In most of the countries, acquired heart disease has been number one killer, and congenital heart diseases occur in approximately 0.9% of total live born neonates. While the postnatal mammalian heart is the least regenerative of organs, the embryonic heart displays tremendous capacity for proliferation and lineage diversification. Therefore, deeper understanding of cardiogenesis, whether developmental or stem cell-based, will provide a potential for novel therapeutic approaches to the heart diseases in adults and children. My research goal is to elucidate how multipotent cardiac progenitors form a diverse set heart tissue through progressive specialization.
The classical view of embryonic cardiogenesis has been modified by the recent discovery of the second heart field. While the classical cardiac crescent give rise to left ventricle first, the second heart field progenitors in the extra-crescent tissue supplies additional myocardium which contribute to right ventricle, atria and cardiac outflow tract. During my postdoc training, I identified Isl1-positive multipotent cardiac progenitors in the second heart field: Innovation of cardiac mesenchymal feeder culture system and use of multiple genetically modified mouse lines allowed us to demonstrate the multipotency of Flk1/Isl1/Nkx2-5+ progenitors that give rise to cardiac, smooth muscle and endocardial cells from single cell (Cell 2006). With more than 500 citations, this work opened a developmental paradigm that major cell populations that constitute the circulatory system arise from single cell level fate decision of multipotent progenitors.
In my own lab at UCLA, we are actively pursuing three major directions.
1. Smooth muscle cell fate decision. A logical question arising from these observations is why and how the cardiac progenitors give rise to smooth muscle. It has been known that aortic smooth muscle cells originate from multiple sources and that cardiomyocytes acquire a smooth muscle phenotype upon culture. However, the biological significance of this phenomenon has been unclear. Our cardiac mesenchymal feeder culture system and newly generated atrial specific SLN-cre line enabled us to analyze smooth muscle differentiation capability of atrial progenitors in vivo and in vitro. Based on these data, we proposed that the multipotency of the cardiac progenitors plays an important role in the formation and structure of heart-vessel boundary (JMCC 2011). Understanding of the mechanism governing heart-vessel junction formation is important because the boundary between heart and great vessels are often affected by aortic dissection and atrial fibrillation, which are both common diseases with no fundamental therapy. We have recently identified two transcription factors that may play important roles in the cell fate decision of atrial vs smooth muscle cells (in preparation).
2. Hemogenic potential in the heart. The circulatory system has evolved to maintain the homeostasis of the organism. Studies of non-vertebrates, lower vertebrates and mammalian ES in vitro differentiation models all suggest a close relationship between cardiac and hematopoietic cells. Identification of mouse Flk1/Isl1/Nkx2.5+ ?cardio-vascular? progenitors has led to the question whether the third component of the circulatory system, the ?hematopoietic? cells, shares clonal origin with Flk1/Isl1/Nkx2.5+ ?cardio-vascular? progenitors. If they do, when, where and how do they diverge into hematopoietic lineage in vivo? Inspired by my past clinical observations of extramedullary hematopoiesis in the hearts of autopsy patients, we have demonstrated in collaboration with Dr. Hanna Mikkola (MCDB) that a specific subpopulation of endocardial cells gives rise to the definitive hematopoietic cells in vitro and in vivo. We also examined the molecular mechanism governing the hematopoietic activity of this new endocardial subpopulation, and found that the cardiac homeobox transcription factor Nkx2.5 plays a key role upstream of multiple hematopoietic transcription factors in the second heart field-derived endocardial cells. Based on these observations, I propose the concept of ?hemogenic endocardium? (Nat Comm, 2013). As Nkx2-5 is known to regulate both cardiogenesis and hematopoiesis in Drosophila, the Nkx2-5-dependent cardio-vasculo-hematopoietic differentiation pathway likely represents phylogenetically conserved mechanism of the development of circulatory system. This is a pioneering work that changes our view of evolution and development of the circulatory system and opens several important questions relevant to basic and translational researches ? among these are where do the hemogenic endocardial cells originate from (multipotent cardiac progenitors or some other source)? What molecular pathway regulates their generation and emergence? What causes the unique anatomical distribution of hemogenic endocardial cells? How does endocardial hematopoiesis interact with other local tissues in the heart? To what extent do mammals need this transient hematopoietic population? Do Nkx2.5-positive cells contribute to adult circulation? Does Nkx2.5+ leukemia in children represent pathological reactivation of this old hematopoietic mechanism?
3. Maturation of the cardiomyocytes. The third direction is to understand how cardiac progenitors mature into rhythmically contracting syncytium of atrial, ventricular and conduction myocardium. While a growing number of reports including ours described above have elucidated the diversity of cardiac lineages, inducing the maturation of ES-derived cardiac progenitors is still a big unmet challenge in the cardiac translational research. As the genetic approaches alone have not been successful in induction of full maturation of in vitro cardiomyocytes, a key to the application of ES cells to human regenerative medicine lies in the understanding the role of in vivo biophysical cues to the cardiac maturation. As demonstrated by us and others, during in vivo cardiogenesis, multipotent progenitors are progressively incorporated into a unique electro-mechanical field generated by already differentiated myocardium. In other words, late-migrating progenitors are gradually exposed to electromechanical force generated by already differentiated myocytes. This unique interaction between progenitors and mature myocardium leads to the hypothesis that the cardiac-specific microenvironment is not only the consequence but also the cause of cardiac maturation. In collaboration with Dr. James K. Gimzewski (School of Engineering), we have reported that matrix elasticity is an independent factor that regulates the cardiac maturation of human ES cells (STAM, in press). We are now testing whether rhythmic contraction induces the cardiac maturation using bioengineering approaches. The effect of Ca modulation on cardiac maturation is also being tested in collaboration with Dr. Jau Chen (MCDB). We have further identified Nkx2-5-Notch pathway that regulates the maturation of working myocardium. Ablation of this pathway induced the overproliferation of late-stage cardiomyocytes and misspecification of cardiac conduction cells (in revision). Our next goals are to understand the role of this genetic pathway in the context of molecular-biophysical feedback mechanism and application of this mechanism to the in vivo cardiac repair. Essential to the translation of these findings is an efficient model of human cardiomyocytes and cardiogenesis. This summer, we sent a postdoc to Kyoto University Stem Cell Center (iCeMS, Dr. Norio Nakatsuji) and established high-yield 2D differentiation protocol for hESC-derived cardiomyocytes in our lab, which consistently yields ~90% cardiac differentiation. This level of efficiency is not available anywhere else in the world (usually ~40% at best). The high-yield 2D differentiation system is extremely powerful tool in the field and opens a broad application to molecular biology, biophysical analyses and transplantation of human cardiomyocytes. Our next goal is to examine how electromechanical cues translate into genetic/epigenetic signals genome-wide in collaboration with Dr Matteo Pellegrini (MCDB).
In summary, I have established a productive research program studying the mechanism of differentiation of mouse and human cardiac progenitors. The smooth muscle project is shedding light on the formation of the heart-vessel junction and pathogenesis of clinically relevant diseases. The hemogenic endocardium concept is novel, and exploring the physiological roles and molecular mechanisms underlying the formation of these cells will be a key idea for various future researches in the field. The cardiac maturation studies will be a step towards the medical translation of our projects. Our human ESC system is very powerful. Combination with our expertise in mouse genetics promises the understanding of biological mechanism of cardiogenesis and its application to the cardiac therapy at unprecedented level. This is an area of research that requires both strong basic and translational components. I hope that my dual degree background makes me extremely well suited for pursuing basic molecular biology and biophysics of the heart relevant to bedside cardiology.
Chen, P. Y.
Orozco, L. D.
Jacobsen, S. E.
Devaskar, S. U.
Pellegrini, M., "Intra-Uterine Calorie Restriction Affects Placental DNA Methylation and Gene Expression", Physiol Genomics 45 (14): 565-576 (2013).
Arshi, A., Nakashima, Y., Nakano, H., Eaimkhong, S.
Evseenko, D. Reed, J., Stieg, A.Z.
Gimzewski, J. K.
Nakano, A., "Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells", Sci Technol Adv Mater 14: (2013). [link]
Sasman, A., Nassano-Miller, C., Shim, K.S., Koo, H.Y., Liu, T., Schultz, K.M., Millay, M., Nanano, A., Kang, M., Suzuki, T., Kume, T.,, "Generation of conditional alleles for Foxc1 and Foxc2 in mice", Genesis 50 (10): 766-774 (2012).
Van Handel, B., Montel-Hagen, A., Sasidharan, R., Nakano, H.
Ferrari, R., Boogerd, C.J., Schredelseker, J., Wang, Y.,
Hunter, S., Org, T., Zhou, J., Li, X., Pellegrini, M.
Chen, J.N., Orkin, S.H., Kurdistani, S.K., Evans, S.M., Nakano, A., Mikkola, H.K., "Scl Represses Cardiomyogenesis
in Prospective Hemogenic
Endothelium and Endocardium", Cell 150: 590-605 (2012).
Nakano H, Williams E, Hoshijima M, Sasaki M, Minamisawa S, Chien KR, Nakano A., "Cardiac origin of smooth muscle cells in the inflow tract", J Mol Cell Cardiol 50 (2): 337-345 (2011).
Laugwitz, K.L., Moretti, A., Caron, L., Nakano, A. and Chien, K.R., "Islet 1 cardiovascular progenitors: a single source for heart lineages?", Development 135 (2): 193-205 (2008).
Moretti, A.*, Caron, L.*, Nakano, A.*, Lam, J.T., Bernshausen, A., Chen, Y., Qyang, Y., Bu, L., Sasaki, M., Martin-Puig, S., Sun, Y., Evans, S.M., Laugwitz, K.L. and Chien, K.R., "Multipotent Embryonic Isll(+) Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification", Cell 127 (6): 1151-1165 (2006).
Sonoda, E., Zhao, G.Y., Kohzaki, M., Dhar, P.K., Kikuchi, K., Redon, C., Pilch, D.R., Bonner, W.M., Nakano, A., Watanabe, M., Nakayama, T., Takeda, S., Takami, Y., "Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair", DNA Repair (Amst) (2006).
Seo, S., Fujita, H., Nakano, A., Kang M., Duarte, A., Kume, T., "The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development", Dev Biol 294 (2): 458-470 (2006).
Ito, H., Nakano, A., Kinoshita, M., Matsumori, A., "Pioglitazone, a peroxisome proliferators-activated receptor-gamma agonist, attenuates myocardial ischemia/reperfusion injury in a rat model", Lab Invest 83 (12): 1715-1721 (2003).
Kitaura-Ikenaga, K., Hara, M., Higuchi, K., Yamamotok K., Yamaki, A., Ono, K., Nakano, A., Kinoshita, M., Sasayama S., Matsumori, A., "Gene expression of cardiac mast cell chymase and tryptase in a murine model of heart failure caused b viral myocarditis", Circ J 67 (10): 881-884 (2003).
Adachi, O., Nakano, A., Sato, O., Kawamoto, S., Tahara, H., Toyoda, N., Yamato, E., Matsumori, A., Tabayashi, K., Miyazaki, J., "Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis", Gene Ther 9 (9): 577-583 (2002).
Nakano, A., Matsumori, A., "Therapeutic use of Interleukin-10 for myocarditis", In: Fulminant Myocarditis (2002).
Nakano, A., Matsumori, A., Kawamoto, S., Tahara, H., Yamato, E., Sasayama, S., Miyazaki, J., "Cytokine Gene Therapy for Myocarditis by in Vivo Electroporation", Hum Gene Ther 12 (10): 1289-1297 (2001).
Kawamoto, S., Nitta, Y., Tashiro, F., Nakano, A., Yamato, E., Tahara, H., Tabayashi, K., Miyazaki, J., "Suppression of Th1 cell activation and prevention of autoimmune diabetes in NOD mice by local expression of viral interleukin-10", International Immunology 13 (5): 685-697 (2001).