Modified langendorff-free method for isolating cardiomyocytes and non-cardiomyocytes from adult mice

Figures

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  Fig. 1. The schematic illustrates the principle of the modified method, which enables direct infusion of the digestion buffer into the aorta using a syringe without relying on the Langendorff perfusion system. (A) By inserting a lumbar puncture needle into the ascending aorta and securing it with a suture, the digestion buffer can be delivered externally via a syringe. (B) This allows the buffer to flow through the coronary circulation (blue arrows), ensuring deep perfusion of the myocardium

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  Fig. 2. Summary of the modified cardiomyocyte isolation protocol. A detailed description of the methodology is provided in Video 1

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  Fig. 3. The Modified method successfully isolated adult mouse cardiomyocytes. (A) Representative images of cells isolated using the three methods (after plating) show that ≈ 80% of the cardiomyocytes are rod-shaped, viable, and have well-organized sarcomeric striations. Scale bars = 100 μm. (B) Total number of rod-shaped cardiomyocytes obtained per mouse using the three separation methods (n = 6 mice per group). (C) Statistical comparison of the percentage of rod-shaped cardiomyocytes obtained using the three methods (n = 6 mice per group). Statistical comparison of the yield of fibroblasts obtained using the three methods (n = 6 mice per group). The cell viability from day 0 to day 21 between cardiomyocytes isolated by the Modified method and Foo method (n = 6 mice per group). Phalloidin staining of cardiomyocytes from day 0 to day 21 of culture. Phalloidin (red), DAPI (blue). Scale bar = 40 μm. For (B, C, D), data are presented as means ± SD, p values were calculated by One-Way ANOVA followed by Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.01, ****P < 0.0001. ns, not significant

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  Fig. 4. Modified isolation and culture of adult mouse cardiomyocytes retain functional characteristics. (A) Schematic diagram of the experimental design. Real-time qPCR analysis of Nppa, Nppb, Myh7, Hk2, Slc2a1, and Hif-1a mRNA levels in cardiomyocytes after 24 h hypoxia (n = 3 mice per group). (C-D) Representative Western blot images and quantification of phosphorylated AKT (p-AKT), total AKT, phosphorylated PLN (p-PLN), total PLN after Cardiomyocytes stimulated with 0.1 µM ISO for 30 min (n = 3 independent experiments). Full-length blots/gels are presented in Supplementary file-Orginal Blots Fig. S1A. (E) Real-time qPCR analysis of Nppa, Nppb, Myh7 mRNA levels after 48 h stimulation with 100 µM PE (n = 3 independent experiments). (F-G) Representative Western blot images and quantification of ANP, BNP, and MYH7 expression after 48-h stimulation with 100 µM PE (n = 3 independent experiments). Full-length blots/gels are presented in Supplementary file-Orginal Blots Fig. S1B. For (B, D, E, G), Data are presented as means ± SD, p values were calculated by Two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.1, **P < 0.01, ***P < 0.01, ****P < 0.0001. ns, not significant

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  Fig. 5. Cardiomyocytes isolated by the Modified method and Langendorff method exhibit normal contractile and diastolic functions, calcium handling capacity and electrophysiological properties. (A-B) Representative sarcomere-length tracings from cardiomyocytes stimulated with or without ISO (0.1 µM) for 30 min. (C–G) Sarcomere fractional shortening (C), time to peak (D), time to half contraction (E), and maximal contraction/relaxation rate (E-G) in cardiomyocytes stimulated with or without ISO (0.1 µM) for 30 min (n = 20 cells/ mice, 3 mice per group). (H-I) Representative Ca²⁺ transient tracings from isolated cardiomyocytes in the indicated groups. (J-M) Pacing-induced Ca²⁺ transient amplitude (J), time to peak (K), time to half decay (L), and tau of Ca²⁺ transient decay (M) in isolated cardiomyocytes (n = 20 cells/ mice, 3 mice per group). For (C, D, E, F, J, K, L, M), data are presented as means ± SD, p values were calculated by Two-Way ANOVA followed by Tukey’s multiple comparisons test. For (F, G), data are presented as means ± SD, p values were calculated by unpaired Student’s t test. ***P < 0.01. ns, not significant

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  Fig. 6. Modified isolation, culture, and stimulation of adult mouse cardiac fibroblasts. (A) Representative immunofluorescence images of cardiac fibroblasts after 4 days of culture. Vimentin (VIM, green), CD31 (red), and DAPI (blue). Scale bar, 200 μm. (B) Real-time qPCR analysis of Tnnt2, Pdgfra, Vimentin, and Pecam1 mRNA levels in cardiomyocytes (CM) and cardiac fibroblasts (CF). (C-D) Representative Western blot images and quantification of ACTA2, Col1a1, phosphorylated ERK (p-ERK), total ERK (t-ERK), phosphorylated Smad3 (p-Smad3), total Smad3 (t-Smad3) after Cardiac fibroblasts were incubated with 10 ng/mL TGF-β for 48 h (n = 3 independent experiments). Full-length blots/gels are presented in Supplementary file-Orginal Blots Fig. S1C. (E) Real-time qPCR analysis of ACTA2, Col1a1, Fibronectin and Pdgfra mRNA levels after 48-h TGF-β treatment (n = 3 independent experiments). For (B, D, E), data are presented as means ± SD, p values were calculated by Multiple t tests followed by Holm-Sidak multiple comparisons test. P < 0.1, **P < 0.01, ***P < 0.01