Abstract The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of contiguous Tm dimers, we used transgenic mice (TmDM) that expressed a mutant α-Tm in the heart; S229E and H276N substitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse cardiac troponin T mutants (TnT1–44Δ and TnT45–74Δ) that have a divergent effect on the overlapping ends of Tm were employed. The S229E-induced instability in the central region of TmDM altered the overlapping ends of TmDM, thereby it negated the attenuating effect of H276N on Ca2+-activated maximal tension. The rate of cross-bridge detachment (g) decreased in TmDM+TnTWT and TmH276N+TnTWT fibers but increased in TmDM+TnT45–74Δ fibers; however, TnT45–74Δ did not alter g, demonstrating that S229E in TmDM had divergent effects on g. The S229E substitution in TmDM ablated the H276N-induced desensitization of myofilament Ca2+ sensitivity in TmDM+TnT1–44Δ fibers. To our knowledge, novel findings from this study show that the structural instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.