Cyclic AMP-Dependent Protein Kinase A Regulates the Alternative Splicing of CaMKIIδ

Ca2+/calmodulin-dependent protein kinase (CaMK) IIδ is predominantly expressed in the heart. There are three isoforms of CaMKIIδ resulting from the alternative splicing of exons 14, 15, and 16 of its pre-mRNA, which is regulated by the splicing factor SF2/ASF. Inclusion of exons 15 and 16 or of exon 14 generates δA or δB isoform. The exclusion of all three exons gives rise to δC isoform, which is selectively increased in pressure-overload-induced hypertrophy. Overexpression of either δB or δC induces hypertrophy and heart failure, suggesting their specific role in the pathogenesis of hypertrophy and heart failure. It is well known that the β-adrenergic-cyclic AMP-dependent protein kinase A (PKA) pathway is implicated in heart failure. To determine the role of PKA in the alternative splicing of CaMKIIδ, we constructed mini-CaMKIIδ genes and used these genes to investigate the regulation of the alternative splicing of CaMKIIδ by PKA in cultured cells. We found that PKA promoted the exclusion of exons 14, 15, and 16 of CaMKIIδ, resulting in an increase in δC isoform. PKA interacted with and phosphorylated SF2/ASF, and enhanced SF2/ASF's activity to promote the exclusion of exons 14, 15, and 16 of CaMKIIδ, leading to a further increase in the expression of δC isoform. These findings suggest that abnormality in β-adrenergic-PKA signaling may contribute to cardiomyopathy and heart failure through dysregulation in the alternative splicing of CaMKIIδ exons 14, 15, and 16 and up-regulation of CaMKIIδC.


Introduction
Altered intracellular Ca 2+ handling plays an important role in the pathogenesis of cardiac hypertrophy and heart failure. Ca 2+ / calmodulin-dependent kinase II (CaMKII) is a critical transducer of Ca 2+ signaling in the heart. Cardiac-specific overexpression of CaMKII induces a hypertrophic phenotype that rapidly transitions to dilated cardiomyopathy with ventricular dysfunction, loss of intracellular Ca 2+ homeostasis, and premature death [1,2,3,4]. Inhibition of CaMKII by either pharmacological or genetic approaches reverses heart failure-associated changes (i.e., arrhythmias, hypertrophy, and dysfunction) in animal models of structural heart disease [5,6]. Upregulation of CaMKII expression and activity have been reported to be a general feature of heart failure in humans and in animal models [7,8,9,10,11].
CaMKII has four isoforms named a, b, c andd. CaMKIId is the predominant isoform in the heart [12] and is required for pathological cardiac hypertrophy and remodeling after pressure overload. Cardiomyocyte expresses three splice variants, A, B and C, of CaMKIId as a result of the alternative splicing of exons 14, 15 or 16 of its pre-mRNA. Inclusion of exon 15 and 16 or exon 14 generates CaMKIIdA or CaMKIIdB. CaMKIIdC is produced by exclusion of all these exons (Fig. 1A,B). The dA isoform was previously described as a neuronal CaMKIId isoform [13] and is associated with the T-tubules. This isoform also is expressed in neonatal heart and begins to switch off 30 days after birth [14].
The B isoform targets CaMKIId to nucleus due to exon 14, which contains a nuclear localization signal, and plays a key role in hypertrophic gene expression [15]. The dC isoform is the cytosolic CaMKII and affects excitation-contraction (EC) coupling through phosphorylation of Ca 2+ -regulatory proteins [16]. Overexpression CaMKIIdA in transgenic mice enhances EC coupling and induces heart failure. Moreover, transgenic mice with overexpression of either CaMKIIdB or CaMKIIdC also develop cardiac hypertrophy or heart failure [17]. Therefore, dysregulation in CaMKIId, including its alternative splicing, may be involved in the pathogenesis of cardiac hypertrophy and heart failure.
Splicing factor 2 or alternative splicing factor (SF2/ASF), also termed serine/arginine-rich splicing factor 1 (SRSF1), regulates both alternative splicing and constitutive splicing of many genes. Cardiac-specific-knockout of SF2/ASF causes the retention of CaMKIIdA in the adult mouse and suppression of dB and dC isoforms, suggesting that it plays critical role in the alternative splicing of CaMKIId [18]. SF2/ASF is a phosphoprotein. Its function and localization is highly regulated by phosphorylation. It is well known that many kinases phosphorylate SF2/ASF and regulate its biological function. We recently found that PKA phosphorylates SF2/ASF in vitro and in cultured cells and regulates its function in tau exon 10 inclusion [19].
b-adrenergic receptor plays a central role in sympathetic regulation of cardiac function. Catecholamines acts on b-adrenergic receptor and activates adenylyl cyclase, which catalyzes cAMP formation, via the stimulatory G protein (Gs). Subsequently, cAMP binds onto the regulatory subunits of PKA (cyclic AMP-dependent protein kinase), resulting in their dissociation from the catalytic subunits and in activation of PKA [20]. Activated PKA phosphorylates regulatory proteins involved in cardiac EC coupling and energy metabolism. It is well known that abnormalities of badrenergic-PKA pathway have been implicated as important determinants of cardiac hypertrophy and heart failure. Chronic heart failure is associated with an increase in circulating catecholamines [21]. Overexpression of b1-AR or Gás in transgenic mice develops cardiomyopathy and heart failure [22,23]. The transgenic mice that express the catalytic subunit of PKA in the heart develop dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlate with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca 2+ -release channel, which enhances Ca 2+ release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca 2+ -ATPase [24]. Therefore, dysregulated PKA plays a specific role for in the pathogenesis of heart failure.
However, the relationship between PKA signaling and CaM-KIId alternative splicing, both of which are related with hypertrophy and heart failure, is unclear. In the present study, by using mini-CaMKIId-gene, consisting of exons 12,13,14,15,16, and 17 and introns 12, 13, 14, 15, 16 and 17, we determined the role of PKA on the alternative splicing of CaMKIId. We found that SF2/ASF promoted the exclusion of exons 14, 15 and 16 and led to an increase of CaMKIIdC expression and a decrease of the expression of CaMKIIdA and dB. Phosphorylation of SF2/ASF by PKA further enhanced SF2/ASF function in the regulation of the alternative splicing of CaMKIId.

Results
Alternative splicing of exons 14, 15 and 16 generates three splicing variants of the mini-CaMKIId genes In order to investigate the regulation of the alternative splicing of CaMKIId in cultured cells, we first constructed a mouse mini-CaMKIId-gene, pCI/CaMKIId E12-E17 , consisting of exons 12,13,14,15,16 and 17, and introns 12, 13, 14, 15, and 16 ( Fig. 1A), and then transfected the mini-gene into HEK-293FT cells. After 48 hr transfection, RNA was extracted and subjected to RT-PCR for measurement of the alternative splicing of exons 14, 15 and 16 of CaMKIId. We observed three alternative splicing products (Fig. 1C). The size of the upper band matched the product of exon 14 exclusion. The middle band was the product of exclusion of exons 15 and 16, and the lower band was the PCR product of exclusion of exons 14, 15 and 16. To confirm these three bands to be the splicing products of the mini-gene of CaMKIId, we sequenced the PCR products. The data showed that these three RT-PCR products were lack of exon 14, of exons 15 and 16 and of exons 14, 15, and 16, respectively (Fig. 1C). These results suggest that the mini-gene of CamKIId, pCI/ CaMKIIdE12-E17, is able to generate three alternative splicing products of CaMKIId exon 14, 15 and 16, which represent CaMKIIdA, B, and C, respectively.

PKA regulates the alternative splicing of CaMKIId
To investigate whether PKA regulates the alternative splicing of CaMKIId, we transfected the HEK293T cells with pCI/ CaMKIId E13-E17 for 40 hr, and treated the cells with various concentration of forskolin to activate PKA for 8 hr. Forskolin treatment increased PKA activity presented by increased phosphorylation of CREB at Ser133 ( Fig. 2A). We measured the alternative splicing products of CaMKIId exons 14, 15, and 16 with RT-PCR. We found that forskolin treatment promoted the exclusion of exons 14, 15, and 16, and suppressed the inclusion of exon 14 and of exons 15 and 16, resulting in an increase in CaMKIIdC and a decrease in CaMKIId A and B expression dosedependently (Fig. 2B). These results suggest that PKA may regulate the alternative splicing of CaMKIId.
To confirm the regulation of forskolin on CaMKIId splicing via activation of PKA, we co-expressed PKA catalytic a subunit (PKA-Ca), a dominant isoform in heart [25], with the pCI/ CaMKIId E13-E17 , and then measured the splicing products of exons 14, 15, and 16 of CaMKIId. Consistent with forskolin treatment, overexpression of PKA-Ca increased PKA activity to phosphorylate CREB (Fig. 2C) and promoted the exclusion of exons 14, 15, and 16, resulting in an increase in the expression of CaMKIIdC, and a decrease in the expressions of CaMKIIdA and CaMKIIdB significantly (Fig. 2D). Previous studies have identified that knockout of SF2/ASF caused deregulation of the alternative splicing of CaMKIId [18].
To determine the regulation of SF2/ASF on the alternative splicing of exons 14, 15, and 16 of the CaMKIId mini-gene, we cotransfected pCEP4/SF2/ASF with pCI/CaMKIId E12-E17 into COS7 cells, and then measured the products of its alternative splicing. We found that overexpression SF2/ASF significantly promoted the exclusion of exons 14, 15 and 16, resulting in an increase of CaMKIIdC and a decrease of CaMKIIdA and B (Fig. 3A, B). Similarly, overexpression of SF2/ASF also promotes CaMKIIdC expression in pCI/CaMKIId E13-E17 transfected HEK293T cells (Fig. 3B, C). These results further confirmed that SF2/ASF could regulate the alternative splicing of exons 14, 15, and 16 and ASF promotes the exclusion of exons 14, 15, and 16 of CaMKIId.

PKA phosphorylates and interacts with SF2/ASF
To elucidate whether PKA regulates SF2/ASF's activity to promote the exclusion of CaMKIId exons 14, 15, and 16, we first determined whether PKA phosphorylates SF2/ASF in vitro. We incubated GST-SF2/ASF or GST with PKA catalytic subunit and [c-32 P]-ATP in reaction buffer for 30 min at 30uC, and then the reaction products were subjected to SDS-PAGE and autoradiograph for detection of 32 P incorporation. We observed that GST-SF2/ASF, but not GST, had 32 P incorporation, suggesting that SF2/ASF is phosphorylated by PKA (Fig. 4A).
To investigate the interaction between PKA and SF2/ASF, we performed GST pull-down assay and co-immunoprecipitation (co-IP). For GST pull down assay, we incubated GST-SF2/ASF or GST alone with rat brain extract. After extensively washing, the proteins pulled down by GST-SF2/ASF or GST were subjected to Western blots with anti-PKA-Ca. We found that PKA-Ca was pulled-down from rat brain extracts by GST-SF2/ASF, but not by GST (Fig. 4B), suggesting that SF2/ASF may interact with PKA-Ca.
Similar results were obtained from co-IP studies. We overexpressed HA tagged SF2/ASF (SF2/ASF-HA) in HEK-293FT cells and immunoprecipitated SF2/ASF with anti-HA. The immuno complex using anti-HA was subjected to Western blot analysis. We found that anti-HA immunoprecipitated SF2/ASF-HA, and that PKA-Ca subunit was co-immunoprecipitated by anti-HA. These results further suggest that SF2/ASF interacts with PKA-Ca (Fig. 4C).
To elucidate the interaction of SF2/ASF with PKA in intact cells, we transfected pCEP4/SF2/ASF into HeLa cells, and then treated the cells with 10 mM Forskolin. Their subcellular localization was determined by confocal microscopy. We observed that, without forskolin treatment, PKA-Ca was mainly located in the cytoplasm and that SF2/ASF was localized extensively in the nucleus (Data not shown). Forskolin treatment appeared to promote PKA catalytic subunit translocation into the nucleus to co-localize with SF2/ASF (Fig. 4D).

PKA enhances SF2/ASF role in the regulation of CaMKIId splicing
To investigated whether PKA affects SF2/ASF-mediated alternative splicing of CaMKIId exons 14, 15, and 16, we co-transfected pCEP4/SF2/ASF with pCI/CaMKIId E13-E17 into HEK293T cells. After 40 hrs transfection, we treated the cells with 10 mM forskolin or 20 mM isoproterenol for 8 hr for activating PKA and then measured the splicing products of CaMKIId exons 14, 15, and 16. We found that both forskolin and isoproterenol treatments enhanced the role of SF2/ASF to promote the exclusion of CaMKIId exons 14, 15, and 16, resulting in a further increase in CaMKIIdC expression (Fig. 5).

Discussion
The alternative splicing of exons 14, 15, and 16 of CaMKIId pre-mRNA generates three different splice variants, A, B and C. SF2/ASF plays an important role in the alternative splicing of CaMKIId [18]. In the present study, we constructed, for first time, two mini-genes of CaMKIId, in which exons 14, 15, and 16 were able to be alternatively spliced to generate the spliced variants  ). B, PKA-Ca was pull-down by SF2/ASF. GST-SF2/ASF or GST coupled onto glutathione-Sepharose or glutathione-Sepharose (GSH-beads) was incubated with rat brain extract. After extensively washing, the bound proteins were analyzed by Western blots developed with anti-GST or anti-PKA-Ca. C, PKA-Ca was co-immunoprecipitated by anti-HA. SF2/ASF tagged with HA were expressed in HEK-293FT cells for 48 h. The cell extracts were immunoprecipitated with anti-HA, and the immunoprecipitates were subjected to Western blots developed with anti-HA and anti-PKA-Ca. D, HeLa cells were transfected with pCEP4/SF2/ASF and treated without (Con) or with forskolin (Fors) for 30 min, followed by triple immunofluorescence staining. doi:10.1371/journal.pone.0025745.g004 corresponding to CaMKIIdA, B and C, respectively. By using these mini-genes, we confirmed the role of SF2/ASF in the promotion of exclusion of exons 14, 15, and 16 of CaMKIId. PKA interacted with and phosphorylated SF2/ASF and further enhanced the SF2/ASF function in promotion of exclusion of CaMKIId exons 14, 15, and 16. Therefore, abnormalities of badrenergic-PKA pathway during pathogenesis of hypertrophy and heart failure may course the dysregulation of the alternative splicing of CaMKIId exons 14, 15, and 16, resulting in an imbalance in the generation of CaMKIIdA, B and C isoforms, which may contribute to the hypertrophy and heart failure.
The expression of CaMKIId isoforms is developmentally regulated in the heart. CaMKIIdB and C are presented in the adult mammalian myocardium, but CaMKIIdA is expressed in neonatal heart and begins to switch off 30 days after birth [18]. These three isoforms of CaMKIId have different subcellular localization, which determines their specific roles in regulating cardiac function. The dA isoform was previously described as a neuronal CaMKIId isoform [13] and associates with the T-tubules where key Ca 2+ channels and activated CaMKIId are concentrated [14]. As a nucleus CaMKIId, the B isoform plays a key role in hypertrophic gene expression [15], whiles the cytoplasmic dC isoform affects EC coupling through phosphorylation of Ca 2+ regulatory proteins [16]. Overexpression of the dB isoform in the nucleus or the dC isoform in the cytoplasm in transgenic mice both give rise to marked decrease in diastolic Ca 2+ , transient amplitude, and caffeine-releasable Ca 2+ content in the sarcoplasmic reticulum, all of which seem to reflect a general heart failure phenotype [4,17].  [18]. The dysregulation of CaMKIId isoforms in the SF2/ASF deficient mice appears to enhance EC coupling and hypercontraction in isolated cardiomyocytes, the phenotype resulting from the targeting of the kinase to the T-tubules where several key Ca 2+ handling proteins are located. In this study, we observed that overexpression of SF2/ASF promoted exclusion of exons 14, 15, and 16, leading to increased expression of CaMKIIdC, which is consistent with above in vivo study.
The function and distribution of SF2/ASF is highly regulated by phosphorylation. Site/regional phosphorylation impacts SF2/ ASF function and subcellular localization differentially. Phosphorylation of SF2/ASF by Clk and SRSK enhances its function in the regulation of alternative splicing [26]. Phosphorylation of SF2/ ASF at Ser227, Ser234 and Ser238 by Dyrk1A suppresses its function on the alternative splicing of tau exon 10 [27]. PKA phosphorylates SF2/ASF and promotes its function in the alternative splicing of CaMKIId. PKA is a Ser/Thr kinase and is activated by the binding of cAMP onto its regulatory subunits. We recently have demonstrated that PKA effectively phosphorylates SF2/ASF and enhances its function in promotion of tau exon 10 inclusion [19]. Here, we found that up-regulation of PKA enhances SF2/ASF-promoted exclusion of CaMKIId exons 14, 15, and 16, resulting in further decrease in dA and dB and increase in dC isoform of CaMKII.
Cytosolic CaMKIIdC regulates cardiac excitation-contraction coupling and maladaptive cardiac remodeling, while the nucleus form of CaMKIId, dB, is involved in the regulation of gene expression. Interestingly, these two major cardiac CaMKII isoforms are inversely regulated in response to oxidative stress and ischemia/reperfusion injury [28]. Overexpression of CaMKII-äC promotes apoptosis, whereas overexpression of CaMKIIdB protects cardiomyocytes against oxidative stress-, hypoxia-, and angiotensin II -induced apoptosis [28]. Overexpression of dC in the transgenic mice develops a dilated cardiomyopathy. Isolated myocytes from the mice are enlarged and exhibit reduced contractility and altered Ca 2+ handing [4]. The expression of dC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload [4], suggesting the involvement of CaMKIIdC activation in the pathogenesis of dilated cardiomyopathy and heart failure. Down-regulation of CaMKIIdC expression may prevent hypertrophy and heart failure caused by dysregulated b-adrenergic-PKA pathway. Therefore, the roles of PKA in the regulation of alternative splicing of CamKIId and in the pathogenesis of cardiomyopathy and heart failure in vivo remain to be elucidated. Construction of mini-CaMKIId-genes, pCI/CaMKIId E12-E17 and pCI/CaMKIId E13-E17 C57BL6 mouse was from the laboratory animal center of Nantong University. The protocol was approved according to the Animal Care and Use Committee of Nantong University and the Jiangsu Province Animal Care Ethics Committee (Approval ID: SYXK(SU)2007-0021). Mouse genomic DNA was extracted from C57BL6 mouse tail with Dneasy Blood and Tissue kit (Qiagen, Germany) and used as a template to amplify genomic DNA of CaMKIId from exon 12 to exon 17 including introns (E12-E17, 12,475 bp) with two pairs of primers (Fig. 1a). Set 1 of primers (forward, 59cgACGCGTatttctttgatagggcgccatcttgacaac39; reverse, 59acgcGTCGACaaagagccttcttctgtgg39) was used to generate 6825 bp fragment. Set 2 of primers (forward, 59acgcGTCGACa-catgggagcttggaatg39; reverse, 59ataagaatGCGGCCGCgcctcaagcc-tacctttcacgtcttcatcc39). The PCR products were digested by Mlu I and Sal I, and Sal I and Not I respectively, and inserted into pCI/ neo vector by restriction sites of Mlu I and Not I, which named pCI/CaMKIId E12-E17 . The sequences of the mini-gene was confirmed by sequencing analysis.

Plasmids, antibodies, and other reagents
By using pCI/CaMKIId E12-E17 as the template, we amplified the genomic DNA of CaMKIId from exon 13 to exon 17 with primers (forward, 59cgACGCGTtcattttgca gcagccaagagtttatt-gaag39; reverse, 59ataagaatGCGGCCGCgcctcaagcctacctttcacgtc ttcatcc39) to amplify the genomic DNA of CaMKIId from exon 13 to exon 17 and inserted into pCI/neo by Mlu I and Not I, which was named pCI/CaMKIId E13-E17 . In vitro phosphorylation of SF2/ASF by PKA GST and GST-SF2/ASF (0.2 mg/ml) was incubated with PKA catalytic subunit in a reaction buffer consisting of 50 mM 40 mM HEPES (pH 6.8), 10 mM b-mercaptoethanol, 10 mM MgCl2, 1.0 mM EGTA and 0.2 mM [c-32 P]ATP (500 cpm/pmol). After incubation at 30uC for 30 min, the reaction was stopped by boiling with an equal volume of 26Laemmli sample buffer. The reaction products were separated by SDS-PAGE. Incorporation of 32 P was detected by exposure of the dried gel to phosphor-image system.

Cell culture and transfection
GST pull-down of PKA by SF2/ASF GST and GST-SF2/ASF were purified by affinity purification with glutathione-Sepharose, but without elution from the beads. These beads coupled with GST and GST-SF2/ASF were incubated with crude extract from rat brain homogenate in buffer (50 mM Tris-HCl, pH 7.4, 8.5% sucrose, 50 mM NaF, 1 mM Na 3 VO 4 , 0.1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin and 10 mg/ml pepstatin). After 4 h incubation at 4uC, the beads were washed with washing buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl and 1 mM DTT) six times, the bound proteins were eluted by boiling in Laemmli sample buffer and the samples were subjected to Western blot analysis.

Co-immunoprecipitation of PKA by SF2/ASF
HEK-293FT cells were transfected with pCEP4-SF2/ASF-HA for 40 h as described above and treated with 10 uM forskolin for 8 h, and then the cells were washed twice with PBS, and lysed by sonication in lysate buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM Na 3 VO 4 , 2 mM EDTA, 1 mM PMSF, 2 mg/ml aprotinin, 2 mg/ml leupeptin and 2 mg/ml pepstatin). The cell lysate was centrifuged at 16,0006g for 10 min and incubated with anti-HA overnight at 4uC, and then protein G beads were added. After 4 h incubation at 4uC, the beads were washed with lysate buffer twice and with TBS twice, and bound proteins were eluted by boiling in Laemmli sample buffer. The samples were subjected to Western blot analysis with the indicated primary antibodies.
Co-localization of PKA with SF2/ASF HeLa cells were plated in 24-well plates onto coverslips one day prior to transfection at 50-60% confluence. The cells were then transfected with pCEP4/SF2/ASF-HA as described above. After 40 h transfection, the cells were treated with 10 mM forskolin for 30 min to activate PKA, and then the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBS, the cells were blocked with 10% goat serum in 0.2% Triton X-100-PBS for 2 h at 37uC, and incubated with rabbit anti-HA antibody (1:200) and mouse anti-PKACa (1:50) overnight at 4uC. The cells were then washed and incubated for 1 h with secondary antibodies (Alexa 488-conjugated goat anti-mouse IgG and Alexa 555-conjugated goat anti-rabbit IgG, 1:1000) plus TO-PRO-3 iodide at room temperature. The cells were washed with PBS, mounted with Fluoromount-G and observed with a Nikon TCS-SP2 laser-scanning confocal microscope.
Quantitation of splicing products of CaMKIId exons 14, 15, and 16 by reverse transcription-PCR (RT-PCR) Total cellular RNA was isolated from cultured cells by using the RNeasy Mini Kit (Qiagen, GmbH, Germany). One microgram of total RNA was used for first-strand cDNA synthesis with Oligo-(dT) 15 -18 by using the Omniscript Reverse Transcription Kit (Qiagen). PCR was performed by using PrimeSTAR TM HS DNA Polymerase (Takara Bio Inc., Otsu, Shiga, Japan) with primers (forward, 59 GGTGTCCACTCCCAGTTCAA 39; reverse, 59 GTCTTCATCCTCAATGGTGG TG 39) to measure alternative splicing of CaMKIId exons 14, 15, and 16. The PCR conditions were: 98uC for 3 min, 98uC for 10 sec and 68uC for 40 sec for 25-30 cycles and then 68uC 10 min for extension. The PCR products were resolved on 1.5% agarose gels. Each splicing product was quantitated by densitometry using the Molecular Imager system (Bio-Rad, Hercules, CA, USA), and the percentage of each splicing form was calculated.

Statistical analysis
All data are expressed as the means6SD. Data points were compared by unpaired two-tailed Student's t test. A value of p,0.05 was considered statistically significant.