Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure

Abstract

Background: Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown.

Methods and results: Here, we show that systemic delivery of an antisense oligonucleotide induces potent and sustained silencing of miR-208a in the heart. Therapeutic inhibition of miR-208a by subcutaneous delivery of antimiR-208a during hypertension-induced heart failure in Dahl hypertensive rats dose-dependently prevents pathological myosin switching and cardiac remodeling while improving cardiac function, overall health, and survival. Transcriptional profiling indicates that antimiR-208a evokes prominent effects on cardiac gene expression; plasma analysis indicates significant changes in circulating levels of miRNAs on antimiR-208a treatment.

Conclusions: These studies indicate the potential of oligonucleotide-based therapies for modulating cardiac miRNAs and validate miR-208 as a potent therapeutic target for the modulation of cardiac function and remodeling during heart disease progression.

Figures

Figure 1

Systemic delivery of antimiR-208a induces potent and sustained silencing of miR-208 in the heart. A, Locked nucleic acid (LNA)–modified antisense oligonucleotide chemistry directed against the 5′end of miR-208a (antimiR-208a). B, Real-time polymerase chain reaction analysis on murine hearts 1 week after intravenous delivery of increasing doses of antimiR-208a shows a dose-dependent reduction in miR-208a levels. C, Northern blot analysis on total RNA from murine hearts 1 week after intravenous delivery of antimiR-208a shows a dose-dependent reduction in miR-208a detection, whereas a control mismatch chemistry (MM) has no effect on miR-208a. U6 serves as a loading control. D, Real-time polymerase chain reaction analysis on cardiac tissue collected at the indicated time points shows that intravenous, intraperitoneal, or subcutaneous delivery of 25 mg/kg antimiR-208a induces potent silencing of miR-208a. In B and D, error bars depict SEM (n=4 for each time point and dose).

Figure 2

miR-208a silencing reduces miR-499 and Myh7. A, Real-time polymerase chain reaction analysis shows that antimiR-208a potently reduces cardiac levels of miR-208a up to 6 weeks after injection, which leads to a time-dependent reduction in miR-499. Dosing with an anti-miR against both miR-208a and miR-499 induces a more rapid reduction in cardiac levels of both miR-208a and miR-499. Control anti-miR shows no robust effect on miR-208a or miR-499 expression. B, Myh7 is reduced 4 weeks after miR-208a inhibition, whereas inhibition of miR-208a and miR-499 reduces Myh7 after 2 weeks, as shown by real-time polymerase chain reaction. Control anti-miR shows no robust effect on Myh7 expression. C, Western blot analysis for Myh7 showing reduced Myh7 expression at the indicated time points after antimiR-208a or antimiR-208a/-499 treatment. GAPDH serves as a loading control. Each lane is a representative animal from B. D and E, Tissue distribution analysis indicates that antimiR-208a is detectable in heart, liver, kidney (D), and plasma (E) up to 6 weeks after injection. In A, B, D, and E, error bars depict the SEM (n=4 for each time point and dose). In A and B, *P<0.05 vs saline at the same time point; #P<0.05 vs control anti-miR at the same time point. In D, *P<0.05 vs week 1 for the same tissue.

Figure 3

Therapeutic silencing of miR-208a is beneficial during heart failure. A, Kaplan-Meier survival curves in the Dahl hypertensive rat model show a pronounced decrease in survival in response to an 8% high-salt (HS) diet for both the HS/saline and HS/control groups, which is significantly improved in response to antimiR-208a treatment. Rats were dosed every 2 weeks at 25 mg/kg starting 1 week after the HS diet. *P=0.0038 vs HS/saline; #P<0.0001 vs HS/control. B, Body weight analysis indicates that Dahl hypertensive rats on an 8% HS diet exhibit reduced weight gain compared with animals on a low-salt (LS) diet, whereas HS/antimiR-208a–treated rats show a significantly better maintenance in weight gain. For A and B, n=6 for LS/saline; n=15 for HS/saline and HS/control; and n=14 for HS/antimiR-208a. n Indicates total survivors remaining at week 8 after the diet. C, Body weight analysis of Dahl rats on the 4% HS diet shows significant reductions in weight gain compared with LS diet controls, whereas both 5- and 25-mg/kg injections every 2 weeks are sufficient to maintain weight gain comparable to that in animals on a normal diet. D, Echocardiography measurements indicate that the increase in isovolumic relaxation time (IVRT) and decrease in mitral valve early to active filling velocity ratio (MV E/A) in response to a 4% HS diet are significantly improved in response to antimiR-208a treatment 8 weeks after the start of the diet. For C and D, n=10 for all groups. E, Representative images of hematoxylin and eosin (H&E)– and Picrosirius Red–stained left ventricular histological sections indicate an increase in cardiomyocyte hypertrophy and perivascular fibrosis in response to the 4% HS diet for 8 weeks, whereas both parameters are reduced in response to antimiR-208a treatment. H&E, scale bar = 50 μm; Picrosirius Red, scale bar =100 μm. F, Bar graph representation of histological quantification showing significantly less hypertrophy and fibrosis in the presence of antimiR-208a. In D and F, error bars depict the SEM. *P<0.05 vs HS/saline; #P<0.05 vs LS/saline.

Figure 4

AntimiR-208a treatment reduces miR-499 and Myh7 in Dahl salt-sensitive rats. All analyses were performed 8 weeks after the start of a 4% high-salt (HS) diet and 7 weeks after the start of anti-miR treatment (n=10 for all groups in A and C). In B and D, each lane is a representative animal from the n=10 group. Rats were dosed every 2 weeks at 25 mg/kg (control) or the indicated dose of antimiR-208a starting 1 week after the HS diet. Red lines on graphs separate the groups. A, Real-time polymerase chain reaction analysis indicates a dose-dependent reduction of miR-208a in both the left ventricle (LV) and right ventricle (RV), which corresponds to a dose-dependent decrease in miR-499. Although miR-208b is increased in response to the HS diet, antimiR-208a significantly blunts this response. Administration of a scrambled control chemistry had no effect on the expression of miR-208a, miR-499, or miR-208b. B, Regulation of miR-499 and miR-208b in response to antimiR-208a treatment shown by Northern blot analysis. C, Real-time polymerase chain reaction analysis shows that the HS diet reduces Myh6 and increases Myh7. AntimiR-208a treatment dose-dependently increases Myh6 expression and reduces Myh7b expression. The HS diet–induced increase in Myh7 is dose-dependently reduced by anti–miR-208a. D, Western blot analysis for Myh7 from ventricular tissue confirms the dose-dependent reduction in response to antimiR-208a treatment. GAPDH is used as a loading control. E, Quantification of HP1β Western blot showing miR-208a target derepression in the presence of antimiR-208a. The full blot in shown in Figure V in the online-only Data Supplement. In A and C, error bars depict the SEM. *P<0.05 vs HS/saline, #P<0.05 vs LS/saline.

Figure 5

AntimiR-208a regulates a specific subset of genes in Dahl salt-sensitive rats. A, Microarray analysis was performed from left ventricular RNA from high salt (HS)/saline, HS/25 mg/kg antimiR-208a, and HS/control at 8 weeks after the start of a 4% HS diet and 7 weeks after the start of anti-miR treatment. Differential expression graphs show the numbers of genes that are differentially expressed when comparing gene expression in hearts of control-treated animals and saline-treated animals (left) or antimiR-208a–treated animals with control-treated animals (right). Dpredicted is the bioinformatically computed reference distribution for the 13 518 transcripts detected on the array. The transcripts highlighted in red are significantly different and due to nonrandom difference, as calculated by Dobserved-Dpredicted. B, Hierarchical clustering and heat map visualization of the 131 differentially expressed genes between control oligo– and antimiR-208a–treated hearts show clustering of treatment groups and few gene changes in the saline-treated vs the control oligo–treated animals. A full list of transcripts in given in Table IV in the online-only Data Supplement. C, Quantification of Dynlt1 Western blot showing miR-208a target derepression in the presence of antimiR-208a. A full blot in given in Figure VIII in the online-only Data Supplement.

Figure 6

miR-499 in plasma serves a biomarker for antimiR-208a efficacy. Real-time polymerase chain reaction (PCR) analysis of the plasma samples indicates an increase in miR-499 in response to a high-salt (HS) diet, whereas antimiR-208a significantly lowers the detection of miR-499 in plasma 8 weeks after the start of a 4% HS diet and 7 weeks after the start of anti-miR treatment. Further miRNA analysis indicates a decrease in plasma-detectable miR-423–5p in response to antimiR-208a. *P<0.05 vs HS saline (n=10 per group).