Cardiomyocyte-restricted MIAT deletion is sufficient to protect against murine myocardial infarction

Abstract

Myocardial infarction-associated transcript (MIAT), an intergenic long noncoding RNA (lncRNA), is conserved between rodents and humans and is directly linked to maladaptive cardiac remodeling in both patients and mouse models with various forms of heart failure (HF). We previously reported attenuation of cardiac stress, apoptosis, and fibrosis in a murine model of myocardial infarction (MI) with global MIAT ablation. Our transcriptomic profiling and mechanistic studies further revealed MIAT-induced activation of maladaptive genes, such as Hoxa4, Fmo2, Lrrn4, Marveld3, and Fat4. However, the source of MIAT and its contribution to MI and HF remain unknown. In this study, we generate a novel cardiomyocyte (CM)-specific MIAT conditional knockout mouse model, which exhibits improved cardiac function after MI. We further report that CM-specific MIAT ablation is sufficient to reduce cardiac damage, apoptosis, and fibrosis following chronic MI. Mechanistically, CM-specific MIAT deletion in mice leads to decreased expression of proapoptotic and pathological profibrotic genes, such as p53, Bak1, Col3a1, Col6a1, Postn, and Snail1 after chronic MI. These results enable us to begin to dissect cell-specific contributions to MIAT signaling and bolster the idea that MIAT plays a direct pathological role in CMs after MI.

Fig. 1. Establishment and confirmation of a novel cardiomyocyte-specific MIAT conditional knockout mouse line.

A Targeting scheme, mouse crossing, and establishment of cardiomyocyte (CM)-restricted conditional knockout (cKO) of MIAT in vivo. Top, Targeting scheme and mouse crossing. Bottom, Representative genotyping results of MIAT^fl/fl and αMHC-Cre mice. Target (tg) and control (ctrl) bands are shown. Neg.= negative control and Pos.= positive control. B–D QRT-PCR analyses of MIAT (B), miR-150-5p (a known direct target of MIAT; C), and Sprr1a (a known direct target of miR-150-5p; D) in left ventricles from adult MIAT^fl/fl or MIAT cKO (i.e., MIAT^fl/fl;αMHC-Cre) mice. N = 3–4 per group. Data are presented as mean ± SEM. Unpaired 2-tailed t-test. *P < 0.05 or **P < 0.01 vs. MIAT^fl/fl mice.

Fig. 2. Cardiomyocyte-restricted MIAT deletion in mice blunts cardiac dysfunction after myocardial infarction.

A–F Transthoracic echocardiography was performed on the four experimental groups (sham and MI of MIAT^fl/fl and CM-specific MIAT cKO) at 0–28 days (d) post-myocardial infarction (MI). Quantification of left ventricular (LV) ejection fraction (EF: A), fractional shortening (FS: B), end-diastolic volume (LVEDV: C), end-systolic volume (LVESV: D), internal diameter in diastole (LVIDd: E), and internal diameter in systole (LVIDs: F) is shown. N = 18–20 per group. Data are presented as mean ± SD. Two-way repeated-measures ANOVA with Bonferroni’s post hoc test. ***P < 0.001 vs. Sham of same genotype (denoted by different colors for sham within same group); ^#P < 0.05, ^##P < 0.01, or ^###P < 0.001 vs. MI MIAT^fl/fl.

Fig. 3. Selective deletion of MIAT in cardiomyocytes reduces damage and the expression of proinflammatory Il-1b in the heart after chronic myocardial infarction.

A Representative hematoxylin and eosin (H&E) staining of heart sections of the peri-ischemic border area at 4 weeks post-MI shows a decrease in disorganized structure in CM-specific MIAT cKO hearts compared to MIAT^fl/fl controls. Scale bars: 100μm. B–D QRT-PCR analysis of Nppa, Nppb, and Myh7 expression representing cardiac damage in ischemic areas from CM-specific MIAT cKO hearts compared to MIAT^fl/fl controls at 4 weeks post-MI. E QRT-PCR analysis of Il-1b expression for cardiac inflammation in ischemic areas from CM-specific MIAT cKO hearts compared to MIAT^fl/fl controls at 4 weeks post-MI. N = 3 per group. QRT-PCR data are shown as fold induction of gene expression normalized to Gapdh. Data are presented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, or ***P < 0.001 vs. sham of same genotype; ^#P < 0.05 or ^##P < 0.01 vs. MI MIAT^fl/fl.

Fig. 4. Cardiomyocyte-specific MIAT loss in mice alleviates cardiac apoptosis following chronic myocardial infarction.

A–B Representative cleaved-caspase 3 staining images in heart sections of the peri-ischemic border area at 4 weeks post-MI (A) and quantification of apoptosis in six 20x fields (B). Scale bars: 50μm. C–D, QRT-PCR analysis of proapoptotic p53 and Bak1 expression in the ischemic areas from CM-specific MIAT cKO hearts compared to MIAT^fl/fl controls at post-MI 4 weeks. QRT-PCR data are shown as fold induction of gene expression normalized to Gapdh. N = 3–7 per group. Data are presented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, or ***P < 0.001 vs. sham of same genotype; ^#P < 0.05, ^##P < 0.01, or ^###P < 0.001 vs. MI MIAT^fl/fl.

Fig. 5. Selective ablation of MIAT in cardiomyocytes suppresses cardiac fibrosis after chronic myocardial infarction.

Representative Masson’s Trichrome staining (A–B) in heart sections from the four experimental groups at 4 weeks post-MI and fibrosis quantification (C). Fibrosis histology images from whole heart sections (A, Scale bars: 1 mm) and zoomed in images of the peri-ischemic border area (B, Scale bars: 100 μm). N = 6–7 per group. Data are presented as the mean ± SEM. Two-way ANOVA with Tukey’s multiple comparison test. *P < 0.05 or ***P < 0.001 vs. sham of same genotype; ^##P < 0.00 vs. MI MIAT^fl/fl.

Fig. 6. Selective knockdown of MIAT in cardiomyocytes decreases the cardiac expression of profibrotic genes post-myocardial infarction.

QRT-PCR analysis of profibrotic Col3a1 (A), Col6a1 (B), Postn (C), or Snail1 (D) expression in ischemic areas from MIAT^fl/fl and CM-specific MIAT cKO mouse left ventricles at 4 weeks post-MI. Data are shown as the fold induction of gene expression normalized to Gapdh. N = 3 per group. Data are presented as the mean ± SEM. Two-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, or ***P < 0.001 vs. sham of same genotype; ^#P < 0.05 or ^##P < 0.01 vs. MI MIAT^fl/fl.

Fig. 7. Generation of a novel MIAT floxed mouse line.

A Targeting strategy of MIAT conditional knockout mouse model. Flox region is hypothetical exon 1–3. B Genotyping strategies to screen embryonic stem (ES) cells. 5’ homologous arm: 9.1 kb fragment should be amplified in the homologous recombinant ES cell clones, and none of fragment should be amplified in the negative ES cell clones. 3’ homologous arm: 5.7 kb fragment should be amplified in the homologous recombinant ES cell clones, and 12.4 kb fragment should be amplified in the negative ES cell clones. As shown in agarose gel electrophoresis of PCR products, six positive homologous recombinant ES clones were identified. C–D Genotyping strategies to screen F1 MIAT^fl/+ mice. The chimeric male mice were crossed with Flp mice to generate F1 mice. 5’ homologous arm: 8.6 kb fragment should be amplified in the homologous recombinant F1 mice, and 11.7 kb fragment should be amplified in the negative F1 mice. 3’ homologous arm: 3.9 kb fragment should be amplified in the homologous recombinant F1 mice, and 12.4 kb fragment should be amplified in the negative F1 mice. By long-PCR identification, seven heterozygous F1 mice were identified. All positive PCR products were confirmed by sequencing. Regions 1 and 2 were for identifying the 5’ homologous recombination, and regions 3 and 4 were for identifying the 3’ homologous recombination (C). For genotyping the offspring, short-PCR was used to identify heterozygous (HE) and wild-type (WT) mice. M: DNA size marker shown in the right (D). Genotyping PCR images show germline transmission of the targeted MIAT floxed allele. Agarose gel electrophoresis of PCR products is shown in the bottom (C–D).