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 4

Delayed maturation of cardiomyocytes in ROCKDNGata5-Cre mutants.

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Delayed maturation of cardiomyocytes in ROCKDNGata5-Cre mutants. (A–E) T...
(A–E) The pattern of α-SMA expression was investigated by immunohistochemistry and visualized by DAB staining, and a representative image of the left ventricular wall, from each age, is shown. In control hearts at E15.5 and E17.5, α-SMA became restricted to the compact myocardium and was no longer expressed in trabeculae (arrows in A and B). Postnatally, expression was further restricted to the smooth muscle of coronary arteries, with no cardiomyocyte staining by P7 (C–E). In contrast, in ROCKDNGata5-Cre mutants, α-SMA staining persisted in trabeculae at E15.5 and E17.5 (arrows in F and G), and there were patches of cardiomyocyte expression postnatally (arrows in H–J). (K) The level of α-SMA expression was measured by ImageJ in trabeculae and in the compact myocardium. The total area of trabeculae or the compact myocardium was measured throughout the heart, and the percentage of this area in which α-SMA was expressed was calculated. This confirmed that α-SMA expression was retained longer in trabeculae in ROCKDNGata5-Cre mutants, as there were significantly higher levels of α-SMA expression at E15.5 and E17.5 in trabeculae of mutants compared with trabeculae of littermate controls. Furthermore, in control hearts at E15.5, there was a significant difference in α-SMA expression levels between trabeculae and the compact myocardium (blue line), confirming, as expected, that the expression was becoming restricted to the compact myocardium. In contrast, in ROCKDNGata5-Cre mutants, there was no significant difference between expression levels in trabeculae and compact myocardium (purple line; NS). By E17.5, there was a significant difference in expression levels in the compact myocardium between controls and mutants, showing that, as expected, expression in control hearts was beginning to decrease in the compact myocardium as the cardiomyocytes matured and that this process was delayed in ROCKDNGata5-Cre mutants. n = 8 for E15.5 and n = 6 for E17.5 for each genotype. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 by 1-way ANOVA with Bonferroni’s correction for multiple comparisons. lv, left ventricle. Scale bars: 100 μm.