Adenovirus-delivered short hairpin RNA targeting contractile function in reconstituted heart PKC alpha improves tissue

Abstract

PKC alpha has been shown to be a negative regulator of contractility and PKCa 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 PKC alpha 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 PKCa (Ad-shPKC alpha) and evaluated its potential to silence PKCa 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 PKC alpha.-mRNA and 50% lower PKC alpha protein in Ad-shPKC alpha-infected cells. EHTs were infected with Ad-shPKCa on day I I and subjected to isometric force measurements in organ baths 4 days later. Mean twitch tension was > 50% higher in Ad-shPKC alpha compared to Ad-shNS-infected EHTs, under basal and Ca2+- or isoprenaline-stimulated conditions. Twitch tension negatively correlated with PKCa mRNA levels. In summary, AV-delivered shRNA mediated highly efficient PKCa knockdown in cardiac myocytes and improved contractility in EHTs. The data support a role of PKC alpha as a negative regulator of myocardial contractility and demonstrate that EHTs in conjunction with AV-delivered shRNA are a useful model for target validation. (c) 2007 Elsevier Inc. All rights reserved.