C3orf58, a Novel Paracrine Protein, Stimulates Cardiomyocyte Cell-Cycle Progression Through the PI3K–AKT–CDK7 PathwayNovelty and Significance
Rationale: The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years.
Objective: We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation.
Methods and Results: Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase–protein kinase B–cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway–specific inhibitors and silencing of the Cdk7 gene.
Conclusions: Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase–protein kinase B–cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.
The heart’s limited ability to regenerate poses one of the most significant challenges in cardiac diseases. Although neonatal cardiomyocytes retain proliferative capacity and are capable of regeneration after injury,1–4 mature cardiomyocytes show very limited capacity for proliferation. As a result, after injury and loss of cardiomyocytes, the adult heart cannot regenerate adequately thereby leading to scar formation, pathological remodeling, and eventually heart failure. To address this problem, considerable efforts have been invested to develop strategies that can stimulate cardiac regeneration by discovering new molecules or pathways that can stimulate cardiomyocytes to enter the cell cycle and undergo division.3,4 Still, only a few such molecules have been identified. These include the p38 mitogen-activated protein kinase inhibitor,5 periostin,6–8 Neuregulin 1,9 as well as tumor necrosis factor–like weak inducer of apoptosis.10–12 However, even with these stimulants, the numbers of dividing cardiomyocytes are, as it is still difficult to achieve sustainable activation of cell-cycle re-entry and division.
Recently, we identified C3orf58 as a novel paracrine factor secreted from mesenchymal stem cells, which we named hypoxia and Akt induced stem cell factor (HASF; Huang et al, article under review). Exogenously administered HASF protein or HASF gene overexpression protected cardiomyocytes from apoptosis and cell death via the selective activation of protein kinase C epsilon both in vitro and in vivo (Huang et al; article under review). HASF is a relatively uncharacterized protein that does not show any similarity to known functional motifs, apart from the presence of a putative signal peptide. HASF shows broad expression in adult mouse tissues, such as heart, brain, liver, etc (Online Figure I). Genetic studies have shown an association of human HASF deletion with human familial autism.13 In the same study, it was suggested that HASF is a downstream target of Mef2c and that HASF is upregulated in rat hippocampal cultures after membrane depolarization with KCl.13
Here, we provide evidence that treatment of rat neonatal cardiomyocytes with HASF recombinant protein increased DNA synthesis through the cell-cycle regulator cycle-dependent kinase 7 (CDK7) complex and promoted mitosis and cell division. These effects were shown to be dependent on the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB also known as AKT) pathway. Importantly, cardiac-specific overexpression of HASF in transgenic mice resulted in enhanced DNA synthesis and karyokinesis of neonatal and adult cardiomyocytes in vivo. Collectively, our data identify HASF as a novel inducer of both cell-cycle re-entry and proliferation of neonatal and adult mammalian cardiomyocytes with potential implications for cardiac repair and regeneration.
Recombinant Protein Generation
Human HASF recombinant protein was produced using bacterial, mammalian, or insect cells. In brief, the open reading frame of human HASF without the predicted N-signal sequence (1158 bp) was cloned in-frame in pMal-2C vector. The fragment was next amplified, using the forward primer (underlined with Nde I restriction site), 5′-ggcggccatatggaccggcgcttcctgcag-3′ and the reverse primer (underlined with BamHI restriction site), 5′-ggcggcggatccctacctcacgttgttacttaattgtgctagg-3′, and cloned in-frame into a pET 15b vector (EMD Biosciences) to generate 6× His tagged HASF recombinant protein. For transfection and protein production using the HEK293 mammalian cell expression system, full-length human HASF cDNA without the stop codon was amplified by polymerase chain reaction and cloned into Gateway Entry vector for sequencing and subsequently recombined into Gateway destination vector 40 (Invitrogen) to generate an 6× His-V5-epitope tagged HASF. In addition, hpc4-tagged HASF recombinant protein was produced from insect cells using Invitrogen’s Drosophila Expression System. Protein was purified from the media using Anti-Protein C Affinity Matrix from Roche (catalog number 11815024001). Protein sequence was confirmed by matrix-assisted laser desorption–ionization mass spectrometry on an Applied Biosystems 4700 Proteomic Analyzer time of flight mass spectrometer (Duke core facility). Positive mode time of flight was used to identify peptides, and individual peptides were sequenced by mass spectrometry/mass spectrometry. All peptide fingerprint data were searched by SwissProt and Mascot search engine.
Isolation and In Vitro Treatment of Rat Ventricular Neonatal Cardiomyocytes
Hearts were harvested from 1- to 2-day-old rat neonates and trypsin-digested overnight (4°C), followed by collagenase Type II (Worthmington) digestion at 37°C. The lysate was then filtered through a 100-μm cell strainer and the cells were pelleted for 5 minutes at 500 rpm. Cardiomyocytes were cultured in the following growth media: Advanced DMEM/ F12 media supplemented with 0.2% BSA, 0.1% ascorbic acid, 2 mmol/L l-glutamine, and 0.5% insulin, transferrin, selenium5,8 supplemented with 5% horse serum and 20 μmol/L ArabinoseC during the first 48 hours.5,8 Cultured cells were stimulated for 7 days in growth media with 10 to 20 nmol/L HASF. Bromodeoxyuridine (BrdU) uptake was used to measure DNA synthesis. BrdU ELISA assay was performed following the manufacturer’s instructions (Roche).
Immunofluorescent Staining of Neonatal Cardiomyocytes In Vitro
Neonatal cardiomyocytes were fixed with 4% parformaldehyde and permeabilized with 1% triton (for BrdU staining) supplemented with 2 mol/L HCl for 30 minutes, rinsed with PBS, and blocked with 10% normal goat serum. Primary antibodies for BrdU (Abcam), cardiac troponin T (Abcam), Ki67 (BD Biosciences), H3P (Abcam), and Aurora B (BD Biosciences) were diluted with blocking solution, and Alexa fluor secondary antibodies (Invitrogen) were used for detection. Confocal images were captured using an LSM 510 Meta DuoScan microscope (Zeiss) and processed using ZEN software, version 2009. Tiled scanned images of 4 or 6 single high-power field images were captured by ZEN software and analyzed.
Western Blot Analysis
Neonatal cardiomyocytes were lysed with 1× lysis buffer (Cell Signaling) in the presence of protease and phosphates inhibitors (Roche). The lysate was centrifuged at 12000g for 5 minutes and total protein concentration was measured with bicinchoninic acid assay (Pierce) assay. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (Invitrogen), transferred to a membrane, blocked with 5% nonfat dry milk (Biorad), and probed with the primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies.
Fluorescence-Activated Cell Sorting
Cells were isolated as before and fixed using the BD Cytoperm/Cytofix kit (BD) according to manufacturer’s instructions. Troponin staining was performed using specific Troponin T or Troponin I antibodies (Abcam). BrdU incorporation was evaluated using a BrdU and 7-amino actinomyosin intracellular staining kit (BD Pharminogen). Cells were analyzed using BD fluorescence-assisted cell sorter caliber.
Gene Expression Quantitative Real-Time Polymerase Chain Reaction
mRNA was isolated from neonatal cardiomyocytes following Qiagen kit (Qiagen) manufacturer instructions. cDNA was produced using high-capacity cDNA transcription kit (Applied Biosciences) with Biorad iCycler. The quantitative expression of each gene was assessed using TaqMan Gene Expression Assays in the StepOnePlus Real-Time Polymerase Chain Reaction System (Applied Biosystems).
Knockdown of Cdk7 Gene Expression
Isolated neonatal cardiomyocytes were cultured as described above. Pools of Accell siRNAs against Cdk7 (Dharmacon) or nontargeting siRNA were used to suppress Cdk77 gene expression in the presence or absence of HASF. siRNA transfection was performed for 3 days using Dharmacon’s protocol. After removal of the siRNA complex the cells were treated with HASF for 4 additional days.
Generation of Transgenic Mice—Cardiac-Specific Overexpression of HASF
For the generation of HASF-Tg mice, human HASF cDNA was placed under the control of the alpha myosin heavy chain (αMHC) promoter in P-Bluescript plasmid as described in the study by Subramaniam et al.14 The DNA was linearized and introduced into friend virus B-type mouse embryos and maintained on a friend virus B-type mouse background for ≥10 generations. Several HASF transgenic lines were established, as described by Huang et al (article under review).
BrdU Injection in HASF Transgenic Neonatal Mice
Wild-type (WT; friend virus B-type mouse) and HASF transgenic mouse neonates that were 1.5 days old received 3 intraperitoneal injections of BrdU (50 mg/kg) every 12 hours and then the hearts of the 3-day-old neonates were harvested. The hearts were fixed in 10% formalin for paraffin embedding. Tissue sections of 5-μm thickness were used for immunohistochemistry.
Induction of Myocardial Infarction in Adult Mice
Permanent ligation of the left anterior descending coronary artery was performed in 8- to 10-week-old male HASF transgenic line (H2) and its related WT (friend virus B-type mouse) mice as previously described.15 Every 24 hours the mice received an intraperitoneal injection of EdU (42 mg/kg per day) for days 2 to 7 and BrdU (50 mg/kg per day for days 9–14 postsurgery).16 On day 14 the hearts were harvested and fixed in 4% paraformaldehyde, embedded in paraffin, and the 5-μm-thick tissue slides were processed for immunohistochemistry fluorescent staining. Animals were handled according to the approved protocols and animal welfare regulations of the Institutional Review Board at Duke University Medical Center.
Quantitative data are given as mean±SEM, and intergroup comparisons were evaluated by ANOVA. P<0.05 was considered statistically significant.
HASF Increases Proliferation and Induces Cell-Cycle Re-Entry in Neonatal Cardiomyocyte In Vitro
To investigate the capacity of HASF to promote DNA synthesis in cardiomyocytes we performed cell-proliferation assays on ventricular cardiomyocytes isolated from 2-day-old rat neonates. Isolated cardiomyocytes were allowed to attach and recover for 48 hours. Cells were then treated daily with 10–20 nmol/L HASF for 7 consecutive days and assayed for DNA synthesis by BrdU incorporation. HASF treatment increased BrdU incorporation in neonatal cardiomyocytes by 1.7±0.1-fold as measured by ELISA assay (Figure 1A), similar to the levels of stimulation by fibroblast growth factor 1 (5.8 nmol/L), a growth factor well established to promote cardiomyocyte proliferation.5,9,17 Consistent with this finding, immunofluorescence confocal microscopy analysis showed that HASF treatment doubled the numbers of BrdU-positive neonatal cardiomyocytes (as identified by costaining of BrdU with cardiac TroponinT) to 4.3±0.6% (Figure 1B and 1C). To confirm these data, we performed BrdU incorporation and fluorescence-assisted cell sorter analysis. For this, 7 days of HASF-stimulated neonatal cardiomyocytes were pulsed with 3 consecutive BrdU (30 μmol/L) treatments every 24 hours for the last 3 days of stimulation. Next day, cells were harvested, fixed, and stained for analysis. As shown in Figure 2A and 2B, 27.8±2.03% of the HASF-treated cardiomyocytes were positive for BrdU, whereas 47.3±1.39% were in the G0/G1 resting phase. In comparison, 12.2±2.0% of the untreated cardiomyocytes were shown to be positive for BrdU, and 55.0±0.61% in the G0/G1 resting phase.18,19 These results indicate that HASF treatment increased DNA synthesis and concurrently reduced the number of cardiomyocytes in resting phase (G0/G1).
To further investigate whether HASF-treated cardiomyocytes show differences in the cell cycle we also performed immunostaining and confocal microscopy imaging of the mitosis marker Ki67.5,20,21 As shown in Figure 2C and 2D, stimulation with HASF resulted in 1.5±0.3% Ki67-positive neonatal cardiomyocytes as compared with 0.5±0.08% in untreated cardiomyocytes, corresponding to a 3- to 4-fold increase in cells in mitosis. During postnatal development cardiomyocytes generally undergo karyokinesis/nuclear division without cytokinesis, resulting in binucleated cells. To enquire about the potential of HASF to induce cardiomyocyte cell division, we evaluated the number of dividing cardiomyocytes using the cytokinesis marker Aurora B (Figure 2E and 2F). During the G2/M phase of the cell cycle, Aurora B is expressed in the nucleus. In metaphase Aurora B is associated to the chromosomes, whereas in anaphase and telophase it is localized to the midzone and midbody, respectively.22 This analysis revealed that 7 days of HASF treatment increased the number of dividing neonatal cardiomyocytes in the anaphase and telophase (2.3-fold as compared with the untreated cardiomyocytes; P≤0.01; Figure 2E and 2F). It is noteworthy to mention that only the mononucleated neonatal cardiomyocytes showed complete cytokinesis and that detection of cytokinesis in binucleated cardiomyocytes was rare. Taken together these data indicate that HASF can significantly induce mitosis and cytokinesis and enhance proliferation of neonatal cardiomyocytes in vitro.
Increased Cardiomyocyte Proliferation in the Neonatal Heart of Transgenic Mice With Cardiac-Specific HASF Overexpression
To explore whether the in vitro effects of HASF could be reproduced in vivo, we measured the levels of proliferation in cardiomyocytes from neonatal mice overexpressing the human HASF gene specifically in cardiac myocytes (αMHC-HASF). These mice show normal cardiac phenotype without any evidence of cardiac hypertrophy or dysfunction (Huang et al, article under review; Online Figure II). For DNA synthesis, neonates received 3 consecutive BrdU injections every 12 hours, and the harvested heart tissue was used for BrdU detection by fluorescence confocal microscopy. In total, 50 to 60 high-power field (40×) images per heart, corresponding to 1.6 to 2 mm2 area of a heart tissue per animal, were captured and analyzed. This analysis revealed that in the neonatal heart, an overexpression of HASF induced 1.5- to 2-fold increase in cardiomyocyte DNA synthesis (Figure 3A and 3B).
Moreover, to determine whether HASF also regulates karyokinesis in cardiomyocytes in vivo, we assayed mitosis by immunofluorescent staining for phosphorylated histone-3 (H3P). H3P at Ser10 is an established marker for chromosome condensation during mitotic prophase in animal cells.23 Immunofluorescent staining of H3P and confocal imaging analysis showed that HASF transgenic (Tg) mice had increased numbers (1.5–2-fold increase) of mitotic cardiomyocytes in the karyokinesis phase as compared with WT neonates (Figure 3C and 3D). Collectively, these data support that endogenous cardiac overexpression of HASF increases the proliferation of neonatal cardiomyocytes in vivo.
Cardiac Overexpression of HASF Results in Increased Adult Cardiomyocyte DNA Synthesis In Vivo
On the basis of the findings in the neonatal heart, we studied whether HASF cardiac overexpression increased cardiomyocyte proliferation in the adult heart at baseline and in response to injury. For this, 8- to 10-week-old male WT and HASF-Tg mice underwent myocardial infarction or sham surgery. Two days postsurgery the mice were administered a daily dose of intraperitoneal injection of EdU or BrdU to measure DNA synthesis and the hearts were harvested after 14 days. For analysis, 50 to 60 high-power field images (40×) per heart were acquired from the peri-infarct area and evaluated. For sham controls, images were acquired at mid-left ventricular area below the sham ligature of the heart tissue, as in previously published reports.8,9 Interestingly, in hearts from the HASF-Tg, cardiomyocyte DNA synthesis was increased at baseline (54% increase sham, HASF-Tg versus WT; P<0.05). Similarly, HASF overexpression showed increased DNA synthesis in adult cardiomyocytes after injury compared with WT controls (54% increase myocardial infarction, HASF-Tg versus WT; P<0.05; Figure 4; Online Figure III). Further analysis using 2-way ANOVA revealed that there was no significant interaction between the effect of HASF and myocardial infarction, suggesting that injury did not affect the proliferative properties of HASF. These data strongly suggest that HASF can regulate cardiomyocyte proliferation in vivo and that these effects are independent of injury.
Cardiac Overexpression of HASF Does Not Affect Cardiac Progenitor Proliferation
It has been reported that the αMHC promoter is active in early committed mouse cardiac stem cells, raising the possibility that HASF may act at least in part by increasing proliferation of these cells. To address this possibility we isolated c-Kit+ mouse cardiac stem cells from the noncardiomyocyte fraction of HASF-Tg and WT animals. As shown in Online Figure IVA, we did not see any expression of αMHC on those cells. In addition, HASF-Tg and WT mouse cardiac stem cells showed no expression of HASF, suggesting that the αMHC promoter is not active on these cells. Moreover, BrdU incorporation data showed that HASF overexpression in the heart did not affect the c-Kit+ progenitors in vitro or in vivo, suggesting that mouse cardiac stem cells are not involved in the generation of the proliferating cardiomyocytes (Online Figure IVB and IVC). Still, our data indicated that CD90+ (Thy1) noncardiomyocyte cells of the neonatal heart, presumably cardiac fibroblasts, have increased levels of DNA synthesis after HASF treatment or HASF cardiac overexpression (Online Figure V). The physiological importance of this observation or the mechanisms involved were not investigated, as they were beyond the focus of this study.
HASF Increases Cardiomyocyte Proliferation via PI3K and AKT Pathways
Our results to date have shown that HASF can stimulate cardiomyocyte proliferation. Next, we studied whether PI3K signaling was involved in HASF-mediated cardiomyocyte proliferation. To test this hypothesis, neonatal cardiomyocytes were treated with 10–20 nmol/L of HASF in a time-dependent manner, and Western blot analysis for the detection of AKT activation was performed. As shown in Figure 5A, HASF stimulation increased the phosphorylation level of AKT (T308, S473) in neonatal cardiomyocytes in vitro compared with the level in untreated cells. Activation of glycogen synthase kinase 3 β or other known Akt downstream targets such mammalian target of rapamycin complex 1– and mammalian target of rapamycin complex 2–associated proteins RAPTOR and RICTOR were not observed (Online Figures VI and VII). Moreover, blocking the PI3K pathway with the pharmacological inhibitors LY294002 or Wortmannin diminished proliferation and abrogated the HASF-induced DNA synthesis (Figure 5B and 5C). These results provide evidence for the direct role of PI3K–AKT pathway and its significance in mediating the HASF-proliferative effects.
HASF Augments the Cell-Cycle Regulator CDK7/Cyclin H Complex in Cardiomyocytes via PI3K–AKT Pathway
In the cell-cycle machinery, DNA synthesis and ultimately cell division is largely regulated by different cell-cycle–dependent kinases (CDK) that can either promote cell-cycle progression or transcription at different phases of the cell cycle.24,25 On the basis of the results mentioned above, we investigated whether HASF treatment had any effect on the major regulating CDKs or their related cyclins. Seven days of HASF treatment in neonatal cardiomyocytes increased the gene-expression levels of several G2/M phase cyclins, (eg, cylinA, cyclinB1, CDK7, and polo-like kinase 1; Figure 6A), further supporting that HASF-treated cardiomyocytes transited into S and G2/M phases leading to cell division. We next focused our analysis on the CDK7 protein and its relation to HASF. For this, we evaluated the protein-expression levels of CDK7 and its binding partner cyclin H on neonatal cardiomyocyte after 7 days of HASF treatment. Western blot analysis, as shown in Figure 6B, revealed that HASF treatment increased the protein expression of both CDK7 and cyclin H (which is part of the CDK7 activity complex), indicating increased CDK7 activity.24,25 Importantly, preincubation with LY294002 abolished the effect of HASF on CDK7 expression (Figure 6C). In contrast, treatment of cardiomyocytes with fibroblast growth factor 1 growth factor in combination with p38-mitogen–activated protein kinase inhibitor (SB2035805; a treatment that has been shown to increase cardiomyocyte proliferation via regulation of the mitogen-activated protein kinase pathway) did not have any effect on CDK7 expression (Figure 6B). Collectively, these data demonstrate that HASF regulates CKD7 expression through the PI3K–AKT pathway.
HASF-Induced Cardiomyocyte Proliferation is Modulated by the Cell-Cycle Regulator CDK7
To establish the role of CDK7 on HASF-induced proliferation, we used RNA interference to suppress Cdk7 gene expression specifically. As shown in Online Figure VIII, siRNA treatment resulted in almost complete depletion of Cdk7 gene expression within 1 day of treatment. Neonatal cardiomyocytes were then treated for 7 days with HASF in the presence and absence of Cdk7 siRNA, and BrdU uptake was measured as the marker for DNA synthesis and proliferation (Figure 6D). Transfection of cardiomyocytes with Cdk7-specific siRNA significantly diminished the HASF effect on BrdU uptake proliferation. No changes were observed when the cells were transfected with control nontargeting siRNA or when treated with fibroblast growth factor 1 instead of HASF (Figure 6D).
There is numerous evidence to indicate that mammalian adult cardiomyocytes harbor signaling circuits capable of resuming their proliferative potential.3,8,9 However, in contrast to teleost fish26,27 and urodele amphibians28 the endogenous levels of proliferation in the mammalian heart are still insufficient for significant regeneration after injury. Enabling cardiomyocyte proliferation represents an important strategy for cardiac regeneration. Here, we report the function and mechanism of C3orf58 (HASF), a novel paracrine protein, in stimulating cell-cycle progression and proliferation in neonatal and adult cardiomyocytes. Our in vivo studies using a murine model with cardiac-specific overexpression of HASF showed increased levels of DNA synthesis, as well as cytokinesis in the hearts of transgenic neonate and adult mice at baseline and after injury. Collectively, these results document the capability of HASF to induce cardiomyocyte proliferation. This discovery is significant, as only a few factors have been shown to possess the capacity of inducing adult cardiomyocyte proliferation. Technical limitations of EdU/BrdU labeling and the in vivo models did not allow us to ascertain whether the proliferating cardiomyocytes in the adult myocardium of HASF-Tg mice result from the maintenance of the amplifying neonatal cardiomyocytes or by induction of cell-cycle re-entry of mature cardiomyocytes. Unfortunately, we did not investigate whether the response to HASF was different among different types of cardiomyocytes. These issues pose interesting questions for future studies.
Cardiac myocytes do proliferate during fetal development. However, soon after birth they undergo an additional round of DNA synthesis without cell division, which results in the majority of cells being binucleated, followed by withdrawal from the cell cycle.8,9,29 The notion that adult cardiomyocytes are incapable of proliferating and are terminally differentiated has been recently challenged.9,30 However, the factors that regulate cardiomyocyte cell cycle and division in the neonatal and adult heart are poorly understood. Our in vitro experiments on rat neonatal cardiomyocytes showed that HASF increased their cell-cycle activity and increased expression of both S and G2/M phase cyclins/and CDKs; cylinA, cyclinB1, CDK7 and polo-like kinase 1. Moreover, the proliferative effect of HASF was dependent on the activation of the cell-cycle regulator, CDK7. CDK7 is a member of the TFIIH complex and is known to be the only CDK capable to drive the cell cycle to complete progression, as well as gene transcription. CDK7 binds to cyclin H for full activation, which binds MAT1, the third subunit, to stabilize the entire TFIIH complex.24,25 Still, the mechanism of CDK7 regulation, in particular, in the cardiomyocyte cell cycle, is poorly understood. Our data suggest that HASF regulates CDK7-mediated cardiomyocyte proliferation by activation of PI3K–AKT pathway. The CDK7 activation by HASF was specific to PI3K–AKT pathway and was not induced by treatment with known cardiomyocyte-proliferation inducers, such as fibroblast growth factor 1 and p38 mitogen-activated protein kinase inhibitor.5 Interestingly, investigation of known Akt downstream targets associated with cardiomyocyte proliferation, such as mammalian target of rapamycin complex 1 and mammalian target of rapamycin complex 2, did not show any activation suggesting alternative Akt-regulated mediators might be involved in increasing CDK7 expression. Our data do not exclude the possibility that other pathways involved in proliferation are stimulated by HASF. Indeed, our previous work on HASF has shown that it can also provide cytoprotection through protein kinase C epsilon-dependent pathways (Huang et al, article under review). The upstream mediators of HASF stimulation of PI3K–AKT–CDK7 are still unknown; future studies to identify HASF receptor(s) and intracellular signaling involved might shed light in this question.
In conclusion, our studies provide evidence that HASF is a novel paracrine factor that induces cardiomyocyte cell-cycle progression and proliferation via PI3K–AKT–CDK7 pathway. These findings may have significant implications in cardiac regeneration biology and therapeutics.
We thank Hui Mu for the original isolation of neonatal cardiomyocytes.
Research conducted in these studies was supported by National Heart, Lung, and Blood Institute grants (RO1 HL35610, HL81744, HL72010, and HL73219 (to V.J. Dzau); the Edna and Fred L. Mandel Jr Foundation (to V.J. Dzau and M. Mirotsou); M. Mirotsou is also supported by an American Heart Association National Scientist Development Award (10SDG4280011). The other authors report no conflict.
In May 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.113.301075/-/DC1.
- Nonstandard Abbreviations and Acronyms
- alpha myosin heavy chain
- protein kinase B (PKB)
- cycle-dependent kinase 7
- hypoxia and Akt induced stem cell factor
- Received January 28, 2013.
- Revision received June 5, 2013.
- Accepted June 19, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Limited proliferative capacity of adult cardiomyocytes translates to limited capacity of the adult heart for regeneration after injury.
The identification of molecules that can stimulate cardiomyocyte proliferation, as well as understanding of the molecular mechanism governing this process, might have important clinical implications for cardiac therapy.
What New Information Does this Article Contribute?
C3orf58, a novel secreted protein, stimulates cardiomyocyte proliferation.
These effects are mediated through activation of the Akt signaling pathway and Cyclin-dependent kinase 7.
Cardiac muscle cells loose their capacity to divide and proliferate shortly after birth. As a result the adult heart shows reduced capacity to replace damaged tissue after injury, such as the one that occurs after myocardial infarction. Currently no therapeutic molecules are available that can stimulate cardiac muscle cells to proliferate. The present work identifies a novel protein, C3orf5, as a possible new modulator of cardiomyocyte proliferation and provides first insights into the molecular pathways modulating these effects.