An optimized Langendorff-free method for isolation and characterization of primary adult cardiomyocytes

Figures

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  Fig. 1. Cardiomyocytes post-isolation. A) Brightfield image of isolated cardiomyocytes after plating. B) Confocal imaging of isolated cardiomyocytes stained with the cell membrane marker wheat germ agglutinin (WGA) (colored purple) and the nuclei marker DAPI (colored blue). C) Confocal imaging of isolated cardiomyocytes stained with the cell membrane and T-tubule marker Di-8-ANEPPS

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  Fig. 2. Analysis of mitochondria morphology, lipid droplet accumulation and autophagy in isolated cardiomyocytes using TEM. A) TEM images of cardiomyocytes isolated from mice that were untreated or treated with LPS (5 mg/kg). Red arrows show autophagic events, the orange arrow shows a lipid droplet, and green arrows show mitochondria with disorganized cristae. Images are representative for n = 3 mice/group. B) Mitochondrial area per 50um2. C) Percentage of cristae occupancy relative to total mitochondria area. D) Number of mitochondria per 50um2.E) Percentage of mitochondria with disorganized cristae relative to the number of mitochondria per 50um2. F) Lipid droplet area per 50um2G) Number of autophagic events per 50um2. In B-C,values were expressed as mean ± SD and analyzed by a Mann-Whitney U Test. In D-G,values were expressed as mean ± SD and analyzed by a Student’s t-test. Significance was determined by a p-value < 0.05. A value of < 0.05 is indicated by a single asterisk (*), while values of < 0.005, < 0.0005, and < 0.00005 aredenoted by two, three, and four asterisks (**, ***, ****), respectively

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  Fig. 3. Assessment of mitochondrial membrane potential in isolated cardiomyocytes. (A) Confocal imaging of JC-1 in in untreated and LPS treated cardiomyocytes (10ng/ml). The scale bar in the upper left panel equals 100 μm and is valid for all panels. Images are representative of 50–70 cardiomyocytes analyzed from n = 3 mice/group. (B) Quantification of JC-1 fluorescence in untreated and LPS treated cardiomyocytes. Values were expressed as mean ± SD and analyzed by a Mann-Whitney U Test. Significance was determined by a p-value < 0.05. **** indicates p < 0.00005

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  Fig. 4. Analysis of ROS production in isolated cardiomyocytes. (A) Confocal imaging of MitoSOX Red and MitoTracker Green in untreated and LPS treated (10 ng/ml) cardiomyocytes. The scale bar in the upper left panel equals 100 μm and is valid for all panels. Images are representative of 50–70 cardiomyocytes analyzed from n = 3 mice/group. (B) Quantification of MitoSOX Red fluorescence in untreated and LPS treated cardiomyocytes. Images are representative of 50–70 cardiomyocytes analyzed from n = 3 mice/group. Values were expressed as mean ± SD and analyzed by a Mann-Whitney U Test. Significance was determined by a p-value < 0.05. **** indicates p < 0.00005

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  Fig. 5. Setup for cardiomyocyte isolation. (A) Surgical tools needed for heart excision, including a surgical table, a 5 ml syringe containing ice-cold EDTA buffer, tweezers, sharp-tipped scissors, Vannas scissors, and curved-ended Reynolds hemostatic forceps. (B) EDTA and digestion buffers in a 37 °C water bath for preparation. (C) Injection needles marked with nail polish to ensure penetration depth of only 3 mm into the heart. (D) A custom-made holder securely positions the injection needle and tubing. (E) An overview of the complete perfusion system

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  Fig. 6. Animal surgical procedure. (A) Chest cavity opened, exposing the heart; (B) Injection of EDTA buffer into the right ventricle; (C) Clamping the aorta

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  Fig. 7. Isolation of cardiomyocytes from the excised heart. A) Excised heart connected to the perfusion system via intraventricular injection. B) Heart tissue disassociation. C) Pellet of healthy cardiomyocytes

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  Fig. 8. Analysis of mitochondria area and cristae surface area from TEM images. (A) Calculating mitochondria area. (B) Calculating area unoccupied by mitochondrial cristae