Advanced Single-Cell Mapping Reveals that in hESC Cardiomyocytes Contraction Kinetics and Action Potential Are Independent of Myosin Isoform

Figures

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  Figure 1. hESC-CM Cultivation and Cell-Mapping Technique (A) Differentiation, enrichment, and cultivation strategy for hESC-CMs. Continuous line, cultivation of hESC-CMs in CBs; dashed line, cultivation of CMs plated on coverslips. (B) From left to right: schematic representation of measurement of electrically stimulated contraction or action potential (AP) of a single CM in experimental “setup 1”, of coordinates of mapped CMs in field of view (blue diamonds), and of sample recordings of twitch contraction or action potential. Reference points for cell-mapping technique (light-blue crosses) and the example cell (orange circle) mapped at the coordinates shown in the displays of the calipers of “setup 1.” This cell is retrieved in the subsequent analysis steps. (C) Scheme of FISH labeling of MYH6 and MYH7-mRNAs, of “setup 2” for FISH analysis, and of new coordinates of cells due to translation and rotation of the coverslip (purple arrow) upon mounting on another microscope. New positions of the CMs (magenta circles), old positions of CMs in the background (blue diamonds), and the actual positions of the two reference points (magenta crosses). Right: four images showing sample cell in three…

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  Figure 2. Heterogeneous Expression of MyHC and MLC2 Isoforms in hESC-CMs (A–D) Immunostaining of (A) plated hESC-CMs, (B) CMs from CBs, and (C) whole CB against β-MyHC (red) and α-MyHC (green). Scale bars, 50 μm. Insets show single CMs (A and B; scale bars, 5 μm). (D) Myofibrils in CBs (arrows) with different MyHC compositions. Scale bars, 5 μm. (E and F) Plated hESC-CMs (E) and CMs from CBs (F) immunostained against MLC2v (magenta) and MLC2a (cyan). Scale bars, 50 μm. Insets show single CMs with different MLC2 compositions. Scale bars, 5 μm. DAPI (blue) staining of nuclei. See also Figures S1, S2, and S3.

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  Figure 3. Heterogeneous α-MyHC versus β-MyHC Protein Expression from Cell to Cell (A) Sample hESC-CMs representing the different categories from pure α-MyHC- to pure β-MyHC-positive CMs with three categories of CMs with mixed α/β-MyHC expression (top to bottom). Left column: merged images of single CMs with marked regions of interest. Scale bars, 10 μm. Middle columns: zoomed regions of sarcomere striation pattern with fluorescent labeling specific for α-MyHC (green) and β-MyHC (red). Scale bars, 5 μm. Right column: merged images (blue, DAPI). Scale bars, 5 μm. Color code indicates protein fluorescence category (α, α > β, α = β, β > α, β) of each displayed CM. (B) Fractions of total number (n) of individual CMs in the different categories of MyHC protein isoform expression as revealed by fluorescent labeling for plated hESC-CMs and hESC-CMs from CBs. Each column represents an independently differentiated batch (Diff.) of hESC-CMs. Each CM was categorized as shown above and explained in Figure S3.

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  Figure 4. FISH Analysis of MYH6-mRNA versus MYH7-mRNA Expression Reveals Highly Variable Fractions and Absolute Counts of Both MYH-mRNAs from Cell to Cell Fractions of (A) MYH6-mRNA (green) and (C) MYH7-mRNA (red) in individual plated hESC-CMs (A) and hESC-CMs from CBs (C). Black-line segments on top of the bar chart indicate individual coverslips on which the cells were analyzed. Absolute counts of MYH6-mRNA spots (green) and MYH7-mRNA spots (red) for the same individual plated hESC-CMs (B) as in (A) and for the same hESC-CMs from CBs (D) as in (C). Dashed lines indicate median values for absolute counts of MYH6 (green) and MYH7 (red). For plated hESC-CMs, n = 61; for hESC-CMs from CBs, n = 49. See also Figure S4.

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  Figure 5. No Strict Relationship between MYH6/MYH7-mRNA and α/β-MyHC Protein Expression per Individual hESC-CM Examples of retrieved hESC-CMs with different MYH-mRNA (left; MYH6-mRNA, green; MYH7-mRNA, red; the same single focal plane for both MYH-mRNAs is shown) and MyHC protein isoform expression (right; α-MyHC, green; β-MyHC, red). (A) Left: single CM with exclusively MYH7-mRNA spots; no MYH6-mRNA specific spots were detected. This hESC-CM showed specific sarcomere staining for β-MyHC isoform only (right). Scale bars, 5 μm; see also zoomed region of interest. (B) Left: CM with both MYH6-mRNA and MYH7-mRNA spots, but specific sarcomere staining for β-MyHC isoform only (right). Scale bars, 5 μm. (C) Left: CM with both MYH6-mRNA and MYH7-mRNA spots and co-expression of both MyHC isoforms (right). Scale bars, 5 μm. (D and E) Fraction of MYH7-mRNA for hESC-CMs with different MyHC protein expression (ranging from pure α-MyHC- to pure β-MyHC-positive CMs with three categories of CMs with mixed α/β-MyHC expression, α > β, α = β, β > α) for plated hESC-CMs (n = 61; D), and hESC-CMs from CBs (n = 49; E). Horizontal lines and numbers above columns indicate mean values. See also Figure S4.

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  Figure 6. Parameters of Twitch Contraction Are Independent of α/β-MyHC Isoform Expression in Individual hESC-CMs (A) Normalized averaged traces of original recordings for twitch contraction of plated CMs (black) and CMs from CBs (gray). (B–D) Time to peak (ttp) of twitch (B), maximum contraction velocity of twitch (Vmax) (C), and half-relaxation time (hrt) of twitch (D) for plated CMs (dark-gray circles) and CMs from CBs (light-gray circles). (E–J) ttp of twitch (E and H), Vmax of twitch (F and I), and hrt of twitch (G and J) of the individual retrieved CMs in the different categories of pure α-MyHC- and pure β-MyHC-positive CMs, with three categories of CMs with mixed α/β-MyHC expression (α > β, α = β, α < β) of plated CMs (n = 90; left column) and CMs from CBs (n = 36; right column). Horizontal lines in (B) to (D), median ± interquartile range (IQR); in (E) to (J), mean values are indicated (horizontal lines and numbers above columns). Statistical significance was tested by Mann-Whitney U tests with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figures S3 and S6.

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  Figure 7. AP Properties Are Independent of α/β-MyHC Isoform Expression in Individual hESC-CMs (A) Representative patch-clamp recordings of APs from hESC-CMs. Upper traces: spontaneous APs. Lower traces: APs evoked by intracellular stimulation from resting membrane potentials (RMP) around −80 mV. Arrows mark stimulus artifacts. Dashed lines indicate 0 mV. All traces recorded at room temperature. (B–E) RMP from CMs (B) spontaneously generating APs (n = 49 for plated CMs; n = 56 for CMs from CBs) and from CMs with evoked APs at physiological resting conditions (with RMP about −80 mV, +Ihold) as adapted by negative holding currents (n = 78 for plated CMs; n = 61 for CMs from CBs). APD50 (C), AP amplitudes (D), and AP upstroke velocities (E) from CMs spontaneously generating APs and from CMs with evoked APs (same cell numbers as in B; dotted lines in C indicate APD50 values of 20 ms and 200 ms). (F and G) APD50 of the individual retrieved CMs in the different categories of pure α-MyHC- and pure β-MyHC-positive CMs, with three categories of CMs with mixed α/β-MyHC expression (α > β, α = β, α < β). (F) Plated CMs with evoked APs (n = 38) and (G) CMs from CBs with evoked APs (n = 27). Scatterplots…