. O’Meara, J.A. Wamstad, R. Gladstone, V.L. Butty, and A. Shrikumar analyzed the data. R.T. Lee and L.A. Boyer contributed reagents/materials/analysis tools. C.C. O’Meara, J.A. Wamstad, R.T. Lee, and L.A. Boyer wrote the article.
In November, 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.96 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.304269/-/DC1.
- Nonstandard Abbreviations and Acronyms
- cardiac myocyte
- days post resection
- isolated cardiac myocytes
- postnatal day 0
- postnatal day 4
- postnatal day 7
- Received April 25, 2014.
- Revision received December 2, 2014.
- Accepted December 4, 2014.
- © 2014 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Neonatal mice have the capacity to regenerate their hearts within the first week of life.
Mammalian heart regeneration is thought to occur by proliferation of existing cardiac myocytes, yet the transcriptional changes that underpin this process have not been fully characterized at the molecular level.
What New Information Does This Article Contribute?
The regenerating mouse heart reveals a transcriptional reversion of the cardiac myocyte differentiation processes.
Systematic analysis of several models of cardiac myocyte differentiation and ex vivo culture identifies potential regulators of cardiac myocyte cell cycle activity and heart regeneration.
Interleukin-13 is a novel upstream regulator of differentially expressed gene networks during regeneration and induces cardiac myocyte cell cycle entry.
Adult mammalian hearts have limited regenerative capacity after injury, such as myocardial infarction, which is likely because of the inability of cardiac myocytes to undergo robust cell cycle activity. However, recent studies demonstrate that neonatal mice regenerate their hearts shortly after birth primarily by proliferation of existing cardiac myocytes. The goal of our study was to determine whether regenerating mouse cardiac myocytes revert the transcriptional phenotype to a less differentiated and proliferative state during regeneration and subsequently identify regulators of this process. We identified differentially expressed genes on injury-induced cardiac myocyte regeneration in mice and compared these to a core network of genes that change during models of in vitro and in vivo cardiac myocyte differentiation and in vitro adult cardiac myocyte explantation. Regenerating mouse hearts display global expression patterns that begin to resemble immature neonatal cardiac myocytes at the transcriptional level, suggesting a transcriptional reversion of gene expression profiles during regeneration. We interrogated differentially expressed gene networks from all data sets to predict factors that might regulate cardiac myocyte cell cycle activity. Among others, interleukin 13 induced cell cycle activity of cultured cardiac myocytes. Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.