Two α-isoforms of the Na⁺,K⁺-ATPase (α₁ and α₂) are expressed in the cardiovascular system, and it is unclear which isoform is the preferential regulator of contractility. Mice heterozygous for the familial hemiplegic migraine type 2 (FHM2) associated mutation in the α₂-isoform (G301R; α₂^+/G301R mice) have decreased expression of cardiac α₂-isoform but elevated expression of the α₁-isoform. We aimed to investigate the contribution of the α₂-isoform function to the cardiac phenotype of α₂^+/G301R hearts. We hypothesized that α₂^+/G301R hearts exhibit greater contractility due to reduced expression of cardiac α₂-isoform. Variables for contractility and relaxation of isolated hearts were assessed in the Langendorff system without and in the presence of ouabain (1 µM). Atrial pacing was performed to investigate rate-dependent changes. The α₂^+/G301R hearts displayed greater contractility than WT hearts during sinus rhythm, which was rate-dependent. The inotropic effect of ouabain was more augmented in α₂^+/G301R hearts than in WT hearts during sinus rhythm and atrial pacing. In conclusion, cardiac contractility was greater in α₂^+/G301R hearts than in WT hearts under resting conditions. The inotropic effect of ouabain was rate-independent and enhanced in α₂^+/G301R hearts, which was associated with increased systolic work.