Integrins are considered to be an important mechanosensor in cardiac myocytes. To test whether integrins can influence cardiac contractile function, the force-frequency relationships of mouse papillary muscle bundles were measured in the presence or absence of a synthetic integrin-binding peptide, GRGDNP (gly-arg-gly-asp-asn-pro). Results demonstrate that in the presence of a "RGD" containing synthetic peptide, contractile force was depressed significantly by, 28% at 4 Hz, 37.7% at 5 Hz and 20% at 10 Hz (n=6, p<0.01). Treatment of myofibers with either protease generated fragments of denatured collagen (Type I) or denatured collagen, that contain the RGD motif, also reduced force production, significantly. An integrin-activating antibody for 1 integrin inhibited the force similar to synthetic RGD peptide. Function blocking integrin antibodies for 5 and 1 integrins reversed the effect of the RGD containing peptide, and 5 integrin also reversed the effect of proteolytic fragments of denatured collagen on contractile force. Whereas experiments with function blocking antibody for 3 integrin did not reverse the effect of RGD peptide. Force - [Ca2+]i measurements showed that the depressed rate of force generation observed in the presence of the RGD containing peptide was associated with reduced [Ca2+]i. Data analyses further demonstrated that force per unit of Ca2+ was reduced, suggesting that myofilament activation process was altered. In addition, inhibition of PKC enzyme using Ro-32-0432 reversed the activity of RGD peptide on papillary muscle bundles. In conclusion, these data indicate that RGD peptide, acting via 51 integrin, depresses the force production from papillary muscle bundles, partly associated with changes in [Ca2+]i and the myofilament activation processes that is modulated by PKC.