The phosphorylation of cardiac troponin I (cTnI) plays an important role in the 
contractile dysfunction associated with heart failure. Human cardiac troponin 
I-interacting kinase (TNNI3K) is a novel cardiac-specific functional kinase that 
can bind to cTnI in a yeast two-hybrid screen. The purpose of this study was to 
investigate whether TNNI3K can phosphorylate cTnI at specific sites and to 
examine whether the phosphorylation of cTnI caused by TNNI3K can regulate 
cardiac myofilament contractile function. Co-immunoprecipitation was performed 
to confirm that TNNI3K could interact with cTnI. Kinase assays further indicated 
that TNNI3K did not phosphorylate cTnI at Ser23/24 and Ser44, but directly 
phosphorylated Ser43 and Thr143 in vitro. The results obtained for adult rat 
cardiomyocytes also indicated that enhanced phosphorylation of cTnI at Ser43 and 
Thr143 correlated with rTNNI3K (rat TNNI3K) overexpression, and phosphorylation 
was reduced when rTNNI3K was knocked down. To determine the contractile function 
modulated by TNNI3K-mediated phosphorylation of cTnI, cardiomyocyte contraction 
was studied in adult rat ventricular myocytes. The contraction of cardiomyocytes 
increased with rTNNI3K overexpression and decreased with rTNNI3K knockdown. We 
conclude that TNNI3K may be a novel mediator of cTnI phosphorylation and 
contribute to the regulation of cardiac myofilament contraction function.