PKCalpha has been shown to be a negative regulator of contractility and PKCalpha gene deletion in mice protected against heart failure. Small interfering (si)RNAs mediate gene silencing by RNA interference (RNAi) and may be used to knockdown PKCalpha in cardiomyocytes. However, transfection efficiencies of (si)RNAs by lipofection tend to be low in primary cells. To address this limitation, we developed an adenoviral vector (AV) driving short hairpin (sh)RNAs against PKCalpha (Ad-shPKCalpha) and evaluated its potential to silence PKCalpha in neonatal rat cardiac myocytes and in engineered heart tissues (EHTs), which resemble functional myocardium in vitro. A nonsense encoding AV (Ad-shNS) served as control. Quantitative PCR and Western blotting showed 90% lower PKCalpha-mRNA and 50% lower PKCalpha protein in Ad-shPKCalpha-infected cells. EHTs were infected with Ad-shPKCalpha on day 11 and subjected to isometric force measurements in organ baths 4 days later. Mean twitch tension was >50% higher in Ad-shPKCalpha compared to Ad-shNS-infected EHTs, under basal and Ca(2+)- or isoprenaline-stimulated conditions. Twitch tension negatively correlated with PKCalpha mRNA levels. In summary, AV-delivered shRNA mediated highly efficient PKCalpha knockdown in cardiac myocytes and improved contractility in EHTs. The data support a role of PKCalpha as a negative regulator of myocardial contractility and demonstrate that EHTs in conjunction with AV-delivered shRNA are a useful model for target validation.