Mechanical and biochemical alterations were investigated in permeabilized cardiomyocytes along with the progression of dilated cardiomyopathy (DCM) in a transgenic mouse line overexpressing the activated Gαq protein (Tgαq*44). The isometric force, its Ca 2+ sensitivity (pCa 50 ) and the turnover rate of the actin–myosin cycle (k tr ) were determined at sarcomere lengths (SLs) of 1.9 μm and 2.3 μm before (at 4 and 10 months of age) and after hemodynamic decompensation (at 14 and 18 months of age) in Tgαq*44 cardiomyocytes and in age-matched control cardiomyocytes. The SL-dependence of pCa 50 was not different in Tgαq*44 and control hearts. In contrast, a significant increase in pCa 50 was observed in the Tgαq*44 cardiomyocytes (ΔpCa 50 : 0.10–0.15 vs. the controls) after 10 months of age that could be diminished by exposures to the catalytic subunit of protein kinase A (PKA). Accordingly, a decline in endogenous PKA activity and decreased troponin I phosphorylation were detected after 10 months in the Tgαq*44 hearts. Finally, the maximal Ca 2+ -activated force (F o ) and k tr were lower and the passive force (F passive ) was higher at 18 months in the Tgαq*44 cardiomyocytes compared to the control. These mechanical alterations were paralleled by a robust increase in β-myosin heavy chain expression in the Tgαq*44 hearts. In conclusion, our data suggested that an initial decrease of PKA signaling and subsequent changes in myofilament protein expression may contribute to the development of dilated cardiomyopathy in Tgαq*44 hearts.