miR-222 inhibits pathological cardiac hypertrophy and heart failure

Abstract

Aims: Physiological cardiac hypertrophy occurs in response to exercise and can protect against pathological stress. In contrast, pathological hypertrophy occurs in disease and often precedes heart failure. The cardiac pathways activated in physiological and pathological hypertrophy are largely distinct. Our prior work demonstrated that miR-222 increases in exercised hearts and is required for exercise-induced cardiac hypertrophy and cardiomyogenesis. Here, we sought to define the role of miR-222 in pathological hypertrophy.

Methods and results: We found that miR-222 also increased in pathological hypertrophy induced by pressure overload. To assess its functional significance in this setting, we generated a miR-222 gain-of-function model through cardiac-specific constitutive transgenic miR-222 expression (TgC-miR-222) and used locked nucleic acid anti-miR specific for miR-222 to inhibit its effects. Both gain- and loss-of-function models manifested normal cardiac structure and function at baseline. However, after transverse aortic constriction (TAC), miR-222 inhibition accelerated the development of pathological hypertrophy, cardiac dysfunction, and heart failure. Conversely, miR-222-overexpressing mice had less pathological hypertrophy after TAC, as well as better cardiac function and survival. We identified p53-up-regulated modulator of apoptosis, a pro-apoptotic Bcl-2 family member, and the transcription factors, Hmbox1 and nuclear factor of activated T-cells 3, as direct miR-222 targets contributing to its roles in this context.

Conclusion: While miR-222 is necessary for physiological cardiac growth, it inhibits cardiac growth in response to pressure overload and reduces adverse remodelling and cardiac dysfunction. These findings support the model that physiological and pathological hypertrophy are fundamentally different. Further, they suggest that miR-222 may hold promise as a therapeutic target in pathological cardiac hypertrophy and heart failure.

Keywords: Heart failure; MicroRNA; Pathological hypertrophy; Transverse aortic constriction (TAC).

Graphical Abstract

Figure 1

Cardiac miR-222 increases in pathological cardiac hypertrophy and heart failure induced by pressure overload. (A and B) HW/TL (A) and LW/TL (B) ratios of sham or TAC mice at 3, 7, and 14 days after TAC (n = 3–4 in each group). (C–E) A qRT-PCR analysis of hypertrophy markers (C), miR-222 (D), and pri-miR-222 (E) in hearts from sham or TAC mice at different time points. Data are shown as fold induction of gene or miR expression normalized to GAPDH (C), U6 (D), and GAPDH (E), respectively (n = 3–4 hearts in each group). (F and G) HW/TL (F) and LW/TL (G) ratios of mice 42 days after sham and TAC surgery (n = 3–4 hearts in each group). (H) A qRT-PCR analysis of miR-222 in hearts from mice 42 days after sham or TAC. Data are shown as fold induction of gene or miR expression normalized to U6 (n = 3–4 hearts in each group). (I) A qRT-PCR analysis of miR-222 in adult cardiomyocytes and non-cardiomyocytes isolated from mice 2 weeks after sham and TAC surgery. Data shown as fold induction of gene or miRNA expression normalized to U6 (n = 5 in each group). The error bars represent SEM. *P < 0.05 compared with respective controls using Student’s t-test or one-way ANOVA with Tukey’s post hoc test.

Figure 2

The inhibition of miR-222 accelerates cardiac dysfunction induced by pressure overload. (A–C) Echocardiography analyses and representative images [A: EF, B: left ventricular posterior wall diastole (LVPWd), C: left ventricular internal dimension diastole (LVIDd)] of scrambled control LNA-anti-miR (ctl-anti) or specific LNA-anti-miR-222 (anti-222)-treated mice after sham or TAC surgery, *P < 0.05 vs. ctl-anti sham, ^#P < 0.05 vs. ctl-anti TAC. These include EF (A) LVPWd (B) LVIDd (C). Representative images are from 3 weeks after TAC. (D) HW/TL ratios of scrambled control (ctl-anti) and LNA-anti-miR-222 (anti-222) mice 1 and 3 weeks after sham or TAC (*P < 0.05 vs. ctl-anti-sham, ^#P < 0.05 vs. ctl-anti-TAC). (E) Quantification of the cardiomyocyte area from heart sections stained with WGA (n = 4, ∼500 cells per animal) demonstrates that miR-222 inhibition exaggerates pathological cardiomyocyte hypertrophy 1 week after TAC. (F) LW/TL ratios of scrambled control (ctl-anti) and LNA-anti-miR-222 (anti-222) mice 1 and 3 weeks after sham or TAC (*P < 0.05 vs. ctl-anti-sham, ^#P < 0.05 vs. ctl-anti-TAC). (G) An increase in the number of TUNEL-troponin I double-positive cells normalized to total 4',6-diamidino-2-phenylindole (DAPI)-labelled cells demonstrates increased apoptosis in miR-222-expressing hearts 3 weeks after TAC (n = 4 hearts in each group). (H) Representative images and quantification of the fibrotic area in Masson trichrome–stained heart sections (n = 3–7 hearts in each group) demonstrating no effect of miR-222 inhibition on fibrosis 1 week after TAC. n = 3 per group in ctl-anti-sham and anti-222 sham; n = 9 per group in ctl-anti-TAC and anti-222 TAC. The error bars represent SEM. *P < 0.05 compared with respective controls using Student’s t-test or one-way ANOVA with Tukey’s post hoc test.

Figure 3

Cardiac-specific expression of miR-222 prevents cardiac dysfunction induced by pressure overload and improves survival. (A–D) Echocardiography analyses and representative images of 4-month-old WT and miR-222 transgenic mice at baseline, 1, 2, and 3 weeks after TAC (n = 6 hearts in each group, *P < 0.05 and **P < 0.01 vs. corresponding WT control group by Student’s t-test). These include EF (A) LVPWd (B) LVIDd (C) representative images (D). These data demonstrate that miR-222 protects against pathological cardiac hypertrophy. (E) HW/TL ratios of 4-month-old WT and miR-222 transgenic mice 2 weeks after sham or TAC surgery (n = 7–9 hearts in each group, *P < 0.05 vs. WT sham, ^#P < 0.05 vs. WT TAC using one-way ANOVA with Tukey’s post hoc test). (F) Quantification of the cardiomyocyte area from heart sections stained with WGA (n = 4, ∼500 cells per animal, *P < 0.05 vs. WT sham, ^#P < 0.05 vs. WT TAC) demonstrate that miR-222 blocks TAC-induced cardiomyocyte hypertrophy. (G) LW/TL ratios of 4-month-old WT and miR-222 transgenic mice 2 weeks after sham or TAC surgery (n = 7–9 hearts in each group, *P < 0.05 vs. WT sham, ^#P < 0.05 vs. WT TAC). (H) Survival analysis shows that 4-month-old miR-222 transgenic mice had improved survival rates compared with WT controls 4 months after TAC (n = 9 in each group, *P < 0.05 vs. WT). (I and J) A qRT-PCR analysis of apoptosis markers (I) and fibrosis markers (J) in hearts from 4-month-old WT and miR-222 transgenic mice 2 weeks after sham or TAC surgery (n = 4–5 hearts in each group). These data demonstrate that miR-222 inhibits fibrosis and pro-apoptosis gene expression. (K) A decrease in the number of TUNEL-troponin I double-positive cells normalized to total DAPI-labelled cells demonstrates reduced apoptosis in cardiomyocytes in miR-222-expressing hearts 2 weeks after TAC (n = 4 hearts in each group). (L) MTS for the fibrosis area from heart sections and quantification (n = 4 hearts in each group) shows that miR-222 expression significantly inhibits fibrosis formation 2 weeks after TAC. *P < 0.05, vs. WT sham; ^#P < 0.05 vs. WT TAC using one-way ANOVA with Tukey’s post hoc test.

Figure 4

miR-222 directly targets HMBOX1, NFATc3, and PUMA in vitro and in vivo. (A) Luciferase assays in COS7 cells co-transfected with a control precursor (ctl scramble) or miR-222 precursor (pre-222) and reporter plasmids containing a fragment of the mouse NFATc3 3′UTR sequence including the WT or mutated miR-222 binding site. These data demonstrate that NFATc3 is a direct target of miR-222. (B and C) Cell viability and cytotoxicity in neonatal rat ventricular cardiomyocytes (NRVMs) treated with scrambled control (Ctl), miR-222 mimic (miR-222), or NFATc3 adenovirus (Ad-NFATc3) in the absence or presence of doxorubicin (Dox). (n = 4 in each group, *P < 0.05 vs. Ctl-Dox, ^#P < 0.05 vs. miR-222-Dox using one-way ANOVA with Tukey’s post hoc test). (D) mRNA expression of ANP, BNP, β/αMHC, and PGC1α in NRVMs treated with Ctl, miR-222, and Ad-NFATc3 in the absence or presence of phenylephrine (PE). (E) Representative images and quantification of NRVMs treated with Ctl, miR-222, and Ad-NFATc3 in the absence or presence of PE. (n = 4 in each group, *P < 0.05 vs. Ctl-PE, ^#P < 0.05 vs. miR-222-PE using one-way ANOVA with Tukey’s post hoc test). (F) Protein levels of the putative miR-222 targets in heart samples taken 1 week after sham or TAC surgery from WT or miR-222 transgenic mice. (G) Protein levels of the putative miR-222 targets in heart samples taken 1 week after sham or TAC surgery from mice treated with specific LNA-anti-miR-222 inhibitor (anti-222) or a scrambled control anti-miR (ctl-anti). Cumulative data are quantified below immunoblots and represented as fold change in protein expression, normalized to vinculin. These data demonstrate that miR-222 reduces the protein levels of all three targets in vivo. *P < 0.05 compared with WT sham or ctl-anti-sham and ^#P < 0.05 compared with WT TAC or ctl-anti TAC using Student’s t-test or one-way ANOVA.