Fig 1.
Cardiomyocyte viability and morphology.
(A) Schematic of experimental design. Three pairs of pigs were subjected to either Langendorff perfusion or tissue slicing-assisted digestion (TSAD) for the isolation of cardiomyocytes from the left ventricle (LV), right ventricle (RV) and left atrial appendage (LAA). Isolated cells were subjected to a series of evaluations. (B) Representative images of cardiomyocytes stained using calcein-AM and EthD-1 following isolation. Scale bar = 100 μm. (C) Quantification of the percentage of viable cardiomyocytes in (B). (D) Quantification of the percentage of rod-shaped cardiomyocytes from (B). (E) Quantification of cell length in (B). Data are mean ± SEM. ** P < 0.01, two-way ANOVA, compared to Langendorff.
Fig 2.
Cardiomyocyte structure and ultrastructure.
(A) Immunofluorescence staining of ACTN2 (red) and DAPI (blue) in isolated porcine cardiomyocytes. Scale bar = 10 μm. (B) Quantification of sarcomere length in (A). (C) Transmission electron microscopy (TEM) of porcine CMs. Scale bar = 2 μm (upper panel); = 500 nm (lower panel). Mitochondrial size and mitochondrial area fraction. (D) were quantified from 3 independent experiments. Data are mean ± SEM. * P < 0.05, two-way ANOVA, compared to Langendorff.
Fig 3.
RNA-seq of isolated cardiomyocytes.
(A) Volcano plots of DEGs between Langendorff and TSAD-isolated porcine CMs. Cyan, genes upregulated in TSAD group; violet, genes upregulated in Langendorff group. (B) Venn diagram showing the number of shared and distinct genes between Langendorff and TSAD groups within the top 500 expressed genes for LV, RV and LAA, respectively. (C) Functional enrichment (KEGG) of shared and distinct genes between cardiomyocytes isolated by Langendorff or TSAD. (D) qPCR validation of cardiomyocyte contraction-, fatty acid metabolism-, and ion channel-related genes. Data are mean ± SEM.
Fig 4.
Action potential recordings of porcine cardiomyocytes.
(A) Whole-cell patch clamping of freshly isolated porcine CMs. (B) Quantification of action potentials. Action potential parameters, including action potential amplitude (APA), peak, rate of membrane depolarization (dV/dtmax), resting membrane potential (RMP), and action potential durations at 30% (APD30), 50% (APD50), 70% (APD70) and 90% (APD90) repolarization, were quantified. Data are mean ± SEM. * P < 0.05, ** P < 0.01, two-way ANOVA, compared to Langendorff.