Prompt coronary catheterization and revascularization have markedly improved the outcomes of myocardial infarction, but have also resulted in a growing number of surviving patients with permanent structural damage of the heart, which frequently leads to heart failure. There is an unmet clinical need for treatments for this condition1, particularly given the inability of cardiomyocytes to replicate and thereby regenerate the lost contractile tissue2. Here we show that expression of human microRNA-199a in infarcted pig hearts can stimulate cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. However, subsequent persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however dosage of this therapy needs to be tightly controlled.
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Tagged cardiac images were analysed using custom software called ‘tagging tool’, based on a previously described method35. This software was implemented by the UOC Magnetic Resonance of Fondazione Toscana Gabriele Monasterio, Pisa, Italy. To request access to this software please contact G.D.A. (firstname.lastname@example.org). This software is only for use in animal studies and clinical use in humans is not permitted.
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This work was supported by the European Research Council (ERC) (Advanced Grants 250124 and 787971) to M.G.; the Leducq Foundation Transatlantic Network of Excellence (grant 14CVD04) to M.G.; the Fondazione CRTrieste (Project CTC), Trieste, Italy and the Italian Ministry of Health (grant RF-2011-02348164 ‘CardioRigen’) to G.S., F.A.R. and M.G. G.P., I.S. and H.A. are supported by an ICGEB Arturo Falaschi pre-doctoral Fellowship. The authors are grateful to M. Dapas and M. Zotti from the ICGEB AAV Unit for AAV vector production.
Nature thanks Roger Hajjar, Eva van Rooij and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, b, AAV6 is the most effective serotype for porcine heart transduction. The graphs show viral genomes (a) and eGFP mRNA (b) levels one month after direct intramyocardial injection of 1 × 1012 viral genome particles of AAV6, AAV8 and AAV9 vectors carrying the eGFP transgene (these three AAV serotypes have been reported to transduce post-mitotic tissues at high efficiency28). Data are mean ± s.e.m.; the number of animals per group is indicated. c, Nucleotide sequence of the miR-199a-1 precursor. Mature miR-199a-5p and miR-199a-3p sequences are shown in green and their seed sequences are shown in blue and red, respectively. d, Mature miR-199a-5p and miR-199a-3p sequences are conserved in human, mouse, rat and pig. The miRNA seed sequences are shown in blue for miR-199a-5p and in red for miR-199a-3p. e, Representative photograph taken during porcine surgery and vector injection. After thoracotomy, the pericardial sac was opened, the LAD was exposed and occluded below its first branch for 90 min. Ten minutes after reperfusion, AAV6-control or AAV6-miR-199a were injected into the infarct border zone.
Extended Data Fig. 2 Systematic assessment of miR-199a-3p expression after AAV6-mediated transduction.
a, Schematic representation of pig heart sectioning for histological and molecular studies. After arrest in diastole, the heart was excised and the pericardial sac removed. AAV injection sites, which were marked with coloured epicardial sutures during surgery, were further traced with a green waterproof paint. Four 1-cm thick transversal slices were cut starting from the base to the apex (1–4). Each slice was subsequently divided into 2–8 regions, each one labelled with a capital letter, and then into additional sub-regions (letters plus numbers) for targeted molecular and histological analyses. Sectors H, T and C corresponded to the infarct border zone, where the vectors were administered, whereas sector L was considered representative of the remote zone. b, Injection and infarct border segments for each slice were divided into smaller fragments (dashed lines) to accurately assess the levels of expression of the transgene at 12 days after transduction. The syringe indicates the injection sites. c, For each slice and segment, the graphs show real-time PCR quantifications of the mature miR-199a-3p expressed as fold change over endogenous levels (AAV6-control). One representative animal is shown out of four analysed in the same systematic manner with comparable results. d, In situ hybridization of pig heart sections for the detection of miR-199a expression at the single-cell level. Each of the sectors indicated in b was tested by in situ hybridization using LNA probes detecting miR-199a-3p or U6 snRNA, or a probe with the same nucleotide composition as the one against miR-199a-3p but with a scrambled sequence (scramble). Expression of miR-199a-3p was robust in cardiomyocytes and specific for the injected areas throughout the LV. One representative animal is shown out of four analysed in the same systematic manner with comparable results. Scale bar, 100 µm.
Extended Data Fig. 3 Downregulation of miR-199a target genes in transduced heart tissue and organ distribution of the AAV6-miR-199a vector.
a, Real-time PCR quantification of both strands of miR-199a in AAV6-control- and AAV6-miR-199a-injected pig hearts (n = 4 and n = 10, respectively) normalized to endogenous 5S rRNA. Data are mean ± s.e.m. b, mRNA levels of predicted and annotated target genes of miR-199a in AAV6-control- and AAV6-miR-199a-treated pig hearts (n = 4 per group) one month after MI and viral transduction. Data are mean ± s.e.m.; *P < 0.05 versus AAV6-control; two-sided t-test. c–e, Predicted target sites of miR-199a-3p in the 3′-untranslated region (UTR) sequences of swine cofilin-2, TAOK1 and βTRC according to TargetScan release 7.2. All three of these genes are verified to be direct targets of this miRNA in rodents; the corresponding 3′ UTR target sites for cofilin-2 and TAOK1 are conserved in swine; for βTRC, two alternative target sites in swine are shown. Other miR-199a-3p target genes originally identified in mice (in particular, homer1 and Clic511,29) are not conserved in the swine genome. In the pig genome, βTRC also has an additional predicted target sequence for miR-199a-5p, which is indicated. f, Predicted target site of miR-199a-5p in the 3′ UTR of pig HIF1A mRNA. g, Quantification of viral genomes in the indicated organs one month after intracardiac injection of AAV6-miR199a. Data are expressed as fold change over liver levels after normalization for cellular DNA content using 18S DNA as a reference (mean ± s.e.m., n = 4 per group). The levels of viral DNA in the myocardium of the injected animals were more than 18 times higher than in the liver and more than 40 times higher than in other organs (spleen, kidney and lung). h, Levels of miR-199a-3p RNA in the indicated organs one month after intracardiac injection of AAV6-miR-199a. Data are shown as fold change over endogenous miRNA levels in the liver of control animals after normalization for cellular 5S rRNA (n = 4 per group). Data are mean ± s.e.m. The amount of hsa-miR-199a-3p RNA was not elevated in any analysed organ, except for the heart. No overt signs of pathology, including hyper-proliferation (assessed by Ki67 staining) were observed.
Extended Data Fig. 4 miR-199a improves global heart function and decreases infarct mass one month after treatment.
a, Graphs showing percentage changes in infarct mass, infarct mass over LV mass and ejection fraction, as indicated, between 2 and 28 days after MI and AAV6-control or AAV6-miR-199a delivery, measured by cMRI. The number of analysed animals were 7 and 8, 7 and 8, and 7 and 9 for infarct mass, infarct mass over LV mass and ejection fraction for the two groups, respectively. Top graphs, cumulative values for all pigs. Data are mean ± s.e.m.; *P < 0.05; two-tailed t-test; bottom graphs, data from individual pigs. b, Infarct healing one month after AAV6-miR-199a injection. The LGE-cMRI images (from apex to base, a to e) are the same as in Fig. 1h, but without red counterstain. The red arrows show the infarcted area in the central plane. c, Gross anatomy of cardiac slices with corresponding LGE-cMRI images in representative AAV6-control- and AAV6-miR-199a-treated pig hearts, at 28 days after MI. d, Heart rate in sham and infarcted animals injected with AAV6-control and AAV6-miR-199a at one month after treatment. Data are mean ± s.e.m.; the number of animals per group and time point are indicated.
a, Representative images of Ki67 and α-actinin immunofluorescence staining of the infarct border (sector H) or remote (sector L) zones of AAV6-control- and AAV6-miR-199a-treated animals (n = 4 and n = 6, respectively; analysis is from at least seven high-resolution images acquired from at least eight different regions of each heart), 12 days after MI. Scale bars, 100 µm. At least six animals were treated. b, High-magnification, representative images of phospho-histone H3 immunostaining in the infarct border zones of four different pigs treated with AAV6-miR-199a, 12 days after MI. Scale bar, 100 µm.
a, Representative images of longitudinal sections stained with WGA to assess the number of nuclei per cardiomyocyte in the infarct border zone of AAV6-control- and AAV6-miR-199a-treated animals (n = 4 and n = 6, respectively; analysis is from at least seven high-resolution images acquired from at least eight different regions of each heart), 12 days after MI. The right panels show the estimated number of nuclei for each cardiomyocyte. Scale bar, 50 µm. b, Additional representative images of mono- or bi-nucleated BrdU+ cardiomyocytes in the infarct border zone of AAV6-control- and AAV6-miR-199a-treated animals, 12 days after MI. Scale bar, 50 µm. c, Cross-sectional area measurements of BrdU+ and BrdU− cardiomyocytes in AAV6-control- and AAV6-miR-199a-treated pigs 12 days after surgery. Data are mean ± s.e.m. from the analysis of four pigs. d, Representative images of BrdU+ and BrdU− cardiomyocytes. Scale bar, 50 µm. The right panels are high-magnification images of the indicated portions of the left images.
Extended Data Fig. 7 Expression of GATA4 in cardiomyocytes in the infarct border zone of AAV6-miR-199a-treated pigs.
a, Left, representative immunohistochemistry images of GATA4+ cells in AAV6-control and AAV6-miR-199a-injected pigs, 30 days after treatment. The bottom panels are high-magnification images of the indicated portions of the top images. The graph on the right shows the quantification of cells showing GATA4 cytoplasmic localization. Data are mean ± s.e.m.; the number of animals per group is indicated. Quantification is from at least seven high-resolution images acquired from at least eight different regions of each heart. *P < 0.05; two-sided Student’s t-test. Scale bars, 100 µm. b, c, Additional low- and high-magnification representative immunohistochemistry images of GATA4+ cells in the infarct border (sector H) or remote zone (sector L) of AAV6-control- and AAV6-miR-199a-injected pigs, 12 days (b) and 30 days (c) after treatment. Scale bars, 100 µm. d, AAV6-miR-199a treatment does not alter the levels of DAB2, SMARCA5 and DESTRIN mRNAs. The graphs show real-time PCR quantifications of the levels of the indicated genes in sham, AAV6-control- and AAV6-miR-199a-injected pig hearts, at 12 and 30 days after surgery; n = 3 per group. Data are mean ± s.e.m.; the number of animals per group and time point is indicated. *P < 0.05 versus AAV6-control at the same time point; two-sided t-test.
a, Real-time PCR quantification of the ratio between α- and β-myosin heavy chain mRNA in sham, AAV6-control- and AAV6-miR-199a-injected pig hearts, at 12 and 30 days after surgery in the H (border zone) and L (remote zone) cardiac sectors. Data are mean ± s.e.m.; the number of animals per group and time point is indicated. *P < 0.05 versus AAV6-control at the same time point; two-way ANOVA with Bonferroni post hoc correction. b, c, Lectin immunofluorescence (b) of sham, AAV6-control- and AAV6-miR-199a-treated pig sections, 30 days after MI and vector administration along with quantification (c) of cardiomyocyte cross-sectional area (μm2). Data are mean ± s.e.m.; the number of analysed animals is indicated. One-way ANOVA with Bonferroni post hoc correction. Scale bar, 50 µm. d, Low- and high-magnification (insets) representative images of infarcted hearts injected with AAV6-control or AAV6-miR-199a after immunohistochemistry to detect desmin (which is essential for maintaining structural and functional integrity of cardiomyocytes40 and was expressed at normally high levels), myogenin (which coordinates skeletal myogenesis and repair41 and was not expressed), endothelin-B receptor (which selectively stained arteriole smooth muscle cells) and WT1 (which was expressed at low levels in the vascular endothelium, but not in cardiomyocytes). Analysis was performed in at least seven high-resolution images acquired from at least eight different regions of the hearts of three pigs per group. Scale bars, 100 µm. e, Real-time PCR quantification of the levels of ANP and BNP in sham, AAV6-control- and AAV6-miR-199a-injected pig hearts, at 12 and 30 days after surgery. Data are mean ± s.e.m.; the number of animals per group and time point are indicated. NS, not significant; *P < 0.05 versus AAV6-control at the same time point. One-way ANOVA with Bonferroni post hoc correction. f, Representative sections of pig hearts treated with AAV6-control and AAV6-miR-199a at day 30 after infarction and vector injection stained with FITC–lectin to visualize vessels and with an α-SMA antibody to detect smooth muscle cells, along with quantification of lectin-positive vessels. No significant difference between the two MI groups was detected in capillary density at either 12 or 30 days. Data are mean ± s.e.m.; the number of animals per group is indicated. Analysis was performed in at least seven high-resolution images acquired from at least eight different regions of the heart. *P < 0.05; Student’s two-sided t-test. Scale bar, 100 µm.
a, The LGE-cMRI images (from apex to base, a–e) are the same as those in Fig. 4a, but without red counterstain. The red arrows show the infarcted area in the central plane c. b, cMRI images from a pig euthanized at week 8 after MI and AAV6-miR-199a treatment. Top, serial images from apex to base at day 2, week 4 and week 8; the infarct area is counterstained in red. Bottom, the same images without counterstaining. The green arrows show the pacemaker-lead attachment sites. c, Gross anatomy of cardiac slices of the pig shown in b after euthanization. The syringe indicates the injected area. The green arrows show the pacemaker-lead attachment sites. Similar cardiac repair results were observed in three pigs treated with miR-199a that survived two months after treatment.
Extended Data Fig. 10 Recording of fatal arrhythmias in two infarcted pigs treated with AAV6-miR-199a-3p.
Initiation of ventricular fibrillation recorded at the moment of death in two AAV6-miR-199a pigs by implanted miniaturized ECG recorders (Reveal, Medtronic, 9529). a, A premature ventricular ectopic beat (red arrow) with a coupling interval of 380 ms during a slowing heart rhythm induced a fast ventricular tachycardia that degenerated in ventricular fibrillation. b, A premature ventricular ectopic beat (red arrow) with coupling interval of 350 ms induced a fast ventricular tachycardia that quickly degenerated in ventricular fibrillation of different amplitudes resembling polymorphic ventricular tachycardia. c. AAV6-mediated, long-term expression of miR-199a did not affect the expression levels of ion channels or associated proteins involved in known arrhythmogenic conditions. In the infarct border zone of pigs treated with AAV6-control or AAV6-miR-199a (n = 6 and n = 4, respectively) at 30 days after transduction, the expression levels of genes known to be involved in the pathogenesis of long QT syndrome (SCN5A, KCNE1, SNTA1, AKAP9 and ANK2), Brugada syndrome (CACNA1, CACNB2 and SCN1B), Carvajal syndrome (DSP), arrhythmogenic right ventricular cardiomyopathy (DSG2 and DSP) and catecholaminergic polymorphic ventricular tachycardia (CASQ2 and RYR2) were assessed. Additional investigated mRNAs were those coding for SERCA2A (which is encoded by ATP2A2 and also served as a positive control since it is depressed during heart failure and was found increased in miR-199a-treated animals), phospholamban (PLN) and connexins 40 and 43 (CX40 (which is encoded by GJA1) and CX43 (which is encoded by GJA5), respectively). The miR-199a-treated pigs in which analysis was performed included one pig that survived eight weeks (pig 50) and three pigs that died from sudden death at seven weeks (pigs 55, 66 and 67). Data are mean ± s.e.m. *P < 0.05 versus AAV6-control; Student’s two-sided t-test.
Extended Data Fig. 11 miR-199a induces formation of proliferating cell clusters with an early myoblast phenotype infiltrating the pig myocardium.
Additional images of cell clusters infiltrating the infarcted hearts injected with AAV6-miR-199a after haematoxylin and eosin staining or immunostaining to detect the indicated antigens. These cells scored negative for the leukocyte common antigen CD45 and for CD34 (excluding their immune, haematopoietic or endothelial origin) and were highly proliferating, as inferred from almost complete positivity for Ki67. These cells also scored negative for markers of muscle differentiation, including desmin (identifying myogenic cells of cardiac, smooth and striated muscle), sarcomeric α-actinin (which labels Z lines in the cardiac and skeletal muscle sarcomere) and HHF35 (a monoclonal antibody recognizing muscle-specific α- and γ-actin); cells were also negative for WT1 (marking several malignancies and the epicardium). The infiltrating cells were positive for GATA4 (which is critical for proper mammalian cardiac development) and myogenin (the reactivation of which characterizes rhabdomyosarcoma cells) as well as the calmodulin-binding protein caldesmon (which regulates smooth muscle contraction and is expressed at high levels in leiomyoma and leiomyosarcoma) and the endothelin-B receptor, normally expressed in smooth muscle cells. The pig identity, treatment, time of analysis and cardiac sector from which the sample was taken are shown for each picture. Scale bars, 100 µm. Clusters of cells were never detected in control-injected animals, although in one animal injected with AAV6-miR-199a clusters of cells were detected in the absence of MI.
Representative cardiac cine MR videos of AAV6-Control (left) and AAV6-miR-199a (right) pig hearts at 28 days post-MI. The short axis view shows LV cross-sections. A pronounced negative remodelling (wall thinning and chamber dilation) and dyskinesia of the LV anterior and septal walls are evident in the heart injected with AAV6-Control, while the heart injected with AAV6-miR-199a displays a markedly preserved morphology and very mild hypokinesia, closely resembling normal heart features.