The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size, and regulating its affinity for actin. We hypothesized that altering the -Tm carboxy terminal overlap end to -TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of -TM in which the first 275 residues are from -TM, and the last nine amino acids are from -TM (-TM9aa). Molecular analyses show that endogenous -TM mRNA and protein are nearly completely replaced with -TM9aa. Working heart preparations data show that the rates of contraction and relaxation are reduced in -TM9aa hearts. Left ventricular pressure and time to peak pressure are reduced (-17% and 19%, respectively). The ratio of dP/dt maximum/minimum is increased, but tau is not changed significantly. Force-intracellular calcium, [Ca²⁺]IC measurements from intact papillary fibers demonstrate that -TM9aa TG fibers produce less force per given [Ca²⁺]IC when compared to NTG fibers. Taken together, data demonstrate the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule the overall carbon backbone is perturbed about 1.5 angstrom unit, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in various TM isoforms coiled coil structure are different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and differentially modulating the myofilament activation processes.