Nano-miR-133a Replacement Therapy Blunts Pressure Overload–Induced Heart Failure
2021; Lippincott Williams & Wilkins; Volume: 144; Issue: 24 Linguagem: Inglês
10.1161/circulationaha.121.055866
ISSN1524-4539
AutoresJessica Modica, Vittoria Di Mauro, Maria Barandalla-Sobrados, S E Pineda Chavez, Pierluigi Carullo, Simona Nemska, Achille Anselmo, Gianluigi Condorelli, Michele Iafisco, Michele Miragoli, Daniele Catalucci,
Tópico(s)RNA Interference and Gene Delivery
ResumoHomeCirculationVol. 144, No. 24Nano-miR-133a Replacement Therapy Blunts Pressure Overload–Induced Heart Failure Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBNano-miR-133a Replacement Therapy Blunts Pressure Overload–Induced Heart Failure Jessica Modica, PhD, Vittoria Di Mauro, PhD, Maria Barandalla-Sobrados, PhD, Samuel Elias Pineda Chavez, BS, Pierluigi Carullo, PhD, Simona Nemska, PhD, Achille Anselmo, PhD, Gianluigi Condorelli, MD, PhD, Michele Iafisco, PhD, Michele Miragoli, PhD and Daniele Catalucci, PhD Jessica ModicaJessica Modica National Research Council–Institute of Genetics and Biomedical Research, Milan Unit, Italy (J.M., V.D.M., M.B.-S., P.C., G.C., M.M., D.C.). Humanitas University, Pieve Emanuele (Milan), Italy (G.C., J.M.). , Vittoria Di MauroVittoria Di Mauro https://orcid.org/0000-0002-4707-4264 National Research Council–Institute of Genetics and Biomedical Research, Milan Unit, Italy (J.M., V.D.M., M.B.-S., P.C., G.C., M.M., D.C.). IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). , Maria Barandalla-SobradosMaria Barandalla-Sobrados https://orcid.org/0000-0003-4498-6230 National Research Council–Institute of Genetics and Biomedical Research, Milan Unit, Italy (J.M., V.D.M., M.B.-S., P.C., G.C., M.M., D.C.). IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). , Samuel Elias Pineda ChavezSamuel Elias Pineda Chavez https://orcid.org/0000-0003-0902-8571 IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). Department of Medicine and Surgery, University of Parma, Italy (S.E.P.C., M.M.). , Pierluigi CarulloPierluigi Carullo https://orcid.org/0000-0003-0812-7770 National Research Council–Institute of Genetics and Biomedical Research, Milan Unit, Italy (J.M., V.D.M., M.B.-S., P.C., G.C., M.M., D.C.). IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). , Simona NemskaSimona Nemska https://orcid.org/0000-0002-6969-6494 IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). , Achille AnselmoAchille Anselmo IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). , Gianluigi CondorelliGianluigi Condorelli National Research Council–Institute of Genetics and Biomedical Research, Milan Unit, Italy (J.M., V.D.M., M.B.-S., P.C., G.C., M.M., D.C.). Humanitas University, Pieve Emanuele (Milan), Italy (G.C., J.M.). , Michele IafiscoMichele Iafisco National Research Council–Institute of Science & Technology for Ceramics, Faenza, Italy (M.I.). , Michele MiragoliMichele Miragoli https://orcid.org/0000-0002-4058-4368 National Research Council–Institute of Genetics and Biomedical Research, Milan Unit, Italy (J.M., V.D.M., M.B.-S., P.C., G.C., M.M., D.C.). IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). Department of Medicine and Surgery, University of Parma, Italy (S.E.P.C., M.M.). and Daniele CatalucciDaniele Catalucci Correspondence to: Daniele Catalucci, PhD, National Research Council (CNR), Institute of Genetic and Biomedical Research (IRGB)–UOS Milan, Italy. Email E-mail Address: [email protected] https://orcid.org/0000-0001-7041-6114 National Research Council–Institute of Genetics and Biomedical Research, Milan Unit, Italy (J.M., V.D.M., M.B.-S., P.C., G.C., M.M., D.C.). IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy (V.D.M., M.B.-S., S.E.P.C., P.C., S.N., A.A., M.M., D.C.). Originally published13 Dec 2021https://doi.org/10.1161/CIRCULATIONAHA.121.055866Circulation. 2021;144:1973–1976Noncoding RNAs for curative purposes have officially entered the clinical field, as shown by the recent US Food and Drug Administration and European Medicines Agency approval of the first 3 liposome-based siRNA drugs: Onpattro (patisiran) for the treatment of a rare life-threatening form of amyloidosis, Givlaari (givosiran) for acute hepatic porphyria, and Leqvio (inclisiran) for lowering cholesterol.1 For all 3 drugs, the liver is the primary target. In cardiac medicine, although several noncoding RNA candidates, especially microRNAs (miRNAs), have been proven to have great therapeutic potentials, the emergence of noncoding RNA therapeutics has not yet translated into any approved drugs. In fact, miRNA-based clinical trials for heart disease are still limited to only diagnostic/prognostic uses of circulating miRNAs; no clinical trials for the potential use of miRNA mimic–based drugs for cardiac treatment are ongoing. A major issue challenging patient-friendly application of an miRNA-based therapy for myocardial treatment is the lack of an efficient delivery system, allowing cardiac-enriched uptake, controlled drug release, reduced unwanted off-target effects, low-toxic side effects, and low-dose administration. Gene therapy via cardiotrophic adeno-associated viral vectors is at present the only valid approach, as shown in recent preclinical trials. However, concerns about uncontrolled long-term expression, difficulties in fine-tuning dosage, immune reaction, risk of random gene integration, and potential cancerogenicity, as well as the need for invasive cardiac administration, still hamper broad, reliable, and safe use in the clinical settings. Here, we provide proof of concept for an unconventional and effective nanotechnology-based inhalation approach for delivery of a synthetic miRNA mimic via biocompatible and biodegradable calcium phosphate–based nanoparticles (CaPs)2 preferentially to cardiomyocytes without significant accumulation in other myocardial cells or organs (Figure [A and B]).Download figureDownload PowerPointFigure. Inhalation of miR-133–loaded nanoparticles contrasts cardiac dysfunction in pressure overload–induced heart failure. A and B, Analysis of synthetic miRNeg5 (miRCURY Negative Control 5, GeneGlobe ID YM00479904, Qiagen) levels in 8-week-old C57BL/6J male mice treated by a single intratracheal administration as indicated. A, Quantitative reverse transcription (qRT)–polymerase chain reaction (PCR) for miRNeg5 (normalized to U6) on heart RNA at different time points after administration (n=3). B, Droplet digital PCR (ddPCR) for miRNeg5 on total RNA from (left) fluorescence-activated cell (FACS)–sorted myocardial cells and (right) peripheral tissues 8 hours after treatment (n=4–6). miRNeg5 molecules were normalized to cell number and expressed as fold change (FC) over empty calcium phosphate–based nanoparticle (CaP) control. C, Schematic overview of mouse treatments. MiR-133a is an miRCURY LNA miRNA Mimic (GeneGlobe ID YM00470572, Qiagen). D, Echocardiographic analyses of treated and untreated mice subjected to transverse aortic constriction (TAC; pressure gradients >70 mm Hg) or sham (n=8–15). E, ddPCR for miR-133a on (left) FACS-isolated cardiomyocytes (normalized to U6) and (right) liver (normalized to the saline control condition) from mice treated as indicated (n=3–5). F, Fluorescence in situ hybridization for miR-133a on heart sections from CaP-miR–treated mice 4 weeks after TAC and sham. Green indicates α-sarcomeric actin (cardiomyocyte marker), vimentin (fibroblast marker), smooth muscle actin (smooth muscle marker), or isolectin B4 (IB4; endothelial marker); red, miR-133a; and cyan, DAPI (nuclei). Scale bar: 10 µm. G, Hematoxylin and eosin– (scale bar: 1 mm), Masson trichrome– (scale bar: 100 µm), and Picro Sirius Red– (scale bar: 100 µm) stained heart sections from mice 4 weeks after TAC or sham, treated as indicated. H, Quantification of fibrotic area (n=5 – 7). I, qRT-PCR for the fibrotic gene Ctgf on left ventricular (LV) RNA normalized to Hprt (n=4 – 7). J, IB4- (isolectin for endothelial cells), wheat germ agglutinin– (WGA; for plasma membrane staining), and DAPI- (nuclei) stained heart sections. Scale bar: 10 µm. K, Quantification of capillary density per cardiomyocyte. L, qRT-PCR for Hif1a on LV RNA normalized to Hprt (n=7–11). M, Terminal deoxynucleotidyl transferase dUTP nick-end labeling– (TUNEL; scale bar: 200 µm) and cleaved caspase-3– (casp-3; scale bar: 20 µm) stained heart sections. N and O, Quantification of percentage TUNEL-positive nuclei (N) and Casp-3–positive signal per signal area (O). All data are represented as mean±SD. Statistical analyses were performed with Prism 8 using the Kruskal-Wallis test with the Dunn multiple-comparisons test (A and N), Mann Whitney test (B), 2-way mixed-effects ANOVA with Tukey multiple-comparisons test after a Shapiro-Wilk test (D), and 1-way ANOVA with Dunnett multiple-comparisons test after a Shapiro-Wilk test (E, H, I, K, L, and O). A.U. indicates arbitrary units. *P<0.05. **P<0.01. ***P<0.005. miR indicates microRNA.The cardiac-enriched miR-133a, which is inversely related to failing heart conditions in human and animals, represents a potential therapeutic molecule to counteract the progression of pathological cardiac remodeling. In a mouse model of ventricular pressure overload induced by transverse aortic constriction (TAC), miR-133a rapidly decreases as early as day 1 after TAC and gradually recovers toward basal levels at 6 to 8 weeks after TAC.3 In a transgenic mouse model for inducible expression of miR-133a, we previously demonstrated that restoration of miR-133a levels mitigates the pathological effects of TAC.4 However, effective and clinically oriented interventions aimed at facilitating cardiac delivery of exogenous synthetic miR-133a or other therapeutic miRNA mimics are currently not available. Here, inhalable CaPs loaded with synthetic miR-133a mimic (CaP-miR) were tested as a replacement therapy approach for the specific restoration of miR-133a levels in cardiomyocytes just within the precise time frame when the miRNA is pathologically repressed. TAC mice were treated with CaP-miR delivered by intratracheal nebulization once a day on alternate days for 4 consecutive weeks starting from the day of TAC (Figure [C]). Saline solution, miR-133 alone (miR), and unloaded CaPs alone served as controls. CaP-miR showed a protective effect against heart failure progression with preservation of left ventricular internal diameter, ejection fraction, and fraction shortening at 14 and 28 days after TAC (Figure [D]). This improvement was associated with effective restoration of physiological levels of miR-133a in fluorescence-activated cell sorting–isolated cardiomyocytes 28 days after TAC as measured by digital polymerase chain reaction (Figure [E], left). No significant accumulation of miR-133a in the liver of CaP-miR133–treated mice versus nontreated mice was found (Figure [E], right). Furthermore, in situ hybridization showed an enrichment of miR-133a in cardiomyocytes from CaP-miR133–treated TAC mice (Figure [F]). Histological analyses revealed reduced fibrosis (Figure [G–I]) in CaP-miR–treated TAC mice, whereas no beneficial effects were observed in mice administered unloaded CaPs or pristine miR-133a. In addition, CaP-miR–treated TAC mice showed increased capillary density per cardiomyocyte, suggesting that intensification of oxygen supply may play a role in preventing heart failure progression (Figure [J–L]). CaP-miR–treated TAC mice consistently showed a marked decrease in the activated programmed cell death (Figure [M and O]), supporting the notion that inhibition of hypoxia-induced apoptosis5 contributes to counteract the pathological effects of TAC. In conclusion, we report that (1) inhalable CaP-miR represents an effective delivery approach for targeting the heart and facilitating the release of miRNA mimics within cardiomyocytes and (2) the delivered miR-133a is functional for the beneficial restoration of cardiac performance and prevention of remodeling in a mouse model of ventricular pressure overload.MiR-133a is a well-established diagnostic circulating biomarker in patients with heart failure, and increased circulating miR-133a levels are inversely correlated with downregulation of the miRNA at the cardiomyocyte level as a result of a stress-induced secretion. Therefore, circulating miR-133a may represent an important biomarker for stratification of patients for a future replacement therapy that is based on inhalable miR-133a–loaded CaPs. Thus, by providing the proof of concept for an innovative application of inhalable nanoparticles for miRNA mimic delivery to the heart, our approach represents an important step forward for noncoding RNA–based precision medicine interventions of the diseased heart.All procedures involving animals were approved by institutional and national authorities. The data that support the findings of this study are available from the corresponding author on reasonable request.Article InformationAcknowledgmentsThe authors thank Marie-Louise Bang for critically reading the article and Etienne Guillot for providing insightful discussions.Sources of FundingThis work was supported by grants to Dr Catalucci from the H2020-NMBP-2016 720834 CUPIDO (www.cupidoproject.eu) and the FISR2019_00320 Leonardo, to Dr Barandalla-Sobrados from the Veronesi Foundation (FUV2018), and to Dr Condorelli from the Proof of Concept PC1, ERC-2015-PoC.Nonstandard Abbreviations and AcronymsCaPcalcium phosphate–based nanoparticlemiRNAmicroRNATACtransverse aortic constrictionDisclosures None.Footnotes*J. Modica and V. Di Mauro contributed equally.https://www.ahajournals.org/journal/circFor Sources of Funding and Disclosures, see page 1975–1976.Correspondence to: Daniele Catalucci, PhD, National Research Council (CNR), Institute of Genetic and Biomedical Research (IRGB)–UOS Milan, Italy. Email daniele.[email protected]itReferences1. Zhang MM, Bahal R, Rasmussen TP, Manautou JE, Zhong XB. The growth of siRNA-based therapeutics: updated clinical studies.Biochem Pharmacol. 2021; 189:114432. doi: 10.1016/j.bcp.2021.114432CrossrefMedlineGoogle Scholar2. Miragoli M, Ceriotti P, Iafisco M, Vacchiano M, Salvarani N, Alogna A, Carullo P, Ramirez-Rodríguez GB, Patrício T, Esposti LD, et al.. Inhalation of peptide-loaded nanoparticles improves heart failure.Sci Transl Med. 2018; 10:eaan6205. doi: 10.1126/scitranslmed.aan6205CrossrefMedlineGoogle Scholar3. Matkovich SJ, Dorn GW, Grossenheider TC, Hecker PA. Cardiac disease status dictates functional mRNA targeting profiles of individual microRNAs.Circ Cardiovasc Genet. 2015; 8:774–784. doi: 10.1161/CIRCGENETICS.115.001237LinkGoogle Scholar4. Castaldi A, Zaglia T, Di Mauro V, Carullo P, Viggiani G, Borile G, Di Stefano B, Schiattarella GG, Gualazzi MG, Elia L, et al.. MicroRNA-133 modulates the β1-adrenergic receptor transduction cascade.Circ Res. 2014; 115:273–283. doi: 10.1161/CIRCRESAHA.115.303252LinkGoogle Scholar5. Li AY, Yang Q, Yang K. miR-133a mediates the hypoxia-induced apoptosis by inhibiting TAGLN2 expression in cardiac myocytes.Mol Cell Biochem. 2015; 400:173–181. doi: 10.1007/s11010-014-2273-2CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails December 14, 2021Vol 144, Issue 24Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.121.055866PMID: 34898244 Originally publishedDecember 13, 2021 Keywordstherapeuticsheart failuremicroRNAsRNAinhalationcardiomyocytesnanoparticlesPDF download Advertisement SubjectsCardiomyopathyHeart FailureTranslational StudiesTreatment
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