Cardiovascular disease is the highest contributor to morbidity and mortality in type 2-diabetic patients. In the heart, insulin has several effects, including altered energy metabolism, regulation of gene transcription, and activation of ion channels. Recently it has been shown that insulin activates a nonspecific cation current (NSCC) in adult cardiomyocytes (Zhang & Hancox, 2003). This NSCC could also be activated by diacylglycerol (DAG). Since DAG is known to activate some canonical transient receptor potential (TRPC) channels, these channels might be involved in the insulin-mediated NSCC. Here we use the whole cell patch-clamp technique and Western blot analysis to study the possible role of TRPC channels in insulin-mediated NSCC in ventricular cardiomyocytes of adult mice. Moreover, we compare the NSCC and TRPC protein expression in control and insulin resistant, obese (ob/ob) mice. The study was approved by the local ethics committee. Mice were killed by cervical dislocation. Single cardiomyocytes were obtained by enzymatic digestion. Western blot analysis was performed on crude lysate from isolated cardiac ventricles. Our data show that expression of TRPC3, 4 & 6 in heart are down regulated by 38%, 34% and 31%, respectively, in ob/ob compared with control mice (n=6 in each group). Application of the membrane permeable DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced an outward rectifying current both in control and ob/ob cardiomyocytes. This OAG-mediated NSCC was significantly smaller (p<0.05, Student's unpaired t test) in ob/ob mice (mean ± SEM; 0.73 ± 0.25 pA/pF at +50 mV, n=8) compared to controls (2.20 ± 0.77, n=6). The current was inhibited by SK&F96365 (10μM) and 2-aminoethoxydiphenyl borate (2-APB; 30μM), both frequently used to inhibit TRPC channels although their action is not specific. Application of insulin gave a dose-dependent NSCC response, which was not altered in ob/ob cardiomyocytes compared to controls. This insulin-mediated NSCC was insensitive to pretreatment with 2-APB but inhibited by SK&F96365 and abolished when phosphatidylinositol 3-kinase was inhibited by wortmannin (0.5μM). In conclusion, DAG activates an NSCC in cardiomyocytes that is decreased in a mouse type 2-diabetes model. Based on pharmacological dissection and protein expression analysis, this DAG-activated NCSS might be mediated via TRPC channels. On the other hand, the insulin-activated NCSS is not different in diabetic and control cardiomyocytes and not inhibited by 2-APB, which suggests that it does not involve TRPC channels.