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

doi:10.1016/j.yjmcc.2007.05.021

Journal of Molecular and Cellular Cardiology

Volume 43, Issue 3, September 2007, Pages 371-376

https://doi.org/10.1016/j.yjmcc.2007.05.021 Get rights and content

Abstract

PKCα has been shown to be a negative regulator of contractility and PKCα 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 PKCα 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 PKCα (Ad-shPKCα) and evaluated its potential to silence PKCα 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 PKCα-mRNA and 50% lower PKCα protein in Ad-shPKCα-infected cells. EHTs were infected with Ad-shPKCα on day 11 and subjected to isometric force measurements in organ baths 4 days later. Mean twitch tension was > 50% higher in Ad-shPKCα compared to Ad-shNS-infected EHTs, under basal and Ca²⁺- or isoprenaline-stimulated conditions. Twitch tension negatively correlated with PKCα mRNA levels. In summary, AV-delivered shRNA mediated highly efficient PKCα knockdown in cardiac myocytes and improved contractility in EHTs. The data support a role of PKCα as a negative regulator of myocardial contractility and demonstrate that EHTs in conjunction with AV-delivered shRNA are a useful model for target validation.

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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3D-cardiomyocyte cultures have evolved to simulate myocardial tissue in vitro, either for experimental applications in drug screening/development or “therapeutic” applications in vivo[13]. Towards this end, we have developed engineered heart tissue (EHT) [14,15]. A key limitation in EHTs, as in other tissue engineering applications, appears to be low cell-seeding efficiency as a consequence of a “metabolic steal effect” and in particular myocyte apoptosis in high density cultures [12].
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PKCα is the predominant PKC isoform expressed in the mouse, human, and rabbit heart [45–47], and PKCα expression and activity is increased in cardiac hypertrophy, dilated cardiomyopathy, ischemic injury, or mitogen stimulation [43,44]. We and others have shown that PKCα functions as a novel regulator of cardiac contractility through effects on Ca2+ handling and myofilament proteins [48–51]. For example, Prkca (PKCα−/−) gene-deleted mice are hypercontractile, while transgenic mice overexpressing PKCα in the heart are hypocontractile.
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¹: Authors contributed equally.

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Journal of Molecular and Cellular Cardiology

Abstract

Introduction

Section snippets

Cell culture and transfection

Feasibility of RNAi in cardiac myocytes using siRNA transfection

Discussion

Conclusions

Acknowledgments

References (17)

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

Eur. J. Cell Biol.

Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs

J. Biotechnol.

Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function

J. Biol. Chem.

In vivo and in vitro analysis of cardiac troponin I phosphorylation

J. Biol. Chem.

Cytoplasmic signaling pathways that regulate cardiac hypertrophy

Annu. Rev. Physiol.

Pharmacological- and gene therapy-based inhibition of protein kinase Calpha/beta enhances cardiac contractility and attenuates heart failure

Circulation

PKC-alpha regulates cardiac contractility and propensity toward heart failure

Nat. Med.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Nature

Cited by (30)

Role of protein phosphatase inhibitor-1 in cardiac beta adrenergic pathway

Life in 3D is never flat: 3D models to optimise drug delivery

Conceptual and technical aspects of transfection and gene delivery

PDGF-BB protects cardiomyocytes from apoptosis and improves contractile function of engineered heart tissue

Gα<inf>q</inf>-mediated activation of GRK2 by mechanical stretch in cardiac myocytes: The role of protein kinase C

With great power comes great responsibility: Using mouse genetics to study cardiac hypertrophy and failure

Brief communicationAdenovirus-delivered short hairpin RNA targeting PKCα improves contractile function in reconstituted heart tissue

Abstract

Introduction

Section snippets

Cell culture and transfection

Feasibility of RNAi in cardiac myocytes using siRNA transfection

Discussion

Conclusions

Acknowledgments

Eur. J. Cell Biol.

J. Biotechnol.

J. Biol. Chem.

J. Biol. Chem.

Cytoplasmic signaling pathways that regulate cardiac hypertrophy

Annu. Rev. Physiol.

Pharmacological- and gene therapy-based inhibition of protein kinase Calpha/beta enhances cardiac contractility and attenuates heart failure

Circulation

PKC-alpha regulates cardiac contractility and propensity toward heart failure

Nat. Med.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Nature

Brief communication
Adenovirus-delivered short hairpin RNA targeting PKCα improves contractile function in reconstituted heart tissue