The Overall Pattern of Cardiac Contraction Depends on a Spatial Gradient of Myosin Regulatory Light Chain Phosphorylation

AbstractEvolution of the human heart has incorporated a variety of successful strategies for motion used throughout the animal kingdom. One such strategy is to add the efficiency of torsion to compression so that blood is wrung, as well as pumped, out of the heart. Models of cardiac torsion have assumed uniform contractile properties of muscle fibers throughout the heart. Here, we show how a spatial gradient of myosin light chain phosphorylation across the heart facilitates torsion by inversely altering tension production and the stretch activation response. To demonstrate the importance of cardiac light chain phosphorylation, we cloned a myosin light chain kinase from a human heart and have identified a gain-in-function mutation in two individuals with cardiac hypertrophy

Measuring Calcium Transient Frequency

<p>Graphical representation of the calcium transient in a beating CPS cell–derived cardiomyocyte (A). Fluorescent intensity is proportional to the amount of calcium binding to fluo-3 dye upon release of calcium from the sarcoplasmic reticulum. Peak intensity (B) and baseline (C) are shown.</p

Transmission EMs Show the Post-Replating Progression of CPS Cells

<div><p>(A) Round, day 3 cells contain disordered myosin filaments. Some of these cells beat while still floating (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030087#sv001" target="_blank">Video S1</a>) and typically have APs as shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030087#pbio-0030087-g006" target="_blank">Figure 6</a>A.</p> <p>(B) Upper box is a blowup taken from lower panel, showing myosin filaments of characteristic 1.6-μm length radiating outward from dense body.</p> <p>(C) Day 14 cell with a single, central nucleus shows a stretching out of the dense bodies into an organizing sarcomere.</p> <p>(D) Day 3 round cells containing copious mitochondria (inset).</p> <p>(E) Elongated day 7 cell containing a dense body (arrowhead).</p> <p>(F) Uninucleate day 14 cell, same cell as in (C).</p> <p>(G) By day 56, a well-defined sarcomere is present, with identifiable A- and I-bands and M- and Z-lines.</p> <p>(H) Sarcomere from a fetal cardiomyocyte is shown for comparison.</p></div

Whole-Cell Voltage Recordings from Spoc Cell–Derived Cardiomyocytes

<div><p>(A) Spontaneous AP firing in a nonbeating, teardrop-shaped cell.</p> <p>(B) Representative AP from recording in (A) on an expanded time scale; AP threshold is –60 mV.</p> <p>(C) Action potential firing in another cell is blocked upon bath perfusion with 0.5 mM cadmium chloride (horizontal bar).</p> <p>(D) Acceleration of AP firing upon perfusion with 25 nM isoproterenol (horizontal bar) is demonstrated, indicating the presence of adrenergic receptors on these cells.</p> <p>(E) Skeletal myotube APs, if present, differ in that their frequency is unaffected by Cd⁺⁺.</p> <p>(F) Isoproterenol also does not affect skeletal muscle AP frequency.</p></div

Cre Expressor/Beta-Galactosidase Reporter Myocardial Infarction Studies

<div><p>(A) Nests of Cre⁺ cells (green) are detected 1 wk after tail-vein injection into an acute infarct model.</p> <p>(B) Nomarski image of (A).</p> <p>(C) Merged image of (A) and (B). The clusters are located near a blood vessel (arrow).</p> <p>(D) Infarcted tissue in a control MI model (infarction surgery but no donor-cell injection) showing a lack of staining for Cre (no green) and GATA-4 (no red). </p> <p>(E) Control X-gal staining of ROSA mouse heart.</p> <p>(F) In a sequential series of tissue sections, odd-numbered sections were immunostained for Cre, yielding the results seen in (A). These two clusters of cells were seen on five sections (sections 1, 3, 5, 7, and 9). Even-numbered sections (sections 2, 4, 6, and 8) were stained for X-gal. No X-gal⁺ cells were found. One slide was immunostained, showing the Cre⁺ cells present. This slide was then stained for X-gal and was found to be X-gal⁻. The lack of X-gal staining of the serial sections indicates that at 1 wk no fusion of donor and host cells has occurred in the infarct.</p> <p>(G) Cluster of Cre⁺ donor cells detected in infarcted heart tissue of a 1-wk-old acute infarct model. Arrowheads indicate cells that also express GATA-4, as shown in (H). </p> <p>(H) GATA-4 (red) is mostly present in some cells in the margin of the cluster. Arrowheads indicate cells that also express Cre, as shown in (G) </p> <p>(I) Merged image showing co-localization (arrowheads) of Cre (green) with GATA-4 (red) in some cells of the cluster of Cre⁺ cells. </p> <p>(J–L) Co-staining of donor cells with anti–Cre antibody (green) and MSC 21 (red) is apparent after 7 d in an acute infarct model.</p> <p>(M) There is a lack of staining with MSC 21 (no red) in the infracted tissue of mice that have not received Spoc cell injections.</p></div

CPS Cells Stain Positive for Cardiac-Specific Proteins

<div><p>(A) GATA-4 in day 7 CPS cells. </p> <p>(B) Nuclear staining with DAPI.</p> <p>(C) Overlay of (A) and (B).</p> <p>(D) Nkx-2.5 is detected in the nuclei of round, day 21 beating cells (green).</p> <p>(E) Noncardiac cells (red arrowheads) do not show nuclear staining for Nkx-2.5.</p> <p>(F) Overlay of (D) and (E).</p> <p>(G) Beating cells, after 28 d in culture, stain positive for cardiac L-type Ca⁺⁺ channel.</p> <p>(H) Connexin 43 (green) in cluster of uninucleate day 21 beating cells in culture.</p> <p>(I) Nomarski light micrograph (differential interference contrast) of cell cluster in (H).</p></div

Sublocalization of GATA-4 in Spoc Cells

<div><p>(A) GATA-4 is detected in the cytoplasm of day 10 Spoc cells (cytospin). </p> <p>(B) Nomarski image of (A), showing DAPI-stained blue nuclei.</p> <p>(C) Merge image showing nuclei and GATA-4 staining. </p> <p>(D) When Spoc cells are incubated with 20 μM isoproterenol for 1 h, the GATA-4 nuclear staining is seen. </p> <p>(E) Nomarski image of (D).</p> <p>(F) Merge of (D) and (E), showing sublocalization of GATA-4 to nuclei. A weaker GATA-4 signal is present in the cytoplasm. </p></div