The Calsequestrin (Csq) transgenic mouse model of cardiomyopathy exhibits wide variation in phenotypic progression dependent on genetic background. Seven heart failure modifier (Hrtfm) loci modify disease progression and outcome. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. Strains with the more susceptible phenotype exhibit high transcript levels while less susceptible strains show dramatically reduced transcript levels. This decrease is caused by an intronic SNP in low-transcript strains that activates a cryptic splice site leading to a frameshifted transcript, followed by nonsense-mediated decay of message and an absence of detectable protein. A transgenic animal overexpressing human TNNI3K alone exhibits no cardiac phenotype. However, TNNI3K/Csq double transgenics display severely impaired systolic function and reduced survival, indicating that TNNI3K expression modifies disease progression. TNNI3K expression also accelerates disease progression in a pressure-overload model of heart failure. These combined data demonstrate that Tnni3k plays a critical role in the modulation of different forms of heart disease, and this protein may provide a novel target for therapeutic intervention.