H11 Kinase Is a Novel Mediator of Myocardial Hypertrophy In Vivo
By subtractive hybridization, we found a significant increase in H11 kinase transcript in large mammalian models of both ischemia/reperfusion (stunning) and chronic pressure overload with hypertrophy. Because this gene has not been characterized in the heart, the goal of the present study was to determine the function of H11 kinase in cardiac tissue, both in vitro and in vivo. In isolated neonatal rat cardiac myocytes, adenoviral-mediated overexpression of H11 kinase resulted in a 37% increase in protein/DNA ratio, reflecting hypertrophy. A cardiac-specific transgene driven by the αMHC-promoter was generated, which resulted in an average 7-fold increase in H11 kinase protein expression. Transgenic hearts were characterized by a 30% increase of the heart weight/body weight ratio, by the reexpression of a fetal gene program, and by concentric hypertrophy with preserved contractile function at echocardiography. This phenotype was accompanied by a dose-dependent activation of Akt/PKB and p70S6 kinase, whereas the MAP kinase pathway was unaffected. Thus, H11 kinase represents a novel mediator of cardiac cell growth and hypertrophy.
H11 kinase is the eukaryotic homologue of the viral protein kinase ICP10, an enzyme involved in the “immortalization” of cells infected by herpes simplex virus 2.1,2⇓ The structure of H11 kinase combines a N-terminal membrane domain,1 the structural motif of the crystallin family of heat-shock proteins,3 and a serine/threonine kinase activity.1 The function of H11 kinase remains unknown so far, but it could probably participate in cellular mechanisms of growth and survival. This hypothesis is based not only on the function of the viral homologue ICP10, but also on the observation that the ectopic expression of H11 kinase in mammalian cells is associated with tumor proliferation, such as melanoma1 and breast cancer.4 Transfection of H11 kinase in normal keratinocytes is sufficient to promote their transformation into melanoma cells, whereas antisense oligonucleotides blocking H11 kinase expression prevent the proliferation of melanoma cells.
Intriguingly, in adult mammals, H11 kinase gene expression is most abundant in heart and skeletal muscle,1,3⇓ yet there is no role or regulatory mechanisms defined so far for this kinase in the heart. Therefore, the goal of the present study was to determine the regulation and function of H11 kinase. Previously, we showed that H11 kinase gene expression is significantly increased in the reversibly ischemic (stunned) myocardium, together with an array of genes participating in cell growth. Based on this observation, we hypothesized that the function of H11 kinase in the heart is related to growth mechanisms. In the present study, we demonstrate such a function by three complementary approaches. We first demonstrate by subtractive hybridization in a large mammalian model that H11 kinase gene and protein expression is increased in response to long-term pressure overload with hypertrophy. Next, we show that overexpression of H11 kinase in vitro in isolated cardiac myocytes promotes cardiac cell hypertrophy. Finally, the function and mechanism of action of H11 kinase were further documented in vivo in a cardiac-specific transgenic model. In particular, our data illustrate that H11 kinase overexpression induces hypertrophy and results in the activation of the Akt pathway, a major signaling pathway of cell growth and survival. Altogether our data illustrate a new mechanism by which the cardiac response to stress (both acute ischemia/reperfusion and chronic pressure overload) is coupled to a stimulation of cell growth.
Materials and Methods
Animal Model of Cardiac Hypertrophy
Chronic, pressure-overload left ventricular hypertrophy (LVH) was induced by aortic banding as described5,6⇓ in mongrel puppies of either sex at 8 to 10 weeks of age. A 1-cm wide polytetrafluoroethylene (Teflon) cuff was placed around the aorta and tightened until a thrill was palpated over the aortic arch and then the chest was closed. The Teflon band creates a fixed supravalvular aortic lesion, which becomes relatively more stenotic as the puppies grow. Five additional pups, in which no banding was performed, were followed as controls. Myocardial samples were taken from LV subendocardium of the beating hearts and were immediately frozen in liquid nitrogen.
cDNA Subtractive Hybridization
Total RNA was first extracted7 from the myocardial samples in both control and LVH conditions. Messenger RNA was isolated, and 2 μg were used for first-strand cDNA synthesis with random primers. After second strand synthesis, the cDNA libraries were digested with RsaI. The subtractive hybridization was performed with the PCR-select cDNA subtraction kit (Clontech), as described before.8,9⇓ Digestion products of the “tester” library were ligated to a specific adapter (T7 promoter), then hybridized with a 30-fold excess of the “driver” library for subtraction. After hybridization, the remaining products were further amplified by PCR. Subtracted products were subcloned into the pGEM-Teasy vector (Promega) and transformed into SURE2 cells (Stratagene). The clones were sequenced by standard procedure (ABI-Prizm 3100 DNA sequencer). Sequences were queried in public database to determine the identity of the genes.
Fifteen micrograms of total RNA was applied on a 1.2%-agarose denaturing gel and transferred overnight. The canine full-length coding sequence of H11 kinase was cloned (Genbank accession number: AF525493) and subsequently used as a probe. Probe was heat-denatured, then digoxin-labeled (Random labeling and Spotlight detection kits, Clontech). Hybridization was performed overnight at 45°C. Washing and detection were performed following the manufacturer’s instructions.
Quantitative RT-PCR (7700 Prizm, Perkin-Elmer/ABI) was performed as described8,10,11⇓⇓ to measure the gene expression of H11 kinase, ANF, BNP, Hsp70, osteoblast-specific factor-2 (OSF-2), the cardiac ankyrin repeat protein (CARP), 36B4, and cyclophilin with specific primers and fluorogenic probes (derived with FAM and TAMRA) designed from the murine sequences available in public databases. For each measurement, the mRNA of interest was reverse-transcribed, and subsequently used for quantitative 2-step PCR. Internal standards were prepared for each transcript from its PCR-amplified cDNA after ligation of the T7 promoter (Ambion).8,11⇓ Due to variation in sample-to-sample loading, PCR data are normalized per number of cyclophilin or 36B4 transcripts, measured as housekeeping genes in each sample.
Tissue was homogenized with a Branson tissue homogenizer in a lysis buffer (in mmol/L: 25 Tris-HCl-pH 8.0, 150 NaCl, 15 mmol/L KCl, 1 EDTA, 1 DTT, 0.5% Triton X-100, and 5% glycerol), containing 1 μg/mL pepstatin-A, 100 μg/mL chymostatin, 2 μg/mL aprotinin, 50 μg/mL antipain, 100 μg/mL PMSF, 1 mmol/L Pefabloc-SC, and 1 μg/mL leupeptin). The homogenates were centrifuged at 12 000g for 20 minutes at 4°C, and 20 to 40 μg protein extract was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes and probed with the different antibodies reported in the Results section. An anti-H11 kinase antibody was generated in the rabbit (Biosource) by injection of a peptide from the rat H11 kinase sequence using the same epitope as described previously.1
Culture of Neonatal Cardiomyocytes
Primary cultures of ventricular cardiac myocytes were prepared from 1-day-old Wistar rats (Charles River Laboratories, Wilmington, Mass), as described previously,12 and plated on gelatin-coated culture dishes at a density of 106 cells/cm2. Cells were cultured in the cardiac myocyte culture medium containing Dulbecco’s modified eagle medium (DMEM)/F12 supplemented with 5% horse serum, 4 μg/mL transferrin, 0.7 ng/mL sodium selenite (GIBCO), 2 g/L bovine serum albumin (fraction V), 3 mmol/L pyruvic acid, 15 mmol/L HEPES, 100 μmol/L ascorbic acid, 100 μg/mL ampicillin, 5 μg/mL linoleic acid, and 100 μmol/L 5-bromo-2′-deoxyuridine (Sigma). After 24 hours, myocytes were cultured in serum-free medium for 48 hours before the experiments. A recombinant adenovirus was constructed by the COS-TPC (terminal protein complex) method using the Adeno-X-system (Clontech).13 The coding sequence of H11 kinase was first amplified by PCR from a mouse heart cDNA library (Clontech) and ligated into pcDNA3.1 (Invitrogen). The insert was subsequently digested and ligated in the Adx vector (Clontech), with or without a Myc-tag. The recombinant adenovirus was prepared in 293 cells by cotransfection of a cosmid containing the adenovirus type 5 genome (devoid of E1 and E3) with the shuttle vectors, using lipofectamine (GIBCO). Titers were determined on 293 cells overlaid with DMEM plus 5% equine serum and 0.5% agarose. An adenovirus harboring LacZ (AdLacZ) was used as a negative control.
Generation of a Transgenic Mouse
A construct was made to generate transgenic mice with the coding sequence of human H11 kinase followed by a C-terminal hemagglutinin tag and the human growth hormone poly-A tail, subcloned downstream of the αMHC promoter (a generous gift of J. Robbins, Children’s Hospital Research Foundation, Cincinnati, Ohio).14 The construct was introduced by pronuclear injection in zygotes of FVB mice. The murine tyrosinase gene was coinjected for easy visual identification of transgenic offspring. Furthermore, transgenic mice were genotyped from tail DNA extracts by PCR using a forward primer specific of the αMHC promoter and a reverse primer corresponding to the HA-tagged C-terminus of H11 kinase.
Heart tissue samples from transgenic mice and wild-type littermates were fixed by immersion in 10% formalin. The fixed tissues were dehydrated, embedded in paraffin, and sectioned at 6-μm thickness. Routine staining included hematoxylin and eosin and Masson’s trichrome. Immunocytochemistry was performed as described before15 with an anti-HA antibody (Clontech). Three hearts from transgenic mice and three hearts from wild-type animals were fixed in methacrylate for measurement of the cross-sectional area of cardiac myocytes.
Results are presented as the mean±SD for the number of samples indicated in each Figure legend. Statistical comparison was performed using the Student’s t test. ANOVA with Bonferroni correction was used when necessary. A value of P<0.05 was considered as significant.
Regulation of H11 Kinase in Hypertrophied Myocardium
To test the genomic regulation involved during chronic, stable LVH, we analyzed a canine model of severe, chronic (2 years), but compensated LVH induced by aortic banding.5,6,16⇓⇓ Table 1 demonstrates that the LVH was severe but stable, ie, cardiac decompensation was not observed. We analyzed by subtractive hybridization the genomic profile of the hypertrophied canine heart compared with control myocardium, and found H11 kinase among other genes as a candidate product for regulation. This regulation was confirmed at both the transcript and protein levels. Northern blot analysis (Figure 1A) showed a marked upregulation of H11 kinase transcript in hypertrophied myocardium, which was quantified as a 4-fold upregulation compared with controls (Figure 1B). This upregulation was correlated with a 3-fold increase in the content of the corresponding protein (Figure 1C).
Overexpression of H11 Kinase Stimulates Hypertrophy In Vitro
Because the physiological function of H11 kinase is unknown in the heart, we overexpressed H11 kinase in rat neonatal cardiac myocytes in culture. Cardiac myocytes were transduced with an adenovirus containing the Myc-tagged H11 kinase vector (AdH11K-Myc) or with a virus containing a control vector harboring the β-galactosidase sequence (AdLacZ). Infection with 3 MOI of AdH11K increased the protein/DNA content of cardiac myocytes by 37% at 48 hours compared with infection with the same dose of AdLacZ (Figure 2A), showing that H11 kinase stimulates cardiac hypertrophy in vitro. The hypertrophy of H11 kinase-transfected cells was confirmed by visual inspection (Figure 2B). No further increase was observed with higher viral load. As expected, the adenovirus-mediated overexpression of H11 kinase in neonatal rat cardiac myocytes increased the mRNA expression of H11 kinase (Figure 2C), as measured by qPCR. This phenotype was accompanied by an increased expression of fetal genes (Figure 2C), such as atrial natriuretic factor (ANF) and brain natriuretic factor (BNP), whose expression is frequently upregulated during cardiac hypertrophy. A small dose of AdH11K virus (5 MOI) was sufficient to fully trigger the reexpression of fetal genes, because infection with a dose 10-fold higher (50 MOI) did not produce additional effects (Figure 2C). Comparison of an adenovirus containing the Myc-tagged H11 kinase with an adenovirus containing the nontagged H11 kinase sequence showed that the tagging did not affect the results.
Expression of H11 Kinase in Mouse Heart In Vivo
As a prelude to the preparation of a transgenic mouse and because of the little information available, we first characterized the expression of H11 kinase in different adult mouse tissues and during development. A multitissue blot in the mouse shows that H11 kinase is robustly expressed in the heart (Figure 3A). A weaker signal was also detected in skeletal muscle and liver. As measured by qPCR, the expression of H11 kinase in the mouse cardiac chambers was 2- to 3-fold higher in both ventricles over atria (Figure 3B). Measurement of the transcript at different stages of development showed that the expression of H11 kinase markedly increases two weeks after birth, then decreases significantly with aging (Figure 3C).
Overexpression of H11 Kinase Stimulates Hypertrophy In Vivo
To ascertain whether H11 kinase is sufficient to produce features of cardiac hypertrophy in vivo, we engineered transgenic mice that express the H11 kinase coding sequence in frame with a hemagglutinin tag under the control of the cardiac-specific αMHC promoter (Figure 4A). We produced 14 transgene-positive F0 founder mice. In three cases, death of the founder precluded the establishment of transgenic lines. In all remaining cases, lines were established and no early mortality was observed. We confirmed the overexpression of the transgene in the heart at the mRNA level by Northern blot (Figure 4B) and at the protein level by Western blot using antibodies against the HA tag and H11 kinase (Figure 4C). Using the anti-HA antibody, different levels of expression (low, medium, and high) were detected (Figure 4C). The total amount of H11 kinase protein in low-, medium- and high-expressing lines was 2-, 4-, and 7-fold superior to control littermates, respectively. Homogenous expression of the transgene was confirmed by immunostaining in the high-expression line (Figure 4D). The increased expression also corresponded to a higher activity of H11 kinase, as shown by phosphorylation of the myelin binding protein (Figure 4E).
The hearts from transgene-positive mice of all lines were analyzed at two months of age. In transgenic mice, there was an increase in the heart weight/body weight ratio that was proportional to the expression level of the transgene, and averaged 30% in the high-expression line over wild-type littermate (Figure 5A). Transgenic hearts were macroscopically bigger than the corresponding wild-type littermates, which was due to a hypertrophy of the four cardiac chambers (Figure 5B). Cross-sectional area of the cardiac myocytes was measured in three transgenic mice with intermediate overexpression and three wild-type mice. An average 27% increase in heart weight/body weight ratio in these three transgenic mice correlated with an average 22% increase in cross-sectional area (275±27 versus 224±12 μm2; P<0.05). Histological analysis of hearts from 2 month-old transgenic mice showed enlarged cardiac myocytes without detectable accumulation of extracellular matrix or fibrosis (Figure 5C). Two-dimensional echocardiographic measurements showed that the transgenic hearts are characterized by a concentric hypertrophy with maintained contractile function and ejection fraction (Table 2). Therefore, a cardiac-specific overexpression of H11 kinase reproduces a dose-dependent pattern of cardiac hypertrophy with preserved contractile function, which confirms the in vitro data showing that this molecule is involved in cardiac cell growth.
At the genomic level, the induction of a hypertrophied phenotype in transgenic mice was accompanied by the reexpression of a fetal gene program, characterized by the upregulation of ANF and BNP (Figure 5D). However, the expression of other markers of cardiac hypertrophy that are upregulated by pressure overload (such as OSF-2, CARP, and HSP70)17–20⇓⇓⇓ was not different between controls and transgenic mice (not shown).
Overexpression of H11 Kinase Activates the Akt/p70S6 Kinase Pathway In Vivo
The data presented illustrate that H11 kinase is a novel mediator of cardiac cell growth and hypertrophy both in vitro and in vivo. To start determining the signaling pathways involved in this function, we examined in transgenic mice whether the activity of two major pathways of cardiac cell growth, the MAP kinase pathway21,22⇓ and the Akt/p70S6K pathway,23,24⇓ is affected by overexpression of H11 kinase. As shown in Figure 6A, overexpression of H11 kinase activated Akt. This effect was proportional to the expression of the transgene, as the activation of Akt was 4-fold higher in high-expression lines over low-expression lines. The kinase p70S6K, a downstream target of Akt which mediates its stimulatory effects on protein synthesis,24,25⇓ was similarly upregulated (Figure 6B). Not only the specific activity of p70S6K was increased in relation with the overexpression level of H11 kinase, but also the p70S6K protein content was 75% higher in high-expression transgenic mice over wild-type littermates (Figure 6B). By contrast, the activity of different MAP kinases, such as ERK-1 and ERK-2 (Figure 6C), and p38 MAP kinase (not shown) was unchanged in the different H11 kinase-overexpressing mice tested. Therefore, activation of the Akt/p70S6K represents a likely mechanism mediating the growth effect of H11 kinase.
In a large mammalian model of severe, stable cardiac hypertrophy in vivo, we found an upregulation at both the gene and protein level of H11 kinase, a molecule not previously characterized in the heart. Our data demonstrate that overexpression of H11 kinase, both in vitro and in vivo, is accompanied by a phenotype of hypertrophy and the reexpression of a cardiac fetal gene program, together with an activation of the Akt/p70S6 kinase pathway. These results demonstrate that H11 kinase is a mediator of cardiac cell growth and hypertrophy.
Upregulation of H11 Kinase Expression in Cardiac Hypertrophy
By functional genomics, we found an increased expression of the gene encoding H11 kinase in a chronic canine model of cardiac hypertrophy. This model has been well characterized5,6,16,26⇓⇓⇓ and reproduces the progressive development of severe but stable hypertrophy, as observed in patients with pressure overload. In particular, it offers the possibility of chronic experimentation, which is more difficult in rodent models. As shown in Table 1, this model induces a 50% increase in LV/BW over two years. Although the contractile function is preserved (Table 1), the model decompensates into heart failure on pacing.27 A marked increase in H11 kinase gene expression was found in this model during hypertrophy (Figure 1). We show that this transcriptional increase is accompanied by an increased content of the corresponding protein, and the experiments conducted in the transgenic model show that the increased expression of the protein results in an increased kinase activity. The mechanism by which the expression of this gene is activated in overloaded canine myocardium, whether neuroendocrine or paracrine/autocrine, remains to be determined. Such mechanism of regulation is intriguing, because kinases participating in signaling cascades are usually regulated by posttranslational mechanisms, such as phosphorylation/dephosphorylation. However, the expression of H11 kinase in heart of large mammals, such as dog and pig, is extremely low in normal conditions and becomes markedly increased during stress, such as overload (Figure 1) or ischemia/reperfusion.8 Therefore, a transcriptional mechanism of regulation may be necessary to rapidly induce the production and activity of this enzyme in stress conditions.
Overexpression of H11 Kinase Stimulates Cardiac Cell Growth
Further experiments were conducted both in vitro and in vivo to confirm our hypothesis that H11 kinase participates in mechanisms of cell growth. The 37% increase in protein/DNA ratio observed in isolated cardiac myocytes infected with an adenovirus containing the H11 kinase vector is relatively similar to the increase observed after stimulation with phenylephrine28 or angiotensin II.25 This effect is already maximal at relatively low viral load (5 MOI), which shows the sensitivity of the system to H11 kinase overexpression. This stimulation of cell growth was confirmed and investigated further in the transgenic model. Importantly, in the transgenic mouse, the overexpression of H11 kinase was accompanied by an increased enzymatic activity of the kinase (Figure 4E). The analysis of these mice shows a correlation between the expression level of H11 kinase and the cardiac phenotype (Figure 5). Cross-sectional measurements showed that the increase in heart mass corresponds to an increase in cell size of the cardiac myocytes. Both the morphological analysis (no extracellular matrix accumulation or myofibril disarrays) and the echocardiographic data (preserved contractile function) indicate that the hypertrophy induced by H11 kinase overexpression is not accompanied by ventricular dysfunction at this age. Further experiments will determine the impact of H11 kinase overexpression in transgenic mice on the cardiac response to stress after ischemia/reperfusion or aortic banding.
The overexpression of H11 kinase in transgenic mice also correlates with the activation of the Akt/p70S6K growth pathway (Figure 6). This activation shows a remarkable correlation with the level of H11 kinase overexpression (Figure 6). As reviewed recently,29 Akt mediates most of the effects of phosphatidylinositol-3-OH kinase (PI3K) on cell growth and survival, protein synthesis, and metabolism. The Akt/p70S6K pathway is primarily involved in the translational adaptation of the heart,30 which eventually leads to increased cell size in response to cardiac overload.22,31⇓ It remains to be determined whether H11 kinase activates the Akt/p70S6K pathway directly or indirectly, via growth factors. In particular, it will be important to test whether the activation of Akt is dependent or independent from PI3K. Further experiments will be conducted both in vitro and in vivo to address that question. In addition, the signaling of H11 kinase does not seem to involve the MAP kinase pathway, as evidenced by the absence of changes in activity of ERK-1, ERK-2 (Figure 6), and p38 MAP kinase.
Upregulation of H11 Kinase Expression in Myocardial Ischemia
We previously showed that H11 kinase gene expression is increased in a swine model of ischemia/reperfusion (stunning).8 The observation that H11 kinase expression is induced in large mammalian models of the two major forms of human heart disease (cardiac hypertrophy and myocardial ischemia) further highlights the relevance of this previously uncharacterized kinase. In addition, the data collected in the stunning model and in the present study show that H11 kinase is induced by both acute and chronic pathophysiological conditions, which illustrates its sensitivity to a wide spectrum of stress stimuli. This increased expression during stress is reminiscent of the family of heat-shock proteins.20 The secondary structure of H11 kinase shows a heat-shock domain,3 which gives further support to its possible role as a stress sensor. If this hypothesis holds true, H11 kinase would represent a novel mechanism by which the cardiac cell can rapidly adapt its growth capacity in response to environmental stress.
In the stunning model, the stimulation of H11 kinase expression was accompanied by the increased expression of a whole array of genes participating in cell growth.8 The physiological relevance of a cardiac program of cell growth in ischemia is strengthened by the observations that repetitive or chronic ischemia in patients leads to the development of cardiac cell hypertrophy despite the absence of changes in workload.32–35⇓⇓⇓ Through the activation of the Akt pathway as illustrated above, such stimulation of cell growth is coupled to protective mechanisms that limit the cell loss induced by ischemia/reperfusion injury.8,36–40⇓⇓⇓⇓⇓ In addition to well-known markers of cardiac cell growth (such as ANF, CARP, and ATF-3), we found during stunning an upregulation of several gene products involved in the growth and proliferation of cancer cells.8 H11 kinase itself, when expressed in extramuscular tissues, has been involved in the development and proliferation of cancer tissues, such as melanoma1 and breast cancer.4 An activation of the Akt pathway is often associated with the progression and aggressiveness of various types of tumors.41,42⇓ Our observations therefore suggest that H11 kinase and its signaling pathway may have a significant impact not only on cardiac hypertrophy and ischemic heart disease, but also in cancer biology.
This work was supported by National Institutes of Health grants HL33065, 5PO1 HL 59139, 1PO1 HL69020, AG 14121 and HL 33107 to S.F.V., and AHA grant 0230017N to C.D. We are indebted to Dr Guiping Yang for the morphometric analysis and to Jim Jetko and Jadwiga Markiewicz for expert technical assistance.
Original received September 11, 2002; revision received October 16, 2002; accepted October 18, 2002.
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