Summary
We have discovered a unique RNA that has the ability to transform non-muscle cells into contracting cardiac muscle tissue using cardiac non-function mutant Mexican axolotl (salamander) embryos as “tools” in the study. In the present study, RNAs derived by molecular cloning of commercially-obtained human fetal heart RNA were tested to determine if human RNA also might have the ability to promote cardiomyocyte differentiation from non-muscle cells. The human fetal heart RNA was cloned, sequenced and synthesized using standard laboratory protocols and then tested initially using the cardiac non-function axolotl embryonic heart bioassay system. This human-derived RNA was able to rescue the non-functional mutant hearts by converting non-muscle cells in their heart walls into contracting myocardial tissue with well-organized sarcomeric myofibrils. Similar results were obtained using undifferentiated mouse embryonic and human-induced pluripotent stem cells in culture. Both mouse- and human-derived pluripotent stem cells transfected with the Cardiac Inducing RNA ( CIR) form into shapes characteristic of early cardiac myocytes in culture and express the cardiac specific contractile protein marker, troponin-T, in addition to tropomyosin and α-actinin as detected by immunohistochemical staining. Expression of these contractile proteins also shows organization into sarcomeric myofibrils characteristic of striated cardiac muscle cells. The active clone producing the cardiac-inducing RNA (CIR) was found to be a fragment of the N-sulfoglucosaminesulfohydrolase and the caspase recruitment domain family member 14 percursor. Computer analyses of the RNA secondary structures of this active CIR using the Genebee Computer Structural Analysis Program developed at the Belozersky Research Institute of Moscow State University, Russia, show significant similarities to the Myocardial Cell Inducing RNA (MIR) from axolotl which also promotes non-muscle cells to differentiate into cardiac muscle. Thus, these two RNAs, salamander MIR and the newly-discovered human-derived Cardiac Inducing RNA reported here, appear to have evolutionarily conserved secondary structures suggesting that both play major roles in vertebrate heart development and, particularly, in the differentiation of cardiomyoctyes from non-muscle cells.
Significance Statement
Of the hundreds of thousands of people who suffer heart attacks each year, in spite of current medical treatments that improve heart function and quality of life for some, many do not significantly recover because connective scar tissue, rather than new muscle, replaces the cardiac muscle in the infarcted area. The research reported here may very well lead to better treatments in the future for patients recovering from myocardial infarctions or other heart diseases that adversely affect the myocardial muscle tissue. Being able to use the Cardiac Inducing RNA (CIR) itself, or in combination with induced pluripotent stem cells derived from that same patient to repair the damaged heart muscle tissue or replace the scar tissue with vigorously contracting normal myocardial cells, could lead to a much better prognosis for cardiac patients and may very well allow them to completely recover and return to pre-heart-attack activity levels.
About the author
Andrei Kochegarov, Ph.D.
Dr. Andrei Kochegarov completed his undergraduate and Master’s degrees in Biology at Saratov State University, Russia and his Ph.D. in Molecular and Cellular Biology at Pushchino State University, Russia. He worked as a Postdoctoral Research Associate for ten years at major research Universities in California, and the last five years, he has been a Research Assistant Professor working on stem cell research and cardiac regeneration at Texas A&M University, Commerce, as well as a lecturer for several Biology courses. He is interested in Regenerative Biology, Cardiac and Neuronal Regeneration, Oxidative Stress, Cellular Aging and Cancer Research.
About the author
Ashley Moses-Arms, B.S., M.S.
Ashley Moses-Arms completed her B.S degree with highest honors (summa cum laude) from Texas A&M University-Commerce Honors College majoring in the Biological Sciences. Her honors thesis research was done under the mentorship of Professor Larry Lemanski and involved studies on a human-derived RNA that promotes cardiac muscle differentiation in cardiac mutant non-function salamanders. She completed a M.S. degree in the Biological Sciences Program also from Texas A&M University-Commerce in Dr. Venugopalan Cheriyath’s laboratory. Her interests have been in the areas of regenerative medicine and cancer research. Currently she is a first year medical student in the College of Medicine at the University of Texas Medical Branch in Galveston.
About the author
Larry F. Lemanski, Ph.D.
Larry F. Lemanski completed his B.S. degree with honors, in Biology and Chemistry, from the University of Wisconsin-Platteville and his M.S. and Ph.D. degrees from Arizona State University, Tempe, Arizona, U.S.A. After four years as an NIH and MDAA Postdoctoral fellow at the University of Pennsylvania, Philadelphia, he joined the medical faculty at the University of California, San Francisco, as an Assistant Professor in residence and then moved to the faculty of the College of Medicine at the University of Wisconsin, Madison, where he went up through the ranks to Full Professor of Anatomy. He then joined the Upstate Medical University in Syracuse, New York, as Professor and Chairman of the Department of Anatomy and Cell Biology. He moved to Texas A&M University as an Associate Vice President for Research, then to Florida Atlantic University as a Vice President for Research, to Temple University as Senior Vice president for Research and Strategic Initiatives, and to Texas A&M University-Commerce as Provost of the University. He has now transitioned to the position of Distinguished Research Professor at Texas A&M University-Commerce. His research interests throughout his career have involved embryonic heart development and cardiac cell differentiation using a variety of morphological, cellular, biochemical, and molecular biology approaches. He and his associates have published numerous papers in the field of embryonic heart development and cardiac cell differentiation. His current work centers around his earlier pioneering discovery that selected RNAs can promote the differentiation of non-muscle cells to form into functional cardiac tissue.
Journal Reference
In Vitro Cell Dev Biol Anim. 2015; 51(7):739-48.
Kochegarov A, Moses-Arms A, Lemanski LF.
Department of Biological and Environmental Sciences, Texas A&M University-Commerce, Commerce, TX, 75429-3011, USA.
Abstract
A specific human fetal heart RNA has been discovered, which has the ability to induce myocardial cell formation from mouse embryonic and human-induced pluripotent stem cells in culture. In this study, commercially obtained RNA from human fetal heart was cloned, sequenced, and synthesized using standard laboratory approaches. Molecular analyses of the specific fetal cardiac-inducing RNA (CIR), revealed that it is a fragment of N-sulfoglucosaminesulfohydrolase and the caspase recruitment domain family member 14 precursor. Stem cells transfected with Cardiac Inducing RNA often form into spindle-shaped cells characteristic of cardiomyocytes,and express the cardiac-specific contractile protein marker, troponin-T, in addition to tropomyosin and α-actinin as detected by immunohistochemical staining. Expression of these contractile proteins showed organization into sarcomeric myofibrils characteristic of striated cardiac muscle cells. Computer analyses of the RNA secondary structures of the active Cardiac Inducing RNA show significant similarities to a RNA from salamander or myofibril-inducing RNA (MIR), which also promotes non-muscle cells to differentiate into cardiac muscle. Thus, these two RNAs, salamander MIR and the newly discovered human-cloned cardiac inducing RNA reported here, appear to have evolutionarily conserved secondary structures suggesting that both play major roles in vertebrate heart development and, particularly, in the differentiation of cardiomyocytes from non-muscle cells during development.
Go To In Vitro Cell Dev Biol Anim.
Acknowledgments: This work is supported by NIH grant (HL061246), NSF grant (1121151), and an American Heart Association grant (10GRNT4530001) awarded to LFL.
Figure Legends
Figure 1. Mouse embryonic stem cells transfected with Cardiac Inducing RNA (CIR), cultured for 8 days and immunostained with antibody against cardiac-specific troponin-T. These CIR-treated cells show significant staining for cardiac troponin-T, indicating they have differentiated into cardiac lineages. The DAPI-stained blue nuclei are difficult to visualize in these cells due to the heavy FITC-staining for cardiac specific troponin-T.
Figure 2. Mouse embryonic stem cell controls which have been cultured for 8 days, stained with antibodies against cardiac-specific troponin-T but not transfected with the cardiac Inducing RNA (CIR). Without the CIR treatment, these cells do not express significant amounts of cardiac troponin-T. Mostly, only the DAPI –stained blue nuclei are visible.
Figure 3. High magnification fluorescent micrograph of a human induced pluripotent stem cell (iPSC) treated with Cardiac Inducing RNA (CIR), cultured for 8 days and stained with cardiac-specific anti-troponin-T antibodies. This spindle-shaped cell, shows staining for cardiac-specific troponin-T which is organized into myofibrillar- like structures characteristic of early developing cardiomyocytes. (From: Kochegarov A., Moses-Arms, A., Lemanski, L.F. 2015 A fetal human heart cardiac inducing RNA (CIR) promotes the differentiation of stem cells into cardiomyocytes. In Vitro Cell Dev Biol-Animal DOI 10.1007/s11626-015-9880-4. With permission of the authors).
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