Myosin binding protein C (MybPC) mutations represent a major cause of hypertrophic cardiomyopathy in humans. Enhanced biomechanical load plays an important role during the development of myocardial hypertrophy. Muscle LIM protein (MLP) is thought to act as a biomechanical stress-sensor. We generated MybPC and MLP double-knockout (Dko) mice and determined their cardiac phenotype at 9 months of age. Double-heterozygous mice were not different from WT mice and served as controls. LV end-systolic and end-diastolic diameters and posterior wall thickness were increased and shortening fraction was decreased in homozygous MybPC k.o. mice, irrespective of their MLP genotype. Likewise, skinned fiber analysis showed that myofilament Ca2+ sensitivity was equally enhanced in homozygous MybPC k.o. mice, irrespective of their MLP genotype. However, myocyte cross-sectional area was reduced in Dko myocardial sections, compared to MybBP k.o./MLP het mice (P<0.001) and RV papillary muscles of MybPC k.o./MLP het mice (n=11) exhibited a negative force-frequency relationship, while that of Dko littermates (n=6) was flat (P=0.028). Myocyte hypertrophy and in-vitro contractile dysfunction appear to be less pronounced if MybPC deficiency is accompanied by complete MLP deficiency. Thus, the disruption of the mechanosensor by MLP knockout appears to exert a protective effect.