Pi3kcb Links Hippo-YAP and PI3K-AKT Signaling Pathways to Promote Cardiomyocyte Proliferation and SurvivalNovelty and Significance
Rationale: Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence–specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined.
Objective: To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart.
Methods and Results: We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110β, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus–mediated Pik3cb expression.
Conclusions: Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival.
Adult mammalian cardiomyocytes largely exit from the cell cycle, thereby limiting the innate regenerative capacity of the mature heart.1 As a result, there are currently no treatments for heart disease that reverse cardiomyocyte loss, a root cause for many cases of heart failure.
Editorial, see p 3
In This Issue, see p 2
Recently, the transcriptional coactivator yes-associated protein (YAP) was found to be essential for fetal cardiomyocyte proliferation, and activated YAP drove adult cardiomyocyte proliferation.2–5 YAP is the nuclear effector of the Hippo kinase cascade, a recently defined pathway that regulates cell proliferation and survival to establish organ size.6 Hippo pathway kinases macrophage stimulating 1/2 and large tumor suppressor kinase 1 phosphorylate YAP, retarding its transcriptional activity by promoting its nuclear export. Hippo kinase inactivation enhanced YAP activity and stimulated fetal and adult cardiomyocyte proliferation.7,8
YAP regulates transcription of its direct target genes by binding to sequence-specific DNA binding proteins, with TEA (transcriptional enhancer activator) domain family members 1–4 (TEAD1-4) being key transcriptional partners.6 However, few direct YAP targets that are essential for its growth promoting activity are known. The phosphoinositide 3-kinase (PI3K)-AKT pathway was reported to be activated downstream of YAP,4 although the link between these pathways was not determined. The PI3K-AKT pathway promotes cardiomyocyte proliferation, survival, and physiological hypertrophy.4,5 Class IA PI3Ks are composed of a p85 regulatory subunit and a p110 catalytic subunit. The p110 catalytic subunit is encoded by 3 different genes, Pik3ca, Pik3cb, and Pik3cd, with most cells expressing Pik3ca and Pik3cb.9 Early lethality of Pik3cb null embryos indicates that Pik3ca and Pik3cb isoforms are not fully redundant.10 However, most work has focused on Pik3ca and much less is known about Pik3cb.
In this study, through an unbiased whole genome screen, we identified Pik3cb as a direct YAP target that links YAP to PI3K-AKT activation. Our data indicate that Pik3cb activation is sufficient to drive adult cardiomyocyte proliferation and is necessary for the mitogenic activity of YAP.
Additional detailed Materials and Methods are provided in the online-only Data Supplement.
All animal procedures were approved by the Institutional Animal Care and Use Committee. Echocardiography and myocardial infarction (MI) surgery were performed blinded to genotype and treatment group.
Hearts were fixed, embedded, cryosectioned, and immunostained as described.12 Antibodies are listed in Online Table I.
HL1 cells overexpressing 3FLAG-YAP[S127A] were used for chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) as described.11 HiSeq 2000 (Illumina) sequencing data were used to identify binding peaks (Online Table II). Chromatin immunoprecipitation followed by quantitative PCR was performed using antibodies and primers listed in Online Tables I and III.
Gene expression was measured by qRT-PCR using primers listed in Online Table III or by microarray (Mouse Gene ST 2.0 array, Affymetrix) using RNA collected from E12.5 mouse heart.
Data are available at GEO (accession number GSE57719) or the Cardiovascular Development Consortium server at https://b2b.hci.utah.edu/gnomex/.
Values are expressed as mean±SEM. To test for statistical significance, we used Student’s t test (2 groups) or ANOVA with the Tukey HSD post hoc test (>2 groups). Tests were performed using JMP10.0 (SAS).
YAP Directly Activates Pik3cb Expression Through TEAD
YAP plays crucial roles in regulating cardiomyocyte proliferation and survival,2–5,7,8 but the direct targets of YAP that mediate its effects are largely unknown. To identify candidate genes that are directly regulated by YAP and that mediate its proliferative activity, we overexpressed activated, FLAG-tagged YAP (3xFlag-YAP[S127A], in which the inhibitory Hippo phosphorylation site serine 127 is mutated to alanine) in HL1 cardiomyocyte-like cells. ChIP and ChIP-seq identified 1340 YAP-bound chromatin regions (Figure 1A; Online Table II). YAP bound to genes enriched for functional terms including cardiovascular system development, regulation of cell proliferation, and cell proliferation (Figure 1B). Motif discovery using these YAP-bound regions yielded the consensus TEAD motif (P=9.7E-47), confirming the predominant interaction of YAP with TEAD and providing validation for the ChIP-seq data set. Scanning the YAP-bound regions for known transcription factor binding motifs also identified the TEAD motif, as well as the signal transducers and activators of transcription (STAT) and v-ets avian erythroblastosis virus E26 oncogene homolog (ETS) motifs, which share sequence similarity to the TEAD motif (Figure 1C).
We used microarray gene expression profiling to identify genes downstream of YAP. We compared E12.5 mouse hearts with TNTCre-mediated YAP inactivation in cardiomyocytes (YAPfl/fl::TNTCre) to Yapfl/+::TNTCre littermate controls (Figure 1A). We also compared adenovirus-mediated YAP[S127A] overexpression in neonatal rat ventricular cardiomyocytes to adenoviral LACZ expression (Figure 1A), which we reported previously.2 In the murine loss-of-function data set, we identified 2200 differentially expressed genes (1137 and 1063 up- and downregulated in knockout, respectively; P<0.05; absolute log2 fold-change >0.2; n=3; Online Table III). In the neonatal rat gain-of-function data set, there were 2091 differentially expressed genes (1030 and 1061 up- and downregulated in Yap overexpression, respectively; P<0.05; absolute log2 fold-change >0.5; n=4; see Ref. 2). There were 217 genes with concordant regulation by YAP in both data sets (Online Table IV). These 217 genes were enriched for functional terms encompassing heart development (P=0.00045).
The intersection of genes associated with YAP-bound chromatin regions and concordantly regulated downstream of YAP in both differential expression data sets contained 26 genes, with 13 activated by YAP and 13 repressed by YAP (Online Table IV). Given that YAP was previously reported to activate the PI3K-AKT pathway though uncertain mechanisms,4 we were interested to find Pi3kcb among the candidates for direct YAP activation. Pik3cb is a little studied isoform that encodes the phosphoinositol-3-kinase (PI3K) catalytic subunit (also referred to as p110β), a key kinase that regulates cell growth and metabolic activity.13,14 Like YAP,2 PIK3CB protein levels decline in the heart with increasing postnatal age (Online Figure I). qRT-PCR of NRVMs confirmed that Ad:YAP[S127A] activated expression of Pik3cb compared with Ad:LacZ (Figure 1D).
The HL1 ChIP-seq data revealed a YAP-bound sequence residing in the first intron of Pik3cb (Figure 2A). We validated YAP binding to the identified sequence by chromatin immunoprecipitation followed by quantitative PCR, using a pair of primers spanning the YAP-bound sequence and a control pair recognizing a sequence 1.3 kb away (Figure 2B). This YAP-bound sequence contained an evolutionarily conserved sequence (AGGAATTCGTGGAATT) containing 2 repeats of a motif that is similar to the TEAD, STAT, and ETS motifs (Figure 2C and 2D). Chromatin immunoprecipitation followed by quantitative PCR to confirmed YAP and TEAD occupancy of this Pik3cb region but not the adjacent control region in neonatal heart (Figure 2E). Although YAP-TEAD interaction is well described, YAP has not been reported to interact with STAT or ETS. Co-IP experiments showed no detectable interaction between YAP and STAT3, STAT5a, STAT6, or ETS1 (Online Figure IIA–IIC). These data suggest that YAP activates the Pik3cb enhancer via TEAD in cardiomyocytes.
To measure the transcriptional activity of the YAP-bound region of Pik3cb, which we refer to as the Pik3cb enhancer, we cloned a 552 bp genomic DNA fragment containing the conserved element and potential TEAD binding sites into a minimal promoter luciferase reporter construct. Co-transfection with Yap in NRVMs showed that Yap stimulates activity of the enhancer by ≈5-fold. Yap stimulation was abrogated by mutation of the core-conserved element (Figure 2F). YAP[S94A], deficient in TEAD interaction,15 failed to activate the Pik3cb enhancer (Figure 2G), indicating that YAP stimulates Pik3cb expression through TEAD. On the other hand, S3I-201, a Stat3 inhibitor which prevents Stat3-binding DNA and suppresses Stat3-dependent transcription,16 did not affect YAP activation of the Pik3cb enhancer or YAP-induced cardiomyocyte proliferation (Online Figure IID and IIE).
Together, these data indicate that YAP binds to an evolutionarily conserved motif in the first intron of Pik3cb through TEAD to upregulate Pik3cb expression in cardiomyocytes.
Yap Is Sufficient and Required to Upregulate Pik3cb/Pik3ca and Activate PI3K-Akt Pathway In Vivo
Having shown that YAP directly binds and activates the Pik3cb enhancer, we next asked whether YAP is necessary and sufficient to stimulate Pik3cb expression in vivo and thereby activate the PI3K-Akt pathway. To overexpress YAP in cardiomyocytes in vivo, we used adeno-associated virus serotype 9 (AAV9), a safe, efficient and cardiotropic vector, for in vivo gene transfer.17,18 We used a cardiomyocyte-specific chicken troponin T promoter (cTNT) to further enhance cardiac selectivity. We validated the cardiomyocyte specificity of this gene transfer system by analyzing the recombination pattern of AAV9:cTNT-iCre, in which the cTNT promoter drives expression of mammalian codon-optimized Cre recombinase. We systemically administered AAV9:cTNT-iCre by subcutaneous injection into 1- to 2-day-old mouse pups harboring the Rosa26fsTRAP allele, in which Cre recombination activates GFP expression. Seven days after virus administration, most TNNI3-positive cardiomyocytes expressed GFP, but TNNI3-negative noncardiomyocytes did not (Online Figure IIIA). These data show that AAV9-cTNT drives cardiomyocyte-selective expression within the heart.
We then replaced the iCre gene with FLAG-tagged, activated YAP (3Flag-YAP[S127A]) to generate AAV9:cTNT-Yap virus (AAV9:Yap). As a negative control, we generated AAV9:cTNT-Luciferase (AAV9:Luci). AAV9:Yap and AAV9:Luci were injected into 1- to 2-day-old pups. Seven days later, the expression of exogenous 3Flag-YAP[S127A] was clearly detected by western blot (Figure 3A). Expression of activated YAP caused >2-fold upregulation of Pik3cb (Figure 3B). Pik3ca was also upregulated, although to a lesser degree compared with Pik3cb (Figure 3B).
Xin et al previously reported that YAP activation in cultured neonatal rat ventricular cardiomyocytes increased the levels of activated AKT (Akt[p-S473]) without changing total AKT protein level.4 YAP was linked to AKT activation in this system by upregulation of IGF1R. In mitotic tissue (skin) and cultured cells, YAP was also shown to activate the PI3K-Akt pathway by suppressing expression of PTEN (phosphatase and tensin homolog deleted on chromosome ten),19 an inhibitor of PI3K-Akt signaling. We found that YAP activation in cardiomyocytes in vivo increased AKT activation without changing total AKT level (Figure 3C). YAP significantly upregulated IGF1R by ≈1.5-fold, but did not alter PTEN level (Online Figure IIIB and IIIC).
We next asked if YAP is required for normal expression of Pik3cb and Pik3ca in the heart. We generated Yapfl/fl::Myh6-Cre mice (YAPcKO), in which cardiomyocyte-specific Myh6-Cre inactivates a conditional YAPflox allele. We confirmed efficient YAP inactivation by western blotting, which showed marked downregulation of cardiac YAP protein (Figure 3D). In YapcKO hearts, Pik3cb and Pik3ca mRNA were both significantly reduced (Figure 3E). Moreover, phosphorylated but not total Akt was reduced in YapcKO mice heart, indicating that Yap is required for maintenance of the normal level of Akt activation (Figure 3F).
The cell cycle inhibitor p27 (CDKN1B) is a direct target of Akt, and Akt-mediated p27 phosophorylation leads to p27 degradation.20 We, therefore, measured p27 protein level as a downstream readout of Akt activation. In AAV9:YAP-treated hearts, p27 protein was downregulated (Figure 3C), while in YapcKO hearts, p27 protein was upregulated (Figure 3F). These data further confirm that Akt activity is governed by YAP activity in cardiomyocytes.
Collectively, both gain- and loss-of-function data indicate that YAP promotes Pik3cb and Pik3ca upregulation and stimulates PI3K-Akt pathway activation in vivo.
Pik3cb Overexpression Activated AKT and Induced Cardiomyocyte Proliferation
To determine if Pik3cb is sufficient to activate Akt and drive cardiomyocyte proliferation, we generated Ad:Pik3cb, an adenovirus that expresses Pik3cb. We validated overexpression of PIK3CB protein in NRVMs by Ad:Pik3cb (Figure 4A). We then treated NRVMs with Ad:Pik3cb and measured the cardiomyocyte proliferation rate using 2 independent markers, phosphorylated histone H3 (pH3), an M phase marker, and bromodeoxyuridine uptake, an S phase marker. Pik3cb overexpression significantly increased the fraction of cardiomyocytes positive for bromodeoxyuridine and pH3 (Figure 4B–4E). These data indicate that Pik3cb is sufficient to stimulate proliferation of cultured neonatal cardiomyocytes in vitro.
To extend these data to an in vivo context, we generated AAV9:cTNT-3Flag-Pik3cb (AAV9:Pik3cb; Figure 4F) and delivered it or control AAV9:Luci systemically to 1- to 2-day-old neonatal mice. Seven days after virus administration (8–9 days of age), we collected hearts for immunoblotting and histological studies. We confirmed expression of Flag-tagged human PIK3CB protein in the heart (Figure 4G). To determine the effect of Pik3cb gain-of-function on cardiomyocyte proliferation, we measured the fraction of cardiomyocytes that were positive for pH3. Compared with AAV9:Luci, AAV9:Pik3cb significantly increased the pH3+ CM fraction (Figure 4H and 4I), suggesting that PIK3CB is sufficient to increase neonatal cardiomyocyte proliferation in vivo.
We next addressed the ability of Pik3cb to stimulate adult cardiomyocyte proliferation in the context of disease. MI was induced in 2-month-old CFW mice by coronary artery ligation (Online Figure IVA). The freshly infarcted myocardium was treated with AAV9:Luci or AAV9:Pik3cb. Four days after MI, mice were treated with 1 dose of 5-ethynyl-2'-deoxyuridine (EdU) to label dividing cardiomyocytes. Five days after MI, hearts were collected for analysis. In the border zone, AAV9:Pik3cb treatment resulted in higher cardiomyocyte EdU labeling index than AAV:Luci treatment (Online Figure IVB and IVC). Cardiomyocyte apoptosis was also significantly reduced in AAV9:Pik3cb treatment compared with control (Online Figure IVD and IVE). These data show that Pik3cb promotes cardiomyocyte cell cycle activity and enhances cardiomyocyte survival after MI.
One mechanism by which a treatment might increase cardiomyocyte proliferation is by promoting dedifferentiation. To assess whether Pik3cb stimulates cardiomyocyte dedifferentiation, we treated P2 neonatal hearts with AAV9-Luci or AAV9-Pik3cb. We then analyzed expression of myocardial differentiation markers at P9. We detected no change in expression of Myh6, Myh7, or Nkx2-5 (Online Figure IVF), suggesting that Pik3cb does not promote cardiomyocyte dedifferentiation.
We next examined the effect of Pik3cb gain-of-function on downstream Akt signaling. Interestingly, Pik3cb overexpression upregulated Pik3ca (Online Figure IVG), suggesting that YAP indirectly upregulates Pik3ca through its direct effect on Pik3cb. AAV9:Pik3cb-treated hearts showed higher level of activated Akt (Akt[pS473]) than AAV9:Luci-treated hearts, whereas the level of total Akt was comparable between groups (Figure 4J). Furthermore, the protein level of p27 was decreased in AAV9:Pik3cb-treated hearts (Figure 4K; P<0.05; quantification in Online Figure IVH). These data demonstrate that overexpression of PIK3CB activates Akt and increases cardiomyocyte proliferation in vivo.
Pik3cb Is Required for YAP-Stimulated Akt Activation and Cardiomyocyte Proliferation
Because YAP directly regulated Pik3cb, which is sufficient to increase cardiomyocyte proliferation, we hypothesized that Pik3cb is required for YAP-induced Akt activation and cardiomyocyte proliferation. To analyze the requirement of Pik3cb downstream of YAP in cultured NRVMs, we synthesized 3 siRNAs and measured their reduction of YAP-stimulated Pik3cb expression. siPik3cb#2 reduced Pik3cb expression to 42% of control values, whereas the other 2 siRNAs were not effective (Figure 5A). Then we stimulated NRVMs with activated YAP and treated them with siPik3cb#2 or control siRNA. siPik3cb#2 partially blocked the YAP-induced increase in cardiomyocyte proliferation markers EdU+ and pH3+ (Figure 5B–5E). These data indicate that Pik3cb is necessary for YAP to fully induce cardiomyocyte proliferation in vitro.
To study the requirement of Pik3cb downstream of YAP in cardiomyocytes in vivo, we followed a previously validated strategy21 and embedded an shRNA directed against Pik3cb into the 3' UTR of AAV9:YAP to simultaneously overexpress Yap and knock down Pik3cb in the mouse heart. We tested 4 different shRNAs, and shPik3cb#3 yielded the greatest Pik3cb knockdown (Online Figure VA). We then cloned this shRNA, or a scrambled negative control shRNA, downstream of YAP or luciferase to yield AAV9:YAP-shPik3cb, AAV9:YAP-sc, and AAV9:Luci-sc (Figure 6A). AAV9:YAP-shPik3cb was designed to overexpress activated YAP and simultaneously knock down Pik3cb, whereas AAV9:YAP-sc was designed to overexpress activated YAP without perturbing Pik3cb expression.
Compared with AAV9:Luci-sc control virus, both AAV9:YAP-shPik3cb and AAV9:YAP-sc strongly upregulated Yap to comparable degrees (≈6.5-fold upregulation). Consistent with our earlier data, AAV9:YAP-sc upregulated Pik3cb by 2.5-fold and Pik3ca by 1.4-fold compared with AAV9:Luci-sc control (Figure 6B). AAV9:YAP-sc robustly stimulated cardiomyocyte proliferation in the neonatal mouse heart, as reflected by pH3 staining (Figure 6C and 6D). In comparison, AAV9:YAP-shPik3cb upregulation of Pik3cb was significantly attenuated. Reduced Pik3cb expression corresponded to less induction of cardiomyocyte proliferation by AAV9:YAP-shPik3cb (Figure 6C and 6D). These changes were not associated with altered expression of Myh6, Myh7, or Nkx2-5, suggesting they were not caused by altered cardiomyocyte differentiation (Online Figure VB). These data indicate that Pik3cb is required for full stimulation of cardiomyocyte proliferation by YAP.
We previously showed that several cell cycle genes, including aurora A kinase (Aurka), cell division cycle 20 (Cdc20), and cyclin A2 (Ccna2), are upregulated by YAP.2 Consistent with these data, AAV9:YAP-sc upregulated these genes compared with AAV9:Luci-sc control (Figure 6E). Reduced Pik3cb expression by AAV9:YAP-shPik3cb interfered with Aurka and Ccna2 upregulation by YAP (Figure 6E). Average Cdc20 expression also decreased, although this did not reach statistical significance. Meanwhile, Cdkn1b (p27) mRNA level was not significantly affected, in keeping with its regulation by its post-transcriptional regulation by PI3K-AKT.20 These results demonstrate that YAP regulation of the expression of a subset of cell cycle genes is dependent on upregulation of Pik3cb.
Because YAP is sufficient to increase Pik3cb expression and activate AKT (Figure 2) and AKT is a downstream target of PIK3CB, we hypothesized that PIK3CB is required for YAP to activate AKT. To test this hypothesis, we measured activated and total AKT, as well as p27 by western blotting of hearts transduced with AAV9:YAP-sc or AAV9:YAP-shPik3cb (Figure 6Fand 6G). Total AKT levels were comparable between groups. YAP-sc elevated activated AKT, and this stimulation was dampened substantially by shPik3cb. Although the p27 mRNA level did not change between AAV9:YAP-sc and AAV9:YAP-shPik3cb groups (Figure 6E), the protein level of p27 was increased by shPIK3cb (Figure 6F), consistent with destabilization of p27 protein by activated AKT.
These data indicate that YAP stimulates AKT at least in part through upregulation of Pik3cb.
Pik3cb Upregulation Improved YapcKO Heart Function
Cardiac-specific depletion of Yap caused pathological hypertrophy and heart failure.3,5 Since YAP is required to activate Pik3cb and to maintain PI3K-AKT activity, we hypothesized that Pik3cb overexpression would restore function of YapcKO hearts. We tested this hypothesis by treating 1-day-old YapcKO or Yapfl/fl pups with either AAV9:Pik3cb or AAV9:Luci (Figure 7A). At 3 weeks of age, we administered 1 dose of EdU to label the cardiomyocytes traversing S phase of the cell cycle. At 4 weeks of age, we measured cardiac function by echocardiography, then we collected the hearts for analysis. Treatment of control (Yapfl/fl) mice with AAV9:Pik3cb did not significantly affect heart function compared with AAV9:Luci, indicating that Pik3cb overexpression is well tolerated (Figure 7B). YAPcKO hearts had severe systolic dysfunction that was partially rescued by AAV9:Pik3cb (Figure 7B). Because of technical difficulties, we were not able to quantify the fraction of cardiomyocytes transduced by AAV9:Pik3cb. It is possible that the incomplete rescue was because of incomplete cardiomyocyte transduction or Pik3cb-independent YAP activities. Nevertheless, our data indicate that decreased Pik3cb and PI3K-AKT signaling is an important contributor to cardiac dysfunction in YAP loss of function.
Next we considered the effect of Pik3cb overexpression on cardiomegaly observed in YAPcKO. AAV9:Pik3cb treatment of control (Yapfl/fl) mice did not significantly affect heart size compared with AAV9:Luci (Figure 7C and 7D), indicating that Pik3cb overexpression does not cause cardiac hypertrophy. YAPcKO hearts showed significant cardiomegaly, likely secondary to cardiac dilation in heart failure. Cardiomegaly was attenuated by AAV9:Pik3cb but not AAV9:Luci (Figure 7C and 7D), consistent with improvement of ventricular function (Figure 7B). We further examined the effect of Pik3cb overexpression on cardiac hypertrophy caused by YAP depletion in the heart at the level of cardiomyocyte size. AAV9:Pik3cb treatment of control (Yapfl/fl) mice did not significantly alter cardiomyocyte cross-sectional area compared with AAV9:Luci treatment (Figure 7E and 7F). Cardiomyocyte cross-sectional area was significantly greater in YAPcKO heart. Pik3cb overexpression by AAV9:Pik3cb significantly blunted this cardiomyocyte hypertrophy compared with AAV9:Luci (Figure 7E and 7F).
Cardiac hypertrophy is often associated with upregulation of the genes Nppa and Myh7. We assessed the effect of AAV9:Pik3cb on the expression of these hypertrophic marker genes in YapcKO heart. AAV9:Pik3cb markedly downregulated Nppa and Myh7 compared with AAV9:Luci (Figure 7G), consistent with attenuation of cardiomyocyte hypertrophy. These data indicate that AAV9:Pik3cb partially mitigated hypertrophic marker gene activation caused by cardiac YAP depletion.
Together these data demonstrate that Pik3cb overexpression attenuates cardiac dysfunction and pathological cardiac hypertrophy of YapcKO mice.
AAV9:Pik3cb Increased Cardiomyocyte Proliferation and Decreased Cardiomyocyte Apoptosis in YapcKO Mice
We investigated potential mechanisms through which AAV9:Pik3cb rescued dysfunction and hypertrophy of YapcKO hearts. YapcKO mice were reported to have more cardiomyocyte apoptosis,3 and their cardiomyocyte regenerative capacity was impaired.5 Because the PI3K-AKT pathway promotes both cell proliferation and survival,22 we hypothesized that Pik3cb overexpression ameliorates the YapcKO phenotype by increasing cardiomyocyte proliferation, decreasing apoptosis, or both.
To investigate the effect on cardiomyocyte proliferation, we measured the fraction of cardiomyocytes that incorporated EdU, administered at 3 weeks of age (experimental timeline shown in Figure 7A). Staining of tissue sections showed that cardiomyocyte EdU uptake was higher in YapcKO mice treated with AAV9:Pik3cb compared with those treated with AAV9:Luci (Figure 8A and 8B). We further confirmed this finding by staining dissociated cardiomyocytes (Figure 8C and 8D), which obviates potential artifacts that can occur in tissue sections. Moreover, AAV9:Pik3cb increased the fraction of mononuclear cardiomyocytes (Figure 8E), which contains the proliferative cardiomyocyte subset,23 and it increased the frequency that we observed cardiomyocytes in cytokinesis, as marked by staining with Aurora B kinase (Figure 8F).23 Together, these data demonstrated that Pik3cb stimulated cardiomyocyte proliferation in YAP-deficient hearts.
To study the effect of Pik3cb overexpression on apoptosis in YAP-deficient cardiomyocytes, we measured the fraction of cardiomyocytes positive for TUNEL-staining, a marker of apoptosis. TUNEL+ cardiomyocytes were less frequent in YapcKO, treated with AAV9:Pik3cb compared with AAV9:Luci, indicating that Pik3cb rescues cardiomyocyte apoptosis caused by YAP-deficiency (Figure 8G and 8H).
Yap activation increased the expression of both Pik3ca and Pik3cb (Figure 3B), and Pik3cb was sufficient to induce the expression of Pik3ca in wild-type mouse heart (Online Figure IVG). These data suggest that Pik3cb functions downstream of Yap to regulate Pik3ca expression, AKT activation, and p27 levels. Indeed, Pik3cb overexpression in the absence of YAP rescued AKT activation without changing total AKT level (Figure 8I). AKT activation corresponded with decreased p27 protein but not mRNA (Figure 8I and IJ).
Together, our data support a model in which YAP directly activates Pik3cb expression through TEAD binding to an enhancer in the first Pik3cb intron. PIK3CB subsequently promotes expression of Pik3ca and activation of AKT, which regulates cardiomyocyte apoptosis and proliferation, in part through p27 (Figure 8K).
Emerging studies have revealed the critical role of Hippo-YAP signaling in heart development, growth, and homeostasis.2–5,7,8 One major pathway through which Hippo-YAP signaling regulates cardiomyocyte growth and survival is the PI3K-AKT signaling axis.3,4 This pathway has well established, pleiotropic effects on cardiomyocyte proliferation, growth, survival, and function.24 However, the mechanistic link between YAP and the PI3K-AKT pathway was not previously known. Our genome-wide screen for directly activated YAP target genes showed that YAP, through its transcriptional partner TEAD, directly activates Pik3cb expression via an enhancer in the first intron of Pik3cb. Our functional analyses demonstrate that YAP requires Pik3cb to promote cardiomyocyte proliferation and activate the AKT pathway. Together, these findings establish Pik3cb as a regulator of cardiac growth, serving as a direct link between Hippo-YAP and PI3K-AKT signaling pathways.
In mammals, most cells express both Pik3ca and Pik3cb, isoforms of the p110 catalytic subunit of Class IA PI3K. These isoforms each have unique functions, as germline inactivation of either Pik3ca or Pik3cb caused embryonic lethality before E10.5.10,25 Compared with Pik3ca, relatively less is known about Pik3cb and how it differs from Pik3ca. Interestingly, Pik3cb is unique among PI3-kinases in signaling downstream of both receptor tyrosine kinases and G-protein-coupled receptors.26 Adult, cardiac-specific inactivation of Pik3cb did not cause a baseline cardiac phenotype, whereas similar inactivation of Pik3ca caused cardiac dysfunction.27 These data showing that Pik3cb is dispensable for adult heart homeostasis are compatible with our observations, as it is likely that Pik3cb regulates physiological heart growth and stress responses downstream of YAP. Indeed, both YAP and PIK3CB protein levels in the heart decline with postnatal age, and our data point to a role of Pik3cb in promoting heart growth downstream of Yap. Additional studies will be required to interrogate further the roles of Pik3cb in heart development, stress responses, and regeneration.
Our work demonstrates that YAP functions directly upstream of Pi3kcb to enhance its expression. However, Pik3cb expression is likely regulated by other factors as well because its expression was reduced but not completely abrogated by YAP knockout. Recently, it was reported that growth factors, such as EGF, signal through receptor tyrosine kinases to PI3K, which then promotes disassembly of the Hippo kinase complex, relieving its inhibition of YAP transcriptional activity.28 Combined, these studies suggest reciprocal, mutually stimulatory cross-talk between YAP and PI3K that establish a feed forward regulatory circuit, in which Yap increases the expression of the PI3K subunit Pik3cb, and PI3K stimulates YAP activity.
YapcKO mice had heart dysfunction.3,5 Several targets of Yap have been identified, such as Ctgf, Birc3, and Birc5, but none have been shown to be essential for the YapcKO phenotype. By activating Pik3cb in 1-day-old YapcKO pups, we largely restored heart function of YapcKO mice, indicating that Pik3cb is a major target of Yap and is important for preserving heart function. This result differs from the observation that Pik3cb is dispensible for normal heart homeostasis.27 We reasoned that the different experimental contexts (normal adult heart versus failing juvenile heart) likely account for the different results. YAP is normally downregulated in the mature adult heart,2 and it is possible that YAP and Pik3cb play more vital roles during heart development and postnatal growth. Furthermore, the YapcKO mouse has systolic dysfunction at birth that progresses over ≈3 months to death. Thus, the YapcKO represents a stressed heart, which may have a different requirement for Pik3cb than a normal heart.
The heart failure phenotype of YapcKO is likely multifactorial. Consistent with published data, we found less proliferation and more apoptosis in the YapcKO mice,3 indicating that the heart dysfunction of YapcKO mice is at least partially because of cumulative effect of cardiomyocyte insufficiency. Pik3cb rescued YapcKO heart function, and our data indicate that this is through both mitogenic and prosurvival activities, in keeping with known roles of PI3K-AKT signaling.The cell cycle inhibitor p27, which is normally downregulated at the protein level by AKT phosphorylation-triggered degradation,20 was upregulated in YapcKO. P27 heterozygous inactivation enhanced cardiomyocyte proliferation,29 suggesting that its aberrant expression in YapcKO heart contributes to reduced cardiomyocyte proliferation seen in these mutants (Figure 8E). At present, we are unable to determine the relative contribution of Pik3cb’s proproliferative versus prosurvival effects to its overall beneficial activity.
Other cardiomyocyte functions are also likely to be regulated by YAP. For instance, TEAD transcription factors are implicated in regulating sarcomere gene expression through its recognition sequence, known as the MCAT motif,30 suggesting a role for YAP in regulation of sarcomere assembly and function. Consistent with this idea, YAP-bound genes in our ChIP-seq data were more highly enriched for functional terms related to cardiovascular system development than terms related to cell proliferation. YAP has also been reported to regulate cell metabolism, a function that intersects with a well-described function of PI3K-AKT signaling. A subset of these YAP activites are likely to be independent of PIK3CB. This, in combination with incomplete transduction of all cardiomyocytes by AAV9:Pik3cb, likely accounts for incomplete Pik3cb rescue of YapcKO hearts.
In summary, we identified Pik3cb as a crucial direct target of YAP that links Hippo-YAP and PI3K-AKT signaling pathways (Figure 8K). YAP, through its transcriptional partner TEAD, increases Pik3cb expression, which further activates AKT. Pik3cb activation downstream of YAP promotes cardiomyocyte proliferation and survival.
We thank Jianming Jiang for sharing with us the protocol of making AAV-shRNA.
Sources of Funding
Z. Lin was supported by an American Heart Association (AHA) postdoctoral fellowship. W.T. Pu. was supported by National Institute of Health HL095712, HL100401, and U01HL098166, by an AHA Established Investigator Award, and by charitable contributions from Gail Federici Smith, Karen Carpenter, Edward Marram, and Dr Edwin A. Boger.
In August, 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.55 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.304457/-/DC1.
- Nonstandard Abbreviations and Acronyms
- adeno-associated virus serotype 9
- cTNT-3Flag-Pik3cb (AAV9:Pik3cb)AAV9 with cTNT promoter expressing Pik3cb with 3 FLAG epitope tags
- AAV9:cTNT-Luci (AAV9:Luci)
- AAV9 with cTNT promoter expressing luciferase
- AAV9 with cTNT promoter expressing luciferase and a scrambled shRNA
- AAV9 with cTNT promoter expressing YAP[S127A] and a scrambled shRNA
- AAV9 with cTNT promoter expressing YAP[S127A] and a Pik3cb-directed shRNA
- Chromatin immunoprecipitation followed by high throughput sequencing
- cardiac troponin T
- v-ets avian erythroblastosis virus E26 oncogene homolog
- myocardial infarction
- neonatal rat ventricular cardiomyocyte
- phosphatidylinositol-4,5-bisphosphate 3-kinase
- Signal transducers and activators of transcription
- TEA-domain family members 1 to 4
- Yes-associated protein
- cardiac specific YAP knockout (Myh6-Cre::Yapfl/fl)
- Received May 26, 2014.
- Revision received September 18, 2014.
- Accepted September 23, 2014.
- © 2014 American Heart Association, Inc.
- Hill JA,
- Olson E
- Zhou B,
- Lin Z,
- Pu WT
- von Gise A,
- Lin Z,
- Schlegelmilch K,
- Honor LB,
- Pan GM,
- Buck JN,
- Ma Q,
- Ishiwata T,
- Zhou B,
- Camargo FD,
- Pu WT
- Del Re DP,
- Yang Y,
- Nakano N,
- Cho J,
- Zhai P,
- Yamamoto T,
- Zhang N,
- Yabuta N,
- Nojima H,
- Pan D,
- Sadoshima J
- Xin M,
- Kim Y,
- Sutherland LB,
- Qi X,
- McAnally J,
- Schwartz RJ,
- Richardson JA,
- Bassel-Duby R,
- Olson EN
- Xin M,
- Kim Y,
- Sutherland LB,
- Murakami M,
- Qi X,
- McAnally J,
- Porrello ER,
- Mahmoud AI,
- Tan W,
- Shelton JM,
- Richardson JA,
- Sadek HA,
- Bassel-Duby R,
- Olson EN
- Heallen T,
- Zhang M,
- Wang J,
- Bonilla-Claudio M,
- Klysik E,
- Johnson RL,
- Martin JF
- Heallen T,
- Morikawa Y,
- Leach J,
- Tao G,
- Willerson JT,
- Johnson RL,
- Martin JF
- He A,
- Kong SW,
- Ma Q,
- Pu WT
- Ciraolo E,
- Iezzi M,
- Marone R,
- et al
- Zhao B,
- Ye X,
- Yu J,
- Li L,
- Li W,
- Li S,
- Yu J,
- Lin JD,
- Wang CY,
- Chinnaiyan AM,
- Lai ZC,
- Guan KL
- Siddiquee K,
- Zhang S,
- Guida WC,
- Blaskovich MA,
- Greedy B,
- Lawrence HR,
- Yip ML,
- Jove R,
- McLaughlin MM,
- Lawrence NJ,
- Sebti SM,
- Turkson J
- Jiang J,
- Wakimoto H,
- Seidman JG,
- Seidman CE
- Bi L,
- Okabe I,
- Bernard DJ,
- Wynshaw-Boris A,
- Nussbaum RL
- Lu Z,
- Jiang YP,
- Wang W,
- Xu XH,
- Mathias RT,
- Entcheva E,
- Ballou LM,
- Cohen IS,
- Lin RZ
- Fan R,
- Kim NG,
- Gumbiner BM
- Poolman RA,
- Li JM,
- Durand B,
- Brooks G
- Yoshida T
Novelty and Significance
What Is Known?
Loss of cardiomyocytes is associated with increased mortality and morbidity.
There is no effective means to replace the lost cardiomyocytes.
The transcriptional coactivator yes-associated protein (YAP) is essential for heart growth and for normal adult heart systolic function. Inactivation of YAP causes lethal dilated cardiomyopathy with reduced cardiomyocyte proliferation and increased apoptosis.
YAP activation increases cardiomyocyte proliferation, albeit modestly.
The direct targets of Yap that convey its mitotic signal have not been defined.
YAP is known to activate the phosphoinositide 3-kinase (PI3K)-AKT pathway, a key regulator of cell proliferation and survival. However, the molecular link is not known.
What New Information Does This Article Contribute?
Through an unbiased, genome-wide screen, we found that YAP directly activates expression of Pik3cb through a conserved enhancer within its first intron. Pik3cb encodes a less-studied isoform of the catalytic subunit of PI3K.
Pik3cb promotes cardiomyocte proliferation and survival by stimulating Pik3ca expression and AKT activation.
YAP-induced cardiomyocyte proliferation requires upregulation of Pik3cb.
Heart failure resulting from inactivation of YAP was mitigated by activation of Pik3cb. Forced expression of Pik3cb normalized cardiomyocyte proliferation and survival in the absence of YAP, pointing out the key role of Pik3cb and the PI3K-AKT pathway in the physiological function of YAP.
This study focused on delineating the mechanism by which YAP regulates cardiomyocyte proliferation and survival. By using unbiased whole genome profiling methods and AAV-mediated gain- and loss-of-function studies, we confirmed that Pik3cb is a major downstream target of YAP, being both sufficient and necessary for YAP activation of AKT. These data show that Pik3cb is a crucial link between Hippo-YAP and PI3K-AKT pathways.