Characterization of a Novel Cardiac Isoform of the Cell Cycle-related Kinase That Is Regulated during Heart Failure

Abstract

Myocardial infarction (MI) is often followed by heart failure (HF), but the mechanisms precipitating the transition to HF remain largely unknown. A genomic profile was performed in a monkey model of MI, from the myocardium adjacent to chronic (2-month) MI followed by 3 weeks of pacing to develop HF. The transcript of the gene encoding the cell cycle-related kinase (CCRK) was down-regulated by 50% in HF heart compared with control (p < 0.05), which was confirmed by quantitative PCR. The CCRK sequence cloned from a heart library showed a conservation of the N-terminal kinase domain when compared with the “generic” isoform cloned previously but a different C-terminal half due to alternative splicing with frameshift. The homology of the cardiac sequence was 100% between mice and humans. Expression of the corresponding protein, measured upon generation of a monoclonal antibody, was limited to heart, liver, and kidney. Upon overexpression in cardiac myocytes, both isoforms promote cell growth and reduce apoptosis by chelerythrine (p < 0.05 versus control). Using a yeast two-hybrid screening, we found an interaction of the generic but not the cardiac CCRK with cyclin H and casein kinase 2. In addition, only the generic CCRK phosphorylates the cyclin-dependent kinase 2, which was accompanied by a doubling of myocytes in the S and G2 phases of the cell cycle (p < 0.05 versus control). Therefore, the heart expresses a splice variant of CCRK, which promotes cardiac cell growth and survival; differs from the generic isoform in terms of protein-protein interactions, substrate specificity and regulation of the cell cycle; and is down-regulated significantly in HF. Myocardial infarction (MI) is often followed by heart failure (HF), but the mechanisms precipitating the transition to HF remain largely unknown. A genomic profile was performed in a monkey model of MI, from the myocardium adjacent to chronic (2-month) MI followed by 3 weeks of pacing to develop HF. The transcript of the gene encoding the cell cycle-related kinase (CCRK) was down-regulated by 50% in HF heart compared with control (p < 0.05), which was confirmed by quantitative PCR. The CCRK sequence cloned from a heart library showed a conservation of the N-terminal kinase domain when compared with the “generic” isoform cloned previously but a different C-terminal half due to alternative splicing with frameshift. The homology of the cardiac sequence was 100% between mice and humans. Expression of the corresponding protein, measured upon generation of a monoclonal antibody, was limited to heart, liver, and kidney. Upon overexpression in cardiac myocytes, both isoforms promote cell growth and reduce apoptosis by chelerythrine (p < 0.05 versus control). Using a yeast two-hybrid screening, we found an interaction of the generic but not the cardiac CCRK with cyclin H and casein kinase 2. In addition, only the generic CCRK phosphorylates the cyclin-dependent kinase 2, which was accompanied by a doubling of myocytes in the S and G2 phases of the cell cycle (p < 0.05 versus control). Therefore, the heart expresses a splice variant of CCRK, which promotes cardiac cell growth and survival; differs from the generic isoform in terms of protein-protein interactions, substrate specificity and regulation of the cell cycle; and is down-regulated significantly in HF. Because the fully differentiated cardiac myocyte has a limited capacity for regeneration, most forms of heart disease, including ischemic heart disease and heart failure (HF), 2The abbreviations used are: HFheart failureCCRKcell cycle-related kinaseMImyocardial infarctionDAPI4′,6-diamidino-2-phenylindole. 2The abbreviations used are: HFheart failureCCRKcell cycle-related kinaseMImyocardial infarctionDAPI4′,6-diamidino-2-phenylindole. are characterized by a loss of cardiomyocytes (1Narula J. Haider N. Virmani R. DiSalvo T.G. Kolodgie F.D. Hajjar R.J. Schmidt U. Semigran M.J. Dec G.W. Khaw B.A. N. Engl. J. Med. 1996; 335: 1182-1189Crossref PubMed Scopus (1227) Google Scholar, 2Olivetti G. Abbi R. Quaini F. Kajstura J. Cheng W. Nitahara J.A. Quaini E. Di Loreto C. Beltrami C.A. Krajewski S. Reed J.C. Anversa P. N. Engl. J. Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1464) Google Scholar, 3Palojoki E. Saraste A. Eriksson A. Pulkki K. Kallajoki M. Voipio-Pulkki L.M. Tikkanen I. Am. J. 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Res. 2003; 58: 126-135Crossref PubMed Scopus (17) Google Scholar). In the present study, we extended this investigation to a model of postischemic cardiomyopathy in monkeys. We reasoned that a nonhuman primate model would be ideal, since it is phylogenetically and genetically closer to humans. Genomic profile by DNA microarrays in that model revealed the down-regulation of the cell cycle-related kinase (CCRK) after development of HF. Although this protein has not been characterized in the heart before, it is known that CCRK plays a major role in promoting the growth of cancer cells (7Caligiuri M. Becker F. Murthi K. Kaplan F. Dedier S. Kaufmann C. Machl A. Zybarth G. Richard J. Bockovich N. Kluge A. Kley N. Chem. Biol. 2005; 12: 1103-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Therefore, the goal of the present study is to characterize the expression of CCRK in the normal and failing heart and to determine whether this kinase is involved in cardiac mechanisms of cell growth and survival. heart failure cell cycle-related kinase myocardial infarction 4′,6-diamidino-2-phenylindole. heart failure cell cycle-related kinase myocardial infarction 4′,6-diamidino-2-phenylindole. CCRK belongs to the family of cyclin-dependent protein kinase-activating kinases. A well characterized cyclin-dependent protein kinase-activating kinase is made of a complex that includes the catalytic subunit p40 MO15 (also called Cdk7 (cyclin-dependent kinase-7)), the regulatory subunit cyclin H, and the assembly factor MAT1 and activates the transcription factor IIH, which phosphorylates RNA polymerase II, as well as multiple components of the transcriptional machinery, including p53, RARα, and Oct-1 (7Caligiuri M. Becker F. Murthi K. Kaplan F. Dedier S. Kaufmann C. Machl A. Zybarth G. 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Cells lacking CCRK were incapable of growth, whereas cells with a reduced level of CCRK grew at a significantly slower rate than control cells (7Caligiuri M. Becker F. Murthi K. Kaplan F. Dedier S. Kaufmann C. Machl A. Zybarth G. Richard J. Bockovich N. Kluge A. Kley N. Chem. Biol. 2005; 12: 1103-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 16Liu Y. Wu C. Galaktionov K. J. Biol. Chem. 2004; 279: 4507-4514Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Most interestingly, it was shown that a of CCRK promotes cell (7Caligiuri M. Becker F. Murthi K. Kaplan F. Dedier S. Kaufmann C. Machl A. Zybarth G. Richard J. Bockovich N. Kluge A. Kley N. Chem. Biol. 2005; 12: 1103-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Our that the heart expresses a splice variant of CCRK, which differs from the “generic” isoform characterized in The expression of the corresponding protein was confirmed upon generation of a monoclonal of the cardiac CCRK in cardiac myocytes promotes cardiac cell and survival. We that this cardiac variant differs from the generic isoform of CCRK in terms of protein-protein interactions, substrate specificity, and regulation of the cell of were The and of have been H. C. K. F. A. Vatner Vatner S.F. Circulation. PubMed Scopus Google Scholar, H. B. A. Vatner Vatner S.F. C. Physiol. PubMed Scopus Google Scholar). Myocardial infarction (MI) was in by of the for of pressure and as well as for of pacing was weeks after a of HF. were used as The transition HF was confirmed by of pressure and Myocardial were from adjacent to the MI and were in of were performed mice was by of the for followed by of as previously C. Wang L. X. H. C. N. A. A. M. L. Wagner T. Gaussin V. Vatner S.F. Circ. Res. 2006; PubMed Scopus (82) Google Scholar). RNA was from of monkey heart the P. N. PubMed Scopus Google Scholar). The of RNA was measured by the at The for RNA was performed as previously C. Wang L. X. H. C. N. A. A. M. L. Wagner T. Gaussin V. Vatner S.F. Circ. Res. 2006; PubMed Scopus (82) Google Scholar). of were used for with the and a was performed with DNA polymerase I. were by DNA was the RNA with the was by and to a for of and of with a different were used to of gene from were to the of the which was at were by A than 0.05 and than or were used to was significantly different between the and with and were the of was from of RNA with the The was used for quantitative of a at and a at the a sequence were for transcript of from after of the C. W. T. S. H. Med. PubMed Scopus Google Scholar). to the in the of the were to the transcript level of used as a gene H. C. K. F. A. Vatner Vatner S.F. Circulation. PubMed Scopus Google Scholar). CCRK and sequence of CCRK was by from both and heart and were by a the was performed with the and sequence were performed with the of a the cardiac CCRK protein sequence were and to the which is by the generic CCRK and the cardiac The only the of the cardiac Using this we both the expression of CCRK the N-terminal and the expression of the cardiac splice variant The were in mice and by an of mice was and with cells for of a monoclonal were at in a and with and and at for at were by and to were at the and and with the antibody, detection was performed by of a CCRK the sequence of the cardiac CCRK or of the generic CCRK was the The was and the in a with to the sequence with at the The was in cells by of a the of and with the the CCRK The was cells with and were performed with an the cardiac myocytes were from (5Depre C. Hase M. Gaussin V. Zajac A. Wang L. Hittinger L. Ghaleh B. Yu X. Kudej R.K. Wagner T. Sadoshima J. Vatner S.F. Circ. Res. 2002; 91: 1007-1014Crossref PubMed Scopus (82) Google by with and K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were a made in and at for were at a of and in with 3 and myocytes were in for cell the myocytes were with chelerythrine for or with for after which the cells were in in for in and in in with for at DNA was performed the followed by The of myocyte was by of the myocytes were cell growth, cells were with of and The cell protein and the DNA were by the and the The protein was by the of DNA to for in cell J. L. T. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). determine the the cell cells were with and at and the was with and followed by at for The DNA was by a with by a was measured myocytes after and of cardiac cells by was measured with the corresponding to the generic and the cardiac of CCRK were as in a yeast two-hybrid performed with the after of sequence in and in the yeast was performed was with a with a heart library in were and L. Zajac A. N. C. Mol. 2004; PubMed Scopus Google Scholar). were by were upon of the of CCRK isoforms and a the sequence of the are as the We used for A of < 0.05 was as of the model of postischemic HF was in as MI was in by of the and HF was by The HF the of HF, including a of the myocardial from overload versus in and HF, < 0.05), an pressure versus < 0.05), and as measured by the versus < of the CCRK DNA showed that were in the HF versus whereas were down-regulated the the of CCRK showed of the most with an down-regulation of in HF versus that CCRK expression is down-regulated in the HF we a of and for quantitative the generic sequence in be with this be due to a in the CCRK sequence between and or due to the that the heart expresses a different transcript compared with the generic transcript in that we cloned the CCRK sequence from both and cardiac showed that the CCRK sequence in heart a in the of the transcript and a in C-terminal half when compared with the generic sequence in whereas the N-terminal which the kinase is between both forms of CCRK in including the liver, confirmed the of the generic sequence in the protein sequence from the of the cardiac transcript in both and is 100% which an role for this variant of the cardiac transcript with the generic sequence that the cardiac isoform the that this isoform from alternative splicing The of the cardiac CCRK from and were in and from the genomic versus in a The of by quantitative was by the that this was A quantitative was the sequence cloned from heart Using that CCRK gene expression be and showed a in the HF versus control Therefore, the down-regulation of this gene in HF found by microarrays is confirmed by quantitative both a of the CCRK goal was to determine whether this alternative transcript of CCRK to a A monoclonal was which the C-terminal half of the cardiac monoclonal was to the N-terminal half of the which is between both shown in was only at an of when the C-terminal antibody, which to the of the novel cardiac variant of CCRK found in the major were when the N-terminal at the of and which to the of the novel cardiac variant of CCRK and of the generic of CCRK Using the C-terminal antibody, we were to the expression of the novel CCRK protein in the monkey heart isoform be in and it was not found in a that homology with the heart in terms of protein the generic of CCRK was in a of including the heart, liver, and Using the C-terminal monoclonal we whether the protein corresponding to the cardiac isoform of CCRK the regulation as the corresponding transcript the transition HF shown in the expression of the cardiac CCRK protein was reduced by half in cardiac from monkey to HF when compared with Because we cloned the cardiac CCRK sequence from a we whether the protein would be down-regulated in a model of were to of followed by of after which CCRK expression was by shown in the protein was reduced by half (p < in the ischemic the in the monkey Therefore, both isoforms be in the heart, and the cardiac CCRK a of regulation after MI, both in and in nonhuman We whether both isoforms of CCRK are in the heart at different of were from and from and Expression of both isoforms of CCRK was measured by with the monoclonal the of the be in the heart but at different The expression of the generic CCRK was in the heart, whereas it after as cardiac myocytes the cardiac isoform showed a of expression a level of expression in the and a expression in the whether the alternative splicing of the cardiac CCRK would the protein-protein of the that a yeast two-hybrid was both the generic and the cardiac as the potential found for the from the library with the generic CCRK showed a protein-protein interaction with casein kinase and cyclin H, which was not found in the library with the cardiac CCRK were confirmed upon overexpression of the corresponding in cells The cells were with an expression the sequence of casein kinase or cyclin H, with an expression the sequence of the generic or the cardiac shown in was a interaction between the generic CCRK and both casein kinase and cyclin H, whereas interaction was not found when the cardiac CCRK of protein with generic and cardiac kinase domain kinase protein isoform in a of in protein-protein we whether the substrate specificity of cardiac CCRK would from the generic A well known substrate for the generic CCRK is (16Liu Y. Wu C. Galaktionov K. J. Biol. Chem. 2004; 279: 4507-4514Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). shown in of significantly after overexpression of the generic CCRK in cardiac myocytes, whereas was not upon overexpression of the cardiac is known that activation of the to transition in cardiac cells H. N. A. B. S. Circ. Res. 2001; PubMed Scopus (82) Google Scholar). Therefore, we whether the overexpression of the generic isoform would the cell cycle in the cardiac The of by the generic CCRK was accompanied by a in the of myocytes in the S and G2 phases of the cell as measured by overexpression of the cardiac CCRK not not the cell cycle expression of the cell was upon overexpression of the generic but not the cardiac CCRK cardiac myocytes with isoform or with the control were by in to determine whether the DNA by the generic isoform the or the of the cardiac shown in myocytes with the generic but not with the cardiac isoform of CCRK showed to a in cells as compared with that both isoforms in terms of substrate specificity, cell cycle DNA and myocyte The CCRK and of this in were performed in to the of the cardiac variant of CCRK and to determine and compared with the generic cardiac myocytes were and The expression of the cardiac CCRK protein was by in the myocytes but not in the cardiac from the which a expression of the variant in cardiac that the down-regulation of the cardiac CCRK HF in the rate of cell that that we whether overexpression of the cardiac isoform of CCRK in cardiac myocytes would cell growth and survival. The myocytes were with an the sequence of the cardiac or the generic isoform of CCRK and compared with a cell growth was measured by the with the the cardiac sequence significantly the of cardiac myocytes by at compared with with the of the of the generic protein in cardiac myocytes showed a of cell growth that was to the of the cardiac variant we showed that the generic CCRK DNA which the of cell growth by Therefore, the of the myocytes was measured in the Although both isoforms of CCRK significantly cell as compared with this was significantly in the with the generic CCRK as compared with the cardiac isoform whether the cardiac CCRK cell survival in cardiac myocytes, chelerythrine or was to the myocytes to apoptosis Cells with the cardiac CCRK showed a 50% in apoptosis after the of chelerythrine or when compared with the this was by the generic isoform in both a and We from that both isoforms of CCRK promote cardiac cell growth and survival. The ischemic cardiomyopathy that MI an of the which includes myocyte cell by both and We previously the genomic that in mechanisms promoting cardiac cell both in the and chronic of reversible myocardial ischemia and C. Y. Gaussin V. Vatner Vatner S.F. Circ. Res. 2004; PubMed Scopus Google Scholar, C. Tomlinson J.E. Kudej R.K. Gaussin V. E. Vatner Topper J.N. Vatner S.F. U. S. A. 2001; PubMed Scopus Google Scholar, C. Vatner S.F. Cardiovasc. Med. 2005; PubMed Scopus Google which to the that than of the that are in ischemic myocardium are involved in different mechanisms of cell including to and cell growth C. Vatner S.F. 12: PubMed Scopus Google Scholar). In the present study, we extended this genomic to a monkey model of myocardial ischemia followed by HF. Using a DNA in this we found that the cardiac expression of a gene encoding a novel isoform of CCRK is down-regulated after MI and after the transition HF, which was confirmed by quantitative PCR. monoclonal were which that the transcript of the cardiac CCRK a protein the of regulation MI and HF. and the CCRK sequence from and heart we found that the CCRK transcript in the heart is a splice variant of the transcript characterized in including a that the sequence of the C-terminal half of the the protein sequence from the of cardiac in both and mice is which an role for this splice variant this study is the to the cardiac only have the and of the generic has been shown that the generic CCRK is essential for activation of which in an activation of the cell cycle in cancer cells (16Liu Y. Wu C. Galaktionov K. J. Biol. Chem. 2004; 279: 4507-4514Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). of the protein the growth of whereas is with survival (16Liu Y. Wu C. Galaktionov K. J. Biol. Chem. 2004; 279: 4507-4514Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). of CCRK is for both and cancer cells (7Caligiuri M. Becker F. Murthi K. Kaplan F. Dedier S. Kaufmann C. Machl A. Zybarth G. Richard J. Bockovich N. Kluge A. Kley N. Chem. Biol. 2005; 12: 1103-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google which the of the role of the from the cell In addition, of CCRK in the growth of M. W. J. L. Y. D. J. PubMed Scopus Google Scholar). of involved in the control of the cardiac cell cycle is of has been shown that the expression of multiple of the cell including and Cdk2, after MI K. Cheng W. A. A. B. Kajstura J. Anversa P. Res. 1996; Scopus Google Scholar). expression of ischemic after MI, cardiac and the transition HF H. Circ. Res. 2005; PubMed Scopus Google Scholar, R.K. T. H. R.J. Y. Wu Circ. Res. 100: PubMed Scopus Google Scholar, Cheng R.K. P. J.E. Circulation. 2006; Google Scholar). In that it be that expression of the generic CCRK be in the heart by promoting the cell of the a previous study has that activation of Cdk2, which is the substrate of the generic CCRK (16Liu Y. Wu C. Galaktionov K. J. Biol. Chem. 2004; 279: 4507-4514Abstract Full Text Full Text PDF PubMed Scopus (51) Google a to pressure overload in the heart H. N. A. B. S. Circ. Res. 2001; PubMed Scopus (82) Google Scholar). a for the cardiac variant of CCRK, which the potential of promoting cardiac cell growth and survival The that the generic isoform of CCRK in the heart may the regulation of both The cardiac isoform a in expression level as the myocytes and the cell whereas the generic isoform a of which with the that the generic but not the cardiac CCRK phosphorylates and promotes a of the cardiac myocytes in the cell the cardiac CCRK as a isoform of CCRK in terms of cell cycle in the it role of promoting cell growth and survival. A was in multiple including liver, heart, and which the of the generic CCRK in a was in heart, liver, and kidney. studies have shown that the expression of in and be to the heart, liver, and T. Yamazaki A. T. S. 2006; 79: PubMed Scopus Google Scholar, G. R. R.A. Res. PubMed Scopus Google Scholar). In addition, the cardiac CCRK molecular that to or the cardiac CCRK is found in the heart but not in the an role of the variant in the heart, gene expression in heart and are Because the cardiac is a splice specificity from the expression of splicing than transcription to the of the cardiac CCRK as a for heart most of the in after MI or be from that both isoforms the of promoting both cardiac cell growth and survival. the that the down-regulation of CCRK expression in a context of HF may cardiac cell survival. the that a activation of survival by CCRK overexpression cell loss in a context of myocardial In study a novel and isoform of CCRK that in mechanisms of both cardiac cell growth and survival but as a isoform in terms of cell cycle regulation in myocytes when compared with the generic isoform of the and the down-regulation of cardiac CCRK expression after MI may be involved in the transition of the ischemic myocardium HF and in the rate of cardiac cell that that of the mechanisms of CCRK in genetically models to a of the promoting cardiac cell growth and survival in the heart, which be to for stem cell with