Abstract
In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.
Highlights
Heart failure is the leading cause of mortality and morbidity in Western countries and a common endpoint of cardiac disorders, including atherosclerosis, ischemic cardiomyopathies, familiar cardiomyopathies, valvular-induced myocardial pathologies and arterial hypertension [1,2]
Silencing efficiency of amiR155-PLBr To develop an improved vector system for modulation of cardiac Ca2+ homeostasis by silencing PLB, an siRNA sequence directed against the rat PLB [11] was embedded into the native environment of the miR-155 stem loop structure and placed under control of the CMV-MLC0.26 promoter, which is active in cardiac cells [43,49]
We show that scAAV6-amiR155-PLBr, which expresses a newly engineered small regulatory RNA directed against the negative sarcoplasmatic reticulum (SR) Ca2+ATPase (SERCA2a) modulatory protein PLB, improves the SERCA2a-catalyzed Ca2+ transport activity of the sarcoplasmatic reticulum (SR) in CM
Summary
Heart failure is the leading cause of mortality and morbidity in Western countries and a common endpoint of cardiac disorders, including atherosclerosis, ischemic cardiomyopathies, familiar cardiomyopathies, valvular-induced myocardial pathologies and arterial hypertension [1,2]. The well-known regulatory role of PLB for the SERCA2a-catalyzed Ca2+-transport of the cardiac SR has qualified this small membrane protein as a promising molecular target for heart failure therapy. In this context, gene therapeutic approaches for inhibiting PLB expression by employment of dominant-negative PLB mutants [7], intracellular inhibitory antibodies targeting PLB [8] or engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous PLB gene [9] have been shown to improve the SERCA2a-catalyzed Ca2+ transport activity in cardiomyocytes and the contractile function of the heart in animals models of heart failure. Long-term and high-level shRNA expression can result in oversaturation of the endogenous cellular microRNA (miR) pathways leading to cytotoxicity and eventually fatality, as reported previously [26,27,28,29]
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