Cardiotrophin 1 (CT-1) Inhibition of Cardiac Myocyte Apoptosis via a Mitogen-activated Protein Kinase-dependent Pathway

Abstract

Cardiac myocyte survival is of central importance in the maintenance of the function of heart, as well as in the development of a variety of cardiac diseases. To understand the molecular mechanisms that govern this function, we characterized apoptosis in cardiac muscle cells following serum deprivation. Cardiotrophin 1 (CT-1), a potent cardiac survival factor (Sheng, Z., Pennica, D., Wood, W. I., and Chien, K. R. (1996) Development (Camb.) 122, 419-428), is capable of inhibiting apoptosis in cardiac myocytes. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 activated both signal transducer and activator of transcription 3 (STAT3)- and mitogen-activated protein (MAP) kinase-dependent pathways. The transfection of a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into myocardial cells blocked the antiapoptotic effects of CT-1, indicating a requirement of the MAP kinase pathway for the survival effect of CT-1. A MEK-specific inhibitor (PD098059) (Dudley, D. T., Pang, L., Decker, S.-J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686-7689) is capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. In contrast, this inhibitor did not block the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Therefore, CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires MAP kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g. Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase. Cardiac myocyte survival is of central importance in the maintenance of the function of heart, as well as in the development of a variety of cardiac diseases. To understand the molecular mechanisms that govern this function, we characterized apoptosis in cardiac muscle cells following serum deprivation. Cardiotrophin 1 (CT-1), a potent cardiac survival factor (Sheng, Z., Pennica, D., Wood, W. I., and Chien, K. R. (1996) Development (Camb.) 122, 419-428), is capable of inhibiting apoptosis in cardiac myocytes. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 activated both signal transducer and activator of transcription 3 (STAT3)- and mitogen-activated protein (MAP) kinase-dependent pathways. The transfection of a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into myocardial cells blocked the antiapoptotic effects of CT-1, indicating a requirement of the MAP kinase pathway for the survival effect of CT-1. A MEK-specific inhibitor (PD098059) (Dudley, D. T., Pang, L., Decker, S.-J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686-7689) is capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. In contrast, this inhibitor did not block the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Therefore, CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires MAP kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g. Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase. INTRODUCTIONCardiac muscle cell survival plays a critical role in maintaining the normal function of the heart and possibly in cardiac development. Adult cardiac muscle cells are terminally differentiated and therefore have lost their proliferative capacity. In contrast to skeletal muscle, the myocardium does not contain satellite heart muscle cells, and irreversible heart injury results in scarring and an eventual decrease in global cardiac function. In response to mechanical stimuli and hemodynamic stress, the adult myocardium activates an adaptive hypertrophic response that is characterized by an increase in myocardial cell size without a concomitant increase in myocyte number (For review, see Refs. 1Chien K.R. Grace A.A. Braunwald E. Heart Disease, A Textbook of Cardiovascular Medicine. W. B. Saunders Co., Philadelphia1996: 1626-1649Google Scholar and 2Chien K.R. Zhu H. Knowlton K.U. Miller-Hance W. van Bilsen M. O'Brien T.X. Evans S.M. Annu. Rev. Physiol. 1993; 55: 77-95Crossref PubMed Scopus (324) Google Scholar). However, during long-standing exposure to hypertension or other forms of hemodynamic stress, a distinct form of myocardial cell hypertrophy can be activated in which the heart becomes dilated and individual cardiac myocytes exhibit an increase in cell length, reflecting the addition of new sarcomeric units in series (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 4Colucci W.S. Braunwald E. Braunwald E. Heart Disease, A Textbook of Cardiovascular Medicine. W. B. Saunders Co., Philadelphia1996: 394-420Google Scholar). This dilatation of the heart is usually accompanied by fibrosis, microscarring, and the loss of viable cardiac myocytes throughout the myocardium. As a result of cardiac dilatation and myocyte dropout, the myocardium ultimately develops an irreversible loss of function and ensuing cardiac muscle failure (4Colucci W.S. Braunwald E. Braunwald E. Heart Disease, A Textbook of Cardiovascular Medicine. W. B. Saunders Co., Philadelphia1996: 394-420Google Scholar). As such, the identification of the signaling pathways that mediate distinct forms of cardiac muscle cell hypertrophy, dysfunction, and cardiac muscle cell survival are critical to the ultimate elucidation of the molecular basis of cardiac muscle failure.By coupling expression cloning with an embryonic stem cell-based model of in vitro cardiogenesis (5Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.-M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar), recent studies have identified cardiotrophin 1 (CT-1), 1The abbreviations used are: CT-1cardiotrophin 1MAPmitogen-activated proteinMEKMAP kinase kinaseSTATsignal transducer and activator of transcriptionPBSphosphate-buffered salineIL-6interleukin 6LIFleukemia inhibitory factorJAKJanus kinaseCNTFciliary neurotrophic factorMLC-2vventricular myosin light chain 2ANFatrial natriuretic factorERKextracellular regulated kinaseTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. a novel cardiac cytokine that was isolated in a search for new factors that induce cardiac myocyte hypertrophy (5Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.-M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar). CT-1 is a new member of the IL-6 family of cytokines that exert their biological effects through the shared signaling subunit gp130 (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 5Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.-M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar, 6Pennica D. Wood W.I. Chien K.R. Cytokine Growth Factor Rev. 1996; 7: 81-91Crossref PubMed Scopus (91) Google Scholar, 7Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1243) Google Scholar) and can activate a distinct form of myocardial cell hypertrophy that is characteristic of volume overload cardiac hypertrophy at the molecular, morphological, and cellular levels (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Importantly, cardiotrophin 1 has been shown to be capable of promoting survival of both embryonic and neonatal rat ventricular muscle cells (8Sheng Z. Pennica D. Wood W.I. Chien K.R. Development (Camb.). 1996; 122: 419-428Crossref PubMed Google Scholar). Recent studies have demonstrated that CT-1 exerts its effects on cardiac muscle cell hypertrophy through promoting the heterodimerization of gp130 with the leukemia inhibitory factor (LIF) receptor β, both of which are required for the activation of the downstream hypertrophic response (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Relatively less is known concerning the mechanisms by which CT-1 promotes cardiac myocyte survival. It is unclear whether this effect is based on a generalized trophic effect or a specific requirement for CT-1 for long-term myocyte survival or whether it reflects the activation of signaling pathways that can act to block programmed cell death of cardiac myocytes, i.e. apoptosis. In addition, it is unknown whether divergent or convergent downstream signaling pathways mediate these two distinct effects of CT-1 on myocyte survival and hypertrophic responses.To address these questions, the current study reports the characterization of an in vitro cardiac muscle assay system in which apoptosis is induced following serum deprivation of myocytes that are plated at a relatively low density. In this assay system, we document the onset of cardiac myocyte cell death via apoptotic pathways by two independent criteria, i.e. scoring for nuclear changes associated with apoptosis and the presence of internucleosomal DNA fragmentation. The addition of CT-1 is capable of promoting cardiac myocyte survival and blocking apoptosis. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 is capable of activating both STAT3- and MAP kinase-dependent pathways. To directly relate the activation of these pathways to the biological functions of CT-1, we transfected a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into neonatal ventricular myocardial cells and found that the mutant was capable of blocking the antiapoptotic effects of CT-1 on individual cardiac myocytes, thereby indicating a requirement of MAP kinase activity for the survival effect of CT-1 on cardiac myocytes. In addition, in studies applying the MEK inhibitor (PD098059) (9Dudley D.T. Pang L. Decker S.-J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2584) Google Scholar), we observed that the inhibitor was capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. The inhibitor displayed specificity for the MAP kinase pathway, as it did not inhibit the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Taken together, these studies indicate that CT-1 promotes cardiac myocyte survival by preventing apoptosis through a signaling pathway that requires MAP kinase. In addition, MAP kinase does not appear to be required for the activation of a CT-1-dependent hypertrophic response, indicating that CT-1 uses divergent signaling pathways for the activation of the survival and hypertrophic responses, the latter of which may be mediated by a JAK/STAT or MEK kinase/c-Jun NH2-terminal protein kinase pathway. INTRODUCTIONCardiac muscle cell survival plays a critical role in maintaining the normal function of the heart and possibly in cardiac development. Adult cardiac muscle cells are terminally differentiated and therefore have lost their proliferative capacity. In contrast to skeletal muscle, the myocardium does not contain satellite heart muscle cells, and irreversible heart injury results in scarring and an eventual decrease in global cardiac function. In response to mechanical stimuli and hemodynamic stress, the adult myocardium activates an adaptive hypertrophic response that is characterized by an increase in myocardial cell size without a concomitant increase in myocyte number (For review, see Refs. 1Chien K.R. Grace A.A. Braunwald E. Heart Disease, A Textbook of Cardiovascular Medicine. W. B. Saunders Co., Philadelphia1996: 1626-1649Google Scholar and 2Chien K.R. Zhu H. Knowlton K.U. Miller-Hance W. van Bilsen M. O'Brien T.X. Evans S.M. Annu. Rev. Physiol. 1993; 55: 77-95Crossref PubMed Scopus (324) Google Scholar). However, during long-standing exposure to hypertension or other forms of hemodynamic stress, a distinct form of myocardial cell hypertrophy can be activated in which the heart becomes dilated and individual cardiac myocytes exhibit an increase in cell length, reflecting the addition of new sarcomeric units in series (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 4Colucci W.S. Braunwald E. Braunwald E. Heart Disease, A Textbook of Cardiovascular Medicine. W. B. Saunders Co., Philadelphia1996: 394-420Google Scholar). This dilatation of the heart is usually accompanied by fibrosis, microscarring, and the loss of viable cardiac myocytes throughout the myocardium. As a result of cardiac dilatation and myocyte dropout, the myocardium ultimately develops an irreversible loss of function and ensuing cardiac muscle failure (4Colucci W.S. Braunwald E. Braunwald E. Heart Disease, A Textbook of Cardiovascular Medicine. W. B. Saunders Co., Philadelphia1996: 394-420Google Scholar). As such, the identification of the signaling pathways that mediate distinct forms of cardiac muscle cell hypertrophy, dysfunction, and cardiac muscle cell survival are critical to the ultimate elucidation of the molecular basis of cardiac muscle failure.By coupling expression cloning with an embryonic stem cell-based model of in vitro cardiogenesis (5Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.-M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar), recent studies have identified cardiotrophin 1 (CT-1), 1The abbreviations used are: CT-1cardiotrophin 1MAPmitogen-activated proteinMEKMAP kinase kinaseSTATsignal transducer and activator of transcriptionPBSphosphate-buffered salineIL-6interleukin 6LIFleukemia inhibitory factorJAKJanus kinaseCNTFciliary neurotrophic factorMLC-2vventricular myosin light chain 2ANFatrial natriuretic factorERKextracellular regulated kinaseTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. a novel cardiac cytokine that was isolated in a search for new factors that induce cardiac myocyte hypertrophy (5Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.-M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar). CT-1 is a new member of the IL-6 family of cytokines that exert their biological effects through the shared signaling subunit gp130 (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 5Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.-M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar, 6Pennica D. Wood W.I. Chien K.R. Cytokine Growth Factor Rev. 1996; 7: 81-91Crossref PubMed Scopus (91) Google Scholar, 7Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1243) Google Scholar) and can activate a distinct form of myocardial cell hypertrophy that is characteristic of volume overload cardiac hypertrophy at the molecular, morphological, and cellular levels (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Importantly, cardiotrophin 1 has been shown to be capable of promoting survival of both embryonic and neonatal rat ventricular muscle cells (8Sheng Z. Pennica D. Wood W.I. Chien K.R. Development (Camb.). 1996; 122: 419-428Crossref PubMed Google Scholar). Recent studies have demonstrated that CT-1 exerts its effects on cardiac muscle cell hypertrophy through promoting the heterodimerization of gp130 with the leukemia inhibitory factor (LIF) receptor β, both of which are required for the activation of the downstream hypertrophic response (3Wollert K.C. Taga T. Saito M. Narazaki M. Kishimoto T. Glembotski C.C. Vernallis A.B. Heath J.K. Pennica D. Wood W.I. Chien K.R. J. Biol. Chem. 1996; 271: 9535-9545Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Relatively less is known concerning the mechanisms by which CT-1 promotes cardiac myocyte survival. It is unclear whether this effect is based on a generalized trophic effect or a specific requirement for CT-1 for long-term myocyte survival or whether it reflects the activation of signaling pathways that can act to block programmed cell death of cardiac myocytes, i.e. apoptosis. In addition, it is unknown whether divergent or convergent downstream signaling pathways mediate these two distinct effects of CT-1 on myocyte survival and hypertrophic responses.To address these questions, the current study reports the characterization of an in vitro cardiac muscle assay system in which apoptosis is induced following serum deprivation of myocytes that are plated at a relatively low density. In this assay system, we document the onset of cardiac myocyte cell death via apoptotic pathways by two independent criteria, i.e. scoring for nuclear changes associated with apoptosis and the presence of internucleosomal DNA fragmentation. The addition of CT-1 is capable of promoting cardiac myocyte survival and blocking apoptosis. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 is capable of activating both STAT3- and MAP kinase-dependent pathways. To directly relate the activation of these pathways to the biological functions of CT-1, we transfected a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into neonatal ventricular myocardial cells and found that the mutant was capable of blocking the antiapoptotic effects of CT-1 on individual cardiac myocytes, thereby indicating a requirement of MAP kinase activity for the survival effect of CT-1 on cardiac myocytes. In addition, in studies applying the MEK inhibitor (PD098059) (9Dudley D.T. Pang L. Decker S.-J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2584) Google Scholar), we observed that the inhibitor was capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. The inhibitor displayed specificity for the MAP kinase pathway, as it did not inhibit the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Taken together, these studies indicate that CT-1 promotes cardiac myocyte survival by preventing apoptosis through a signaling pathway that requires MAP kinase. In addition, MAP kinase does not appear to be required for the activation of a CT-1-dependent hypertrophic response, indicating that CT-1 uses divergent signaling pathways for the activation of the survival and hypertrophic responses, the latter of which may be mediated by a JAK/STAT or MEK kinase/c-Jun NH2-terminal protein kinase pathway.