It is poorly understood how mechanical stimuli influence in vivo myocardial remodeling during chronic hemodynamic overload. Combined quantitation of ventricular mechanics and expression of key proteins involved in mechanotransduction can improve fundamental understanding. Adult anesthetized dogs (n = 20) were studied at sinus rhythm (SR) and 0, 3, 10, and 35 days of complete atrioventricular block (AVB). Serial left ventricular (LV) myofiber mechanics were measured. Repeated LV biopsies were analyzed for mRNA and/or protein expression of beta(1D)-integrin, melusin, Akt, GSK3beta, muscle LIM protein (MLP), four-and-a-half LIM protein 2 (fhl2), desmin, and calpain. Upon AVB, increased ejection strain (0.29 +/- 0.01 vs. 0.13 +/- 0.02, SR) and end-diastolic stress (4.8 +/- 1.1 vs. 2.7 +/- 0.4 kPa) dominated mechanical changes. Brain natriuretic peptide plasma levels were correspondingly high (33 +/- 4 vs. 19 +/- 1 pg/ml, SR). beta(1D)-Integrin protein expression increased chronically after AVB. Melusin was temporarily overexpressed (+33 +/- 9%, 3 days AVB vs. SR), followed by elevated ratios of phosphorylated (P)-Akt to Akt and P-GSK3beta to GSK3beta (+26 +/- 6% and +30 +/- 8% at 10 days AVB vs. SR). These changes corresponded to peak hypertrophic growth at 3 to 10 days. MLP increased gradually to maxima at chronic AVB (+36 +/- 7%). In contrast, fhl2 (-22 +/- 3%, 3 days) and desmin (-30 +/- 9%, 10 days AVB) transiently declined but recovered at chronic AVB. Calpain protein expression remained unaltered. In conclusion, volume overload after AVB causes a transient compromise of cytoskeletal integrity based, at least partly, on transcriptional downregulation. Subsequent cytoskeletal reorganization coincides with the upregulation of melusin, P-Akt, P-GSK3beta, and MLP, indicating a strong drive to compensated hypertrophy.