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. 2012 May;33(9):1067-75.
doi: 10.1093/eurheartj/ehs043. Epub 2012 Feb 23.

SERCA2a gene therapy restores microRNA-1 expression in heart failure via an Akt/FoxO3A-dependent pathway

Regalla Kumarswamy  1 , Alexander R Lyon, Ingo Volkmann, Adam M Mills, Julia Bretthauer, Aanchal Pahuja, Cornelia Geers-Knörr, Theresia Kraft, Roger J Hajjar, Kenneth T Macleod, Sian E Harding, Thomas Thum
Affiliations

SERCA2a gene therapy restores microRNA-1 expression in heart failure via an Akt/FoxO3A-dependent pathway

Regalla Kumarswamy et al. Eur Heart J. 2012 May.

Abstract

Aims: Impaired myocardial sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) activity is a hallmark of failing hearts, and SERCA2a gene therapy improves cardiac function in animals and patients with heart failure (HF). Deregulation of microRNAs has been demonstrated in HF pathophysiology. We studied the effects of therapeutic AAV9.SERCA2a gene therapy on cardiac miRNome expression and focused on regulation, expression, and function of miR-1 in reverse remodelled failing hearts.

Methods and results: We studied a chronic post-myocardial infarction HF model treated with AAV9.SERCA2a gene therapy. Heart failure resulted in a strong deregulation of the cardiac miRNome. miR-1 expression was decreased in failing hearts, but normalized in reverse remodelled hearts after AAV9.SERCA2a gene delivery. Increased Akt activation in cultured cardiomyocytes led to phosphorylation of FoxO3A and subsequent exclusion from the nucleus, resulting in miR-1 gene silencing. In vitro SERCA2a expression also rescued miR-1 in failing cardiomyocytes, whereas SERCA2a inhibition reduced miR-1 levels. In vivo, Akt and FoxO3A were highly phosphorylated in failing hearts, but reversed to normal by AAV9.SERCA2a, leading to cardiac miR-1 restoration. Likewise, enhanced sodium-calcium exchanger 1 (NCX1) expression during HF was normalized by SERCA2a gene therapy. Validation experiments identified NCX1 as a novel functional miR-1 target.

Conclusion: SERCA2a gene therapy of failing hearts restores miR-1 expression by an Akt/FoxO3A-dependent pathway, which is associated with normalized NCX1 expression and improved cardiac function.

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Figures

Figure 1
Figure 1
(A and B) miRNA expression profiling in pooled RNA samples from left ventricular tissue from controls (CTR), rats with heart failure, and heart failure rats after sarcoplasmic reticulum calcium ATPase 2a gene therapy (HF + SERCA). MicroRNAs deregulated greater than three-fold (HF vs. CTR) are depicted in green and the effects on individual microRNA expression up on sarcoplasmic reticulum calcium ATPase 2a gene therapy in shown in red. (B) Validation of miR-1 expression in left ventricular tissue from controls (CTR), rats with heart failure, and heart failure rats after sarcoplasmic reticulum calcium ATPase 2a gene therapy (HF + SERCA). n = 6–10 experiments/animals per group for validation experiments. Triplicate polymerase chain reactions for each animal/sample were used. Data are mean ± SD. *P < 0.05.
Figure 2
Figure 2
(A) miR-1 expression 48 h after treatment of adult rat cardiomyocytes with phenylephrine (PE, 100 µM) or placebo (CTR). (B) Adult rat cardiomyocytes were transfected with scrambled control, pre-miR-1, or anti-miR-1 and treated with phenylephrine (100 μM) as indicated. Cells were fixed with paraformaldehyde and stained for α-actinin. (C) Cardiomyocyte surface area was measured using NIS-Elements BR software (version 3.2). Cell size of 13–15 cardiomyocytes was counted for each condition. miR-1 expression in adult rat cardiomyocytes 48 h after transfection with scrambled control, pre-miR-1 (D), or anti-miR-1 (E) (each 100 nM). RNU6b served as a housekeeping control. **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
(A and B) Akt, p-Akt, FoxO3a, and p-FoxO3a protein expression in left ventricular tissue from controls (CTR), rats with heart failure, and heart failure rats after sarcoplasmic reticulum calcium ATPase 2a gene therapy (HF + SERCA). See Figure 4D for housekeeping control GAPDH. (C) Time course of p-Akt and p-FoxO3a expression after Akt activation (PDGF, 100 ng/mL, 24 h) in cultured cardiomyocytes. (D) FoxO3a total levels in cytosolic and nucleic fractions of adult cardiomyocytes 24 h after Akt stimulation (PDGF, 100 ng/mL). Gapdh (cytosolic localization) and Sp1 (nuclear localization) levels served as controls. (E) Time course of p-Akt and p-FoxO3a expression and miR-1 expression (F) after sarcoplasmic reticulum calcium ATPase inhibition (thapsigargin, 100 μM) in cultured cardiomyocytes. (G) Effects of viral sarcoplasmic reticulum calcium ATPase 2a transduction of freshly isolated adult cardiomyocytes from healthy control rats (Healthy) and rats with heart failure due to myocardial infarction. (H) Typical examples of intracellular Ca2+ levels (inferred from the ratio of fluorescence emitted at 510 nm after excitation at 340 nm and 380 nm) immediately after thapsigargin (TSG) addition in stimulated (1 Hz) adult rat cardiomyocytes. (I) miR-1 expression levels in adult rat cardiomyocytes treated with the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin (TSG, 100 µM, 24 h) or with TSG and the calcium scavenger BAPTA (100 µM, 24 h) or (K) TSG and the calmodulin-dependent protein kinase kinase inhibitor STO609 (10 µM, 24 h). n = 3–10 experiments/animals per group. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; #P = 0.06.
Figure 4
Figure 4
(A) Evolutionarily conserved miR-1 binding sites in the 3′UTR of the NCX1 gene. (B) NCX1 protein expression 48 h after transfection of adult cardiomyocytes with miR-1 precursor molecules (pre-miR-1; 100 nM) or scrambled controls (Scr, 100 nM). (C) Luciferase gene reporter assay of a 3′UTR region of the NCX1 gene harbouring an miR-1-binding site and co-transfection with miR-1 or scrambled controls (Scr). (D) NCX1 protein expression in left ventricular tissue from controls (CTR), rats with heart failure, and heart failure rats after sarcoplasmic reticulum calcium ATPase 2a gene therapy (HF + SERCA). (E) Time constant of decay (τ) of caffeine transients in cardiomyocytes transfected with miR-1 inhibitors (anti-miR-1) or scrambled controls (Scr). n = 3–8 experiments/animals per group. Data are mean ± SEM. *P < 0.05; ***P < 0.001.
Figure 5
Figure 5
Scheme of the proposed mechanism: high intracellular cytoplasmatic calcium (Ca2+) concentrations during heart failure lead to a Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)-dependent activation of Akt, which phosphorylates FoxO3a, thus affecting miR-1 expression. FoxO3a phosphorylation leads to FoxO3a export from the cardiomyocyte nucleus, thus resulting in decreased miR-1 transactivation. This leads to a de-repression of the direct miR-1 target sodium–calcium exchanger 1 (NCX1). These detrimental mechanisms are reversed by sarcoplasmic reticulum calcium ATPase 2a gene therapy.
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References

    1. Lyon AR, Bannister ML, Collins T, Pearce E, Sepehripour AH, Dubb SS, Garcia E, O'Gara P, Liang L, Kohlbrenner E, Hajjar RJ, Peters NS, Poole-Wilson PA, Macleod KT, Harding SE. SERCA2a gene transfer decreases SR calcium leak and reduces ventricular arrhythmias in a model of chronic heart failure. Circ Arrhythm Electrophysiol. 2011;4:362–372. doi:10.1161/CIRCEP.110.961615. - DOI - PMC - PubMed
    1. Miyamoto MI, del Monte F, Schmidt U, DiSalvo TS, Kang ZB, Matsui T, Guerrero JL, Gwathmey JK, Rosenzweig A, Hajjar RJ. Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure. Proc Natl Acad Sci USA. 2000;97:793–798. doi:10.1073/pnas.97.2.793. - DOI - PMC - PubMed
    1. Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B, Yaroshinsky A, Zsebo KM, Dittrich H, Hajjar RJ Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) Investigators. Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation. 2011;124:304–313. doi:10.1161/CIRCULATIONAHA.111.022889. - DOI - PMC - PubMed
    1. Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology. Nature. 2011;469:336–342. doi:10.1038/nature09783. - DOI - PMC - PubMed
    1. Bauersachs J, Thum T. Biogenesis and regulation of cardiovascular microRNAs. Circ Res. 2011;109:334–347. doi:10.1161/CIRCRESAHA.110.228676. - DOI - PubMed

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