Brief communicationAdenovirus-delivered short hairpin RNA targeting PKCα improves contractile function in reconstituted heart tissue
Introduction
The PKC family of Ca2+ and/or lipid-activated serine–threonine kinases functions downstream of many membrane-associated signal transduction pathways [1]. In the human heart, PKCα was shown to be the dominantly expressed conventional PKC isoform [2]. PKCα has been recently identified as an important regulator of cardiac contractility and Ca2+ handling [3]. PKCα gene-deleted mice were shown to be hypercontractile, whereas transgenic mice overexpressing PKCα were hypocontractile. Furthermore, enhancement in cardiac contractility associated with PKCα knockout or with pharmacological PKCα inhibition protected against heart failure in several experimental models [2], [3]. These data suggest that inhibition of PKCα may serve as a novel therapeutic strategy for enhancing cardiac contractility in heart failure.
Given the limited specificity of current small molecule inhibitors of PKC isoforms, RNA interference (RNAi)-mediated gene silencing [4] could serve as an alternative approach. RNAi utilizes sequence-specific double-stranded small interfering RNA (siRNA) to silence gene expression in mammalian cells [5]. To overcome notoriously low transfection efficiencies of siRNAs in primary cardiac cells and even more in intact heart tissues, we developed a novel adenoviral vector (AV) driving transcription of short hairpin RNA (shRNA). Its efficacy was tested both in neonatal rat cardiac myocytes and three-dimensional engineered heart tissues (EHT) to monitor contracile consequences of the shRNA-mediated gene manipulation.
Section snippets
Cell culture and transfection
Neonatal rat cardiac myocytes (NRCM) were isolated from 1–3 day old neonates as described previously [6]. NRCM were infected with Ad-EGFP 48 h before transfection as described earlier [6]. NIH/3T3 cells (mouse fibroblasts) were grown in DMEM supplemented with 10% FCS and transfected at 50–80% confluency. Transfection of NIH/3T3 and NRCM was carried out using Polyfect (Qiagen) with 3′-rhodamine-labeled control siRNA (UUCUCCGAACGUGUCACGUdTdT, Qiagen) or 3′-rhodamine-labeled EGFP-22 siRNA
Feasibility of RNAi in cardiac myocytes using siRNA transfection
NRCM were infected with a GFP-only AV (MOI 3, 24 h) followed by Polyfect-mediated transfection of rhodamine-labeled siRNA targeting GFP. Several conditions including different siRNA (0.6–15 μg), Polyfect amounts (10–20 μl) and incubation times (24–48 h) were tested. However, neither condition allowed GFP silencing in NRCM (Fig. 1A), likely because the siRNA was not taken up in NRCM (intracellular rhodamine-signal after 8 h; Fig. 1B, top). The general feasibility of the siRNA approach was
Discussion
RNAi is the process of sequence-specific, posttranscriptional gene silencing mediated by double-stranded RNA [4], [5]. Target validation using RNAi requires efficient siRNA introduction into cells. A limitation for the use of RNAi in cardiac cells is the low efficiency of liposome-mediated transfection of naked siRNA [11]. This limitation will be even more pronounced for any in vivo application [12], [13]. In the present study, we also failed to effectively transfect cultured neonatal rat
Conclusions
The study demonstrates that AV-delivered shRNAs mediate efficient PKCα knockdown in cardiac myocytes. This was accompanied by improved contractility of cardiomyocytes in EHT supporting the notion that PKCα negatively regulates myocardial contractility and that PKCα knockdown may be a useful strategy to enhance contractile performance in failing hearts. Our data further demonstrate that EHT in conjunction with AV-delivered shRNA is a useful model for target validation.
Acknowledgments
These studies were supported by the Deutsche Forschungsgemeinschaft (FOR 604 to A.E.A. and T.E.), by the European Commission (EUGeneHeart to T. E.) and by the German Ministry for Education and Research (BMBF 01GN 0520 to W.H.Z.).
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Authors contributed equally.