Transformation of jaw muscle satellite cells to cardiomyocytes

Abstract

In the embryo a population of progenitor cells known as the second heart field forms not just parts of the heart but also the jaw muscles of the head. Here we show that it is possible to take skeletal muscle satellite cells from jaw muscles of the adult mouse and to direct their differentiation to become heart muscle cells (cardiomyocytes). This is done by exposing the cells to extracellular factors similar to those which heart progenitors would experience during normal embryonic development. By contrast, cardiac differentiation does not occur at all from satellite cells isolated from trunk and limb muscles, which originate from the somites of the embryo. The cardiomyocytes arising from jaw muscle satellite cells express a range of specific marker proteins, beat spontaneously, display long action potentials with appropriate responses to nifedipine, norepinephrine and carbachol, and show synchronized calcium transients. Our results show the existence of a persistent cardiac developmental competence in satellite cells of the adult jaw muscles, associated with their origin from the second heart field of the embryo, and suggest a possible method of obtaining cardiomyocytes from individual patients without the need for a heart biopsy.

Keywords: BMP; Cardiomyocytes; Muscle satellite cell; Myogenesis; Second heart field; WNT.

Fig.1. Satellite cells from jaw muscles are derived from the secondary heart field and express cardiac transcription factors in response to BMP4

A. Isl1-Cre;mT/mG mouse, frontal section through the head of E13.5 embryo showing that jaw muscles are derived from Isl1-positive precursors. High power views are shown on the right. Myofibers, immunostained for MF20, and satellite cells, immunostained for PAX7, are both derived from Isl1-positive precursors. White boxes on low magnification image indicate the location of the high magnification images on the right. The masseter (ma) consists of both deep and superficial masses; (ad) anterior digastric; (my) mylohyoid; (t) tongue; (tm) temporalis; (eom) extraocular muscles; (br) brain. Scale bars: 100μm. B. Schematic of representative muscles of adult mice used to prepare satellite cells, and typical sequential FACS plot. C. Absence of difference between jaw and trunk-derived satellite cells in myogenic transcription factor gene expression in the presence of BMP4 (day 3). D. Upregulation of cardiogenic transcription factor genes in jaw but not trunk satellite cells in the presence of BMP4 (day 3).

Fig.2. Formation of cardiomyocyte precursors from jaw muscle satellite cells

A. Scheme of the growth and differentiation protocol. B. Percentage of cTnT-positive cells generated by the end of the differentiation period, with and without serum. Error bars indicate ± standard error of the mean. C. qRT-PCR of expression of myogenic and cardiogenic transcription factors during the first 7 days. Error bars indicate ± standard error of the mean. D. NKX2.5 (red) is found only in those cells expressing PAX7 (green) at day 3 cultures. Scale bar 25μm. E. At 7 days of culture, myogenin (green) is not co-expressed with GATA4 (red). Scale bar 25μm.

Fig.3. Characterization of induced cardiomyocytes

A-C. Induced cardiomyocytes visualized by immunostaining at day 14 of culture. A. Cardiac troponin T (cTnT). B. α-myosin heavy chain. C. Connexin 43 showing gap junctions between adjacent cells D. Expression of five genes for cardiomyocyte terminal differentiation markers (qRT-PCR). Error bars indicate ± standard error of the mean. E. Typical spontaneous action potentials for E13.5 embryonic cardiomyocytes; induced cardiomyocyte; and evoked action potential for skeletal myotube. The duration of the induced cardiomyocyte action potentials is similar to those of the embryonic cardiomyocytes and much longer than those of the myotubes. F. Effects of norepinephrine, carbachol and nifedipine on spontaneous action potentials. Colored overlayed traces indicate spontaneous action potential after addition of each drug. G. Calcium transients in induced cardiomyocytes visualized with Fluo3AM. The traces indicate the signal for the four indicated cells, which are highly synchronized. Scale bar 20μm.All measurements were repeated several times as indicated, with standard errors, in the text.

Fig.4. Cell lineage studies

A. Evidence that induced cardiomyocytes are derived from satellite cells. Pax7 expressing cells were labeled by treating the Pax7-CreER;mT/mG mice with tamoxifen. The green label indicates that cTnT-positive cardiomyocytes formerly expressed Pax7. Scale bar 25μm. B. Evidence that induced cardiomyocytes are derived from the secondary heart field. Satellite cells were prepared from Isl1-Cre;mTmG mice and then sorted for GFP expression, and subjected to the cardiomyocyte differentiation procedure. The NKX2.5 and cTnT-positive cells must be derived from cells that had expressed Isl1 in vivo. Scale bar: 25μm.