b-adrenergic stimulation modulates cardiac contractility through protein kinase A (PKA), which phosphorylates cellular proteins, such as cardiac troponin I (cTnI) and cardiac C-protein (cMyBP-C). The relative contributions of cTnI and cMyBP-C to the regulation of myofilaments Ca²⁺ sensitivity are still controversial. In the present work we studied the PKA effect on myofilament Ca²⁺ sensitivity of left ventricular skinned myocytes isolated from young (5-weeks old) and old (55-weeks old) wild type mice (WT) and cMyBP-C deficient mice (KO) at two sarcomere lengths (SL: 1.9 and 2.3 mm). Without PKA stimulation and at the shorter SL, Ca²⁺ sensitivity was higher in KO than in WT. The difference disappeared at the longer SL. No difference in passive tension or maximal active tension was observed. PKA stimulation induced a desensitization of WT myofilaments at both SL but had almost no effect on young and old KO myofilaments. The results suggest that cMyBP-C contributes to the regulation of cardiac contraction and that TnI phosphorylation alone by PKA was not sufficient to induce myofilament desensitization.