Two important charge differences between the α‐ and β‐tropomyosin (TM) isoforms are the exchange of a serine residue in the inner‐core region at position 229, and a histidine residue at the carboxy‐terminal end at position 276, with glutamic acid and asparagine, respectively. We have recently shown that altering these two residues in α‐TM to their β‐TM counterparts in transgenic (TG) mouse hearts causes a depression in both +dP/dt and −dP/dt and a decrease in calcium sensitivity. In this study, we address whether independent charge changes at these two residues in α‐TM modulate cardiac function differentially. To test this hypothesis we generated two TG lines: α‐TMSer229Glu and α‐TMHis276Asn. Molecular analyses show that 98% of native α‐TM is replaced by mutated protein in α‐TM229 hearts whereas α‐TM276 hearts show 82% replacement with the mutated protein. Isolated working heart data show that α‐TM229 TG hearts exhibit a significant decrease in both +dP/dt (7%) and −dP/dt (8%) compared with nontransgenics (NTGs) and time to peak pressure (TPP) is also reduced in α‐TM229 hearts. α‐TM276 hearts show a decrease only in −dP/dt (14%) and TPP is increased. pCa2+–tension relationships in skinned fibre preparations indicate decreased calcium sensitivity in α‐TM229 but no change in α‐TM276 preparations. Force–[Ca2+]IC measurements from intact papillary fibres indicate that α‐TM276 fibres produce more force per given [Ca2+]IC when compared to NTG fibres, while α‐TM229 fibres produce less force per given [Ca2+]IC. These data demonstrate that changing charged residues at either the inner‐core domain or the carboxyl end of TM alters sarcomeric performance differently, suggesting that the function of TM is compartmentalized along its length.