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Aims: Germ-line mutations in genes encoding components of the RAS/mitogen-activated protein kinase (MAPK) pathway cause developmental disorders called RASopathies. Hypertrophic cardiomyopathy (HCM) is the most common myocardial pathology and a leading cause of death in RASopathy patients. KRAS mutations are found in Noonan and cardio-facio-cutaneous syndromes. KRAS mutations, unlike mutations of RAF1 and HRAS, are rarely associated with HCM. This has been attributed to the fact that germ-line KRAS mutations cause only a moderate up-regulation of the MAPK pathway. Highly bioactive KRAS mutations have been hypothesized to cause severe cardiomyopathy incompatible with life. The aim of this study was to define the impact of KRAS(G12D) expression in the heart., Methods and Results: To generate mice with endogenous cardiomyocyte-specific KRAS(G12D) expression (cKRAS(G12D) mice), we bred mice with a Cre-inducible allele expressing KRAS(G12D) from its endogenous promoter (Kras2(LSL)) to mice expressing Cre under control of the cardiomyocyte-specific α-myosin heavy chain promoter (αMHC-Cre). cKRAS(G12D) mice showed high levels of myocardial ERK and AKT signalling. However, surprisingly, cKRAS(G12D) mice were born in Mendelian ratios, appeared healthy, and had normal function, size, and histology of the heart., Conclusion: Mice with cardiomyocyte-specific KRAS(G12D) expression do not develop heart pathology. These results challenge the view that the level of MAPK activation correlates with the severity of HCM in RASopathies and suggests that MAPK-independent strategies may be of interest in the development of new treatments for these syndromes.

Aims: Several mutations in the ventricular myosin regulatory light chain (RLC) were identified to cause familial hypertrophic cardiomyopathy (FHC). Based on our previous cellular findings showing delayed calcium transients in electrically stimulated intact papillary muscle fibres from transgenic Tg-R58Q and Tg-N47K mice and, in addition, prolonged force transients in Tg-R58Q fibres, we hypothesized that the malignant FHC phenotype associated with the R58Q mutation is most likely related to diastolic dysfunction., Methods and Results: Cardiac morphology and in vivo haemodynamics by echocardiography as well as cardiac function in isolated perfused working hearts were assessed in transgenic (Tg) mutant mice. The ATPase-pCa relationship was determined in myofibrils isolated from Tg mouse hearts. In addition, the effect of both mutations on RLC phosphorylation was examined in rapidly frozen ventricular samples from Tg mice. Significantly, decreased cardiac function was observed in isolated perfused working hearts from both Tg-R58Q and Tg-N47K mice. However, echocardiographic examination showed significant alterations in diastolic transmitral velocities and deceleration time only in Tg-R58Q myocardium. Likewise, changes in Ca(2+) sensitivity, cooperativity, and an elevated level of ATPase activity at low [Ca(2+)] were only observed in myofibrils from Tg-R58Q mice. In addition, the R58Q mutation and not the N47K led to reduced RLC phosphorylation in Tg ventricles., Conclusion: Our results suggest that the N47K and R58Q mutations may act through similar mechanisms, leading to compensatory hypertrophy of the functionally compromised myocardium, but the malignant R58Q phenotype is most likely associated with more severe alterations in cardiac performance manifested as impaired relaxation and global diastolic dysfunction. At the molecular level, we suggest that by reducing the phosphorylation of RLC, the R58Q mutation decreases the kinetics of myosin cross-bridges, leading to an increased myofilament calcium sensitivity and to overall changes in intracellular Ca(2+) homeostasis.