Journal: iScience

Article Title: α- and β-myosin II can be non-uniformly distributed within the cardiac sarcomere

doi: 10.1016/j.isci.2025.112233

Figure Lengend Snippet: α- and β-myosin II can exhibit unique distributions within cardiac sarcomeres (A) Representative example of IF of endogenous β-myosin II (top) and expression of mEGFP- MYH6 (middle) in a hiCM; bottom shows overlay. Scale bars: left 10 μm, right 2 μm. (B) Line scan from yellow boxes in (A). (C) Average of all line scans (20 cells, 772 sarcomeres, 3 independent experiments). Lines show population-averaged mean value while shaded bands show ± standard error of the mean. (D) Top: expression of mNeon- MYH7 and IF of endogenous α-myosin II in a representative hiCM. Bottom: Average of all line scans (15 cells, 456 sarcomeres, 3 independent experiments). Lines show population-averaged mean value while shaded bands show ± standard error of the mean. White brackets denote individual filament stacks. Scale bar: 2 μm. (E) Distance each myosin II extended from filament center in hiCMs (β-myosin II IF: 14 cells, 280 measurements; α-myosin II IF: 15 cells, 300 measurements; mNeon- MYH7 (β expressed): 15 cells, 300 measurements; mEGFP- MYH6 (α expressed): 15 cells, 300 measurements). p -values in grey (Welch's t-test). (F) Simplified model of the potential arrangement of β-myosin II (magenta) and α-myosin II (green) in thick filaments of iPSC-derived cardiac myocyte sarcomeres. (G) Left: IF using antibodies against β-myosin II (top) and α-myosin II (bottom) in different sections of human ventricle; right: distance the fluorescent signal of each myosin extended from the filament center (β-myosin II: 280 measurements; α-myosin II: 321 measurements). Scale bar: 2 μm. p -values in grey (Welch's t-test). (H) Left: individual β-myosin II (ventricle) and α-myosin II (atrium) whole-mount immunofluorescence stains in 72 h post-fertilization zebrafish embryos; right: distance the fluorescent signal of each myosin extended from the filament center (β-myosin II: 14 embryos, 222 measurements; α-myosin II: 13 embryos, 102 measurements). White brackets in each image denote individual filament stacks. Scale bar: 2 μm. p -values in grey (Welch's t-test); (I) AlphaFold3-predicted model of α-myosin II/β-myosin II heavy chain ( MYH6 / MYH7 genes, respectively) motors + motor/lever arms, modeled with MYL2 / MYL3 (gray), which encode the ventricular essential/regulatory myosin light chains (a similar AlphaFold3-predicted model, but modeled with atrial essential/regulatory myosin light chains instead of ventricular light chains, is shown in ). N1, 2, and 3 in (E), (G), and (H) represent individual biological replicates, color-coded by replicate.

Article Snippet: In brief, the hiCMs were thawed and plated in 100uL per well Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics; catalog #R1151) at a density of 50,000 cells per well in 96-well polystyrene cell-culture plates pre-coated for 2 h with gelatin (EMD Millipore; catalog # ES-006-B).

Techniques: Expressing, Derivative Assay, Immunofluorescence