(A–C) Immunofluorescence staining of ph-cTnI (green) in Cre-activated/GFP⁺ cells (red staining) in cardiomyocytes of control E10.5 hearts (yellow arrows). (D–F) There was a reduction in ph-cTnI staining in Cre-activated cardiomyocytes in ROCKDN^Gata5-Cre hearts (white arrows) compared with control samples (yellow arrows). n = 3 for each genotype. (G and H) Co-IP using protein extracts from whole heart lysates from E16.5 embryos showed that in control hearts, ROCK1 (158kDa) and ROCK2 (161 kDa), bind to cTnT (40 kDa) (G) and to cTnI (26 kDa) and ssTnI (22 kDa) (H). (I) In ROCKDN^Gata5-Cre mutant hearts, there was a significant reduction in binding between the ROCK proteins and the troponin proteins. Co-IP with TnI showed a significant reduction in expression of ROCK1 and ROCK2, and conversely, in the same samples, co-IP with ROCK1 gave a significant reduction in cTnI and ssTnI expression in ROCKDN^Gata5-Cre mutants. n = 4 for each genotype. (J–X) Primary cardiomyocytes were cultured from E15.5 control and ROCKDN^Gata5-Cre mutants. GFP antibody was used to identify the Cre-activated (Cre+ve) cells (red staining), and cTnI antibody confirmed which cells were cardiomyocytes by labeling sarcomeres (green). Addition of IBMX to control cells did not have any effect (M–O). Cre-activated cardiomyocytes from ROCKDN^Gata5-Cre mutants, lacked sarcomeric structure (P–R). Addition of IBMX rescued the sarcomeric structure in Cre-activated cardiomyocytes from ROCKDN^Gata5-Cre mutants (S–U). Similar to the control samples, Cre^– cells from ROCKDN^Gata5-Cre mutants had normal sarcomeric organization that was not altered following the addition of IBMX (V–X). (Y) The average number of sarcomeres per cardiomyocyte, from a total of 10 cells per well, was determined. There were no significant differences between control Cre⁺ cardiomyocytes with and without IBMX. There was a significant difference in sarcomere number between ROCKDN^Gata5-Cre Cre⁺ cardiomyocytes without IBMX compared with both control samples. Addition of IBMX to ROCKDN^Gata5-Cre Cre⁺ cardiomyocytes significantly increased sarcomeres, to a level that was no longer significantly different relative to both control groups; thus, addition of IBMX rescues the sarcomeric phenotype of ROCKDN^Gata5-Cre Cre⁺ cardiomyocytes. n = 3 for each genotype. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 by unpaired t test (I) and ****P < 0.0001 by 1-way ANOVA with Bonferroni’s correction for multiple comparisons (Y). Scale bars: 50 μm (A–F) and 10 μm (J–X).