Disruption of embryonic ROCK signaling reproduces the sarcomeric phenotype of hypertrophic cardiomyopathy
Kate E. Bailey, Guy A. MacGowan, Simon Tual-Chalot, Lauren Phillips, Timothy J. Mohun, Deborah J. Henderson, Helen M. Arthur, Simon D. Bamforth, Helen M. Phillips
Kate E. Bailey, Guy A. MacGowan, Simon Tual-Chalot, Lauren Phillips, Timothy J. Mohun, Deborah J. Henderson, Helen M. Arthur, Simon D. Bamforth, Helen M. Phillips
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Research Article Cardiology

Disruption of embryonic ROCK signaling reproduces the sarcomeric phenotype of hypertrophic cardiomyopathy

Abstract

Sarcomeric disarray is a hallmark of gene mutations in patients with hypertrophic cardiomyopathy (HCM). However, it is unknown when detrimental sarcomeric changes first occur and whether they originate in the developing embryonic heart. Furthermore, Rho kinase (ROCK) is a serine/threonine protein kinase that is critical for regulating the function of several sarcomeric proteins, and therefore, our aim was to determine whether disruption of ROCK signaling during the earliest stages of heart development would disrupt the integrity of sarcomeres, altering heart development and function. Using a mouse model in which the function of ROCK is specifically disrupted in embryonic cardiomyocytes, we demonstrate a progressive cardiomyopathy that first appeared as sarcomeric disarray during cardiogenesis. This led to abnormalities in the structure of the embryonic ventricular wall and compensatory cardiomyocyte hypertrophy during fetal development. This sarcomeric disruption and hypertrophy persisted throughout adult life, triggering left ventricular concentric hypertrophy with systolic dysfunction, and reactivation of fetal gene expression and cardiac fibrosis, all typical features of HCM. Taken together, our findings establish a mechanism for the developmental origin of the sarcomeric phenotype of HCM and suggest that variants in the ROCK genes or disruption of ROCK signaling could, in part, contribute to its pathogenesis.

Authors

Kate E. Bailey, Guy A. MacGowan, Simon Tual-Chalot, Lauren Phillips, Timothy J. Mohun, Deborah J. Henderson, Helen M. Arthur, Simon D. Bamforth, Helen M. Phillips

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Figure 3

Reduced proliferation of cardiomyocytes followed by increased cardiomyocyte hypertrophy leads to abnormal ventricular wall development in ROCKDNGata5-Cre mutants.

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Reduced proliferation of cardiomyocytes followed by increased cardiomyoc...
(A and B) Transverse sections from control and ROCKDNGata5-Cre mutant embryos collected from mothers injected with BrdU were stained with BrdU antibody to label proliferating cells (green staining) and GFP antibody to stain Cre-activated cells (red staining). The total number of Cre-activated cardiomyocytes and total number of proliferating Cre-activated cardiomyocytes, in which ROCK was downregulated, were determined in each sample, from which the percentage of proliferating Cre-activated cardiomyocytes was calculated. A representative image of the left ventricle from E11.5 embryos is shown. (C) No significant difference in the percentage of proliferating Cre-activated cardiomyocytes was detected in the left or right ventricle at E10.5. At E11.5 there was a significant decrease in the percentage of proliferating Cre-activated cardiomyocytes in ROCKDNGata5-Cre mutants, in both the left and right ventricles compared with controls. n = 6 for each genotype at both time points. (D) No significant difference in cardiomyocyte proliferation was identified in ROCKDNGata5-Cre hearts at either E15.5 or E16.5. n = 3 for each genotype at both time points. (E) The average number of cardiomyocytes in the compact myocardium, per field of view, was calculated at E15.5, E16.5, and E17.5 and showed a significant decrease in the number of cardiomyocytes in ROCKDNGata5-Cre hearts at E16.5 and E17.5 in both the left and right ventricle, compared with controls. n = 3 for each genotype at all time points. (F and G) Wheat germ agglutinin (WGA) immunofluorescence was used to determine cardiomyocyte cell size in control and ROCKDNGata5-Cre hearts. The cardiomyocytes in ROCKDNGata5-Cre hearts (G) were visibly larger than those in control hearts (F). In ROCKDNGata5-Cre hearts, both Cre-activated/GFP+ (GFP+ve) (green staining) (example cell outlined in yellow in H–J) and GFP– (GFP-ve; example cell outlined in white cell in H–J) cardiomyocytes were hypertrophic. (K and L) In ROCKDNGata5-Cre hearts, the cardiomyocytes in the compact myocardium were significantly larger compared with control hearts, from E16.5 to P21 in the left ventricle (K) and from E15.5 to P7 in the right ventricle (L). n=5 for E15.5 and E16.5; n = 4 for E17.5 and P21; n = 3 for P0, P4, and P7 for each genotype. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA with Bonferroni’s correction for multiple comparisons. lv/LV, left ventricle; RV, right ventricle. Scale bars: 100 μm (A and B), 50 μm (F and G), and 20 μm (H–J).