Fig. 3.

Cardiac miR-128 deletion promotes postnatal CM proliferation in vivo. a Schematic diagram depicting the generation of cardiac-specific miR-128 knockout (miR-128^−/−) mice. Control mice were miR-128^fl/fl mice, and miR-128^−/− mice were Nkx2.5^Cre; miR-128^fl/fl mice. b The expression level of miR-128 during heart development (n = 6) analyzed by qPCR, including embryonic day 10.5 (E10.5), E14.5, postnatal day 7 (P7), and P28. c Masson trichrome staining of mouse hearts at P7. Scale bars, 2.0 mm. d Comparison of cardiac function between Ctrl and miR-128^−/− hearts analyzed by echocardiography at P7, and measured by EF and FS (n = 6). e Measurement of HW to BW ratio in Ctrl and miR-128^−/− mice (n = 6). f Evaluation of CM size in P7 Ctrl and miR-128^−/− hearts assessed by WGA and cardiac troponin T (cTnT) staining (n = 5 mice, ~250 CMs/heart). Scale bars, 50 µm (yellow); 10 µm (white). g Assessment of CM proliferative activity and sarcomere structure in P7 hearts by immunofluorescence of cTnT and Ki67. Arrows indicate Ki67-positive CMs with sarcomere disassembly. Scale bars, 500 µm (yellow); 25 µm (white). h–j Quantification of Ki67⁺ CMs, sarcomere disassembled CMs and Ki67⁺ disassembled CMs (n = 4860 CMs pooled from six mice). k CM apoptosis analyzed by TUNEL staining. l Schematic diagram depicting the protocol for EdU intraperitoneal (i.p.) injection at P7 mice to label proliferating CMs in vivo. m Analysis of CM proliferation by EdU incorporation assay in Ctrl and miR-128^−/− hearts at P14 (n = 6 mice, ~250 CMs/heart). Scale bars, 50 µm. n Schematic diagram depicting the protocol for EdU intraperitoneal (i.p.) injection at P14 to label proliferating CMs in vivo. o Comparison of EdU⁺ CMs in Ctrl and miR-128^−/− hearts at P21 (n = 6 mice, ~200 CMs/heart). Statistical significance was calculated using Student’s t-test. Data are represented as means ± SEM. *P < 0.05