MicroRNA-214 protects the mouse heart from ischemic injury by controlling Ca²⁺ overload and cell death

Abstract

Early reperfusion of ischemic cardiac tissue remains the most effective intervention for improving clinical outcome following myocardial infarction. However, abnormal increases in intracellular Ca²⁺ during myocardial reperfusion can cause cardiomyocyte death and consequent loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Therapeutic modulation of Ca²⁺ handling provides some cardioprotection against the paradoxical effects of restoring blood flow to the heart, highlighting the significance of Ca²⁺ overload to IR injury. Cardiac IR is also accompanied by dynamic changes in the expression of microRNAs (miRNAs); for example, miR-214 is upregulated during ischemic injury and heart failure, but its potential role in these processes is unknown. Here, we show that genetic deletion of miR-214 in mice causes loss of cardiac contractility, increased apoptosis, and excessive fibrosis in response to IR injury. The cardioprotective roles of miR-214 during IR injury were attributed to repression of the mRNA encoding sodium/calcium exchanger 1 (Ncx1), a key regulator of Ca²⁺ influx; and to repression of several downstream effectors of Ca²⁺ signaling that mediate cell death. These findings reveal a pivotal role for miR-214 as a regulator of cardiomyocyte Ca²⁺ homeostasis and survival during cardiac injury.

Figure 1. miR-214 genomic structure and genetic deletion.

(A) Schematic representation of the mouse miR-214 locus and its host gene, Dnm3. Boxes represent exons of the Dnm3 gene. miR-214 and miR-199a-2 are clustered on the opposite strand within the non-coding RNA Dnm3os. Conservation of miR-214 is shown, and the seed region is highlighted. (B) miR-214 expression levels in the heart at various embryonic and postnatal stages according to miR-214–specific RT and qPCR. Data were normalized to RNU6B and expressed relative to levels at P1. (C) Northern blots show the relative expression level of miR-214 in WT adult mouse tissues. E10.5 heart RNA is included as a reference. U6 is a loading control. Blot is representative of 2 different sets of tissues analyzed. (D) Absence of miR-214 and preservation of miR-199a expression in KO hearts as shown by representative Northern blot from heart tissue of WT, heterozygous (HET), or miR-214 KO mice. U6 is a loading control. Sk, skeletal.

Figure 2. Normal cardiac structure and function in miR-214 KO mice at baseline.

(A) Heart sections stained with H&E from WT and miR-214 KO mice at 8 weeks of age. Heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios shown are representative of adult mice at 3 different ages. Mean values ± SEM; n = 3. Scale bar: 2 mm. (B) qPCR expression analysis of cardiac stress response genes in miR-214 KO hearts relative to WT. n = 3 per group; data are representative of 2 separate experiments. Data are shown as fold induction of gene expression normalized to 18S and expressed as mean ± SEM. (C) Representative electron microscopy images from a miR-214 heterozygote and 2 miR-214 KO hearts at 8 weeks of age highlighting sarcomeric structure and mitochondria. Scale bars: 1 μm (top row) and 200 nm (bottom row).

Figure 3. miR-214 protects the heart against MI and IR.

(A) Survival curve following MI (permanent LAD ligation) in miR-214 KO mice or WT littermates. n = 13–15, data represent mice from 3 different experiments. *P < 0.05. (B) Northern blotting and quantification of miR-214 expression in WT hearts at baseline and following IR. miR-214 levels were normalized to U6 loading control and expressed relative to baseline. *P = 0.04, **P < 0.01. (C) Cardiac function in WT and miR-214 KO mice before and after IR (7 days reperfusion). Quantification of left ventricular internal diameter in systole or diastole (LVIDs or LVIDd), fractional shortening (FS) and ejection fraction (EF), is shown. n = 6; data represent mean ± SEM of 3 independent experiments. **P < 0.01, ***P < 0.001. (D) TUNEL staining in heart sections following IR. Representative images at 24 hours and 7 days of reperfusion are shown. Scale bar: 200 μm. The percentage of TUNEL-positive nuclei was calculated. For each mouse, sections at 3 different levels (6–8 fields per section) were counted. Bottom: Simultaneous TUNEL (green) and desmin staining (red) from miR-214 KO heart sections following IR. Scale bar: 40 μm. (E) Masson’s trichrome staining on transverse heart sections after 7 days of reperfusion. Representative images shown at two different magnifications. Scale bars: 2 mm (top), 100 μM (bottom). The area of blue staining was quantified (3 section levels per heart) and expressed as a percentage of total area. For D and E, mean ± SEM; n = 3; *P < 0.05.

Figure 4. miR-214 regulates NCX1.

(A) Predicted miR-214 binding sites in the 3′-UTR of Ncx1 mRNA. Ncx1 contains 3 conserved sites. Site position relative to beginning of the 3′-UTR is indicated above. Seed and target sequences are highlighted in red, and base pairing between miR-214 and target site marked by vertical lines. (B) Ability of miR-214 to directly repress activity of the luciferase reporter construct that contains the portion of the Ncx1 3′-UTR that includes site 3 (pmiR-Ncx1 site 3). WT and mutant Ncx1 3′-UTR sequences were tested. Black triangles indicate increasing amounts of transfected miR-214 expression plasmid (0, 50, 100, and 200 ng). Luciferase activity was normalized to β-galactosidase activity and compared with empty vector measurements (pmiR empty). Luciferase assays were performed in triplicate and are representative of 2–3 independent experiments. Data are mean ± SEM. **P < 0.01, ^#P < 0.001. (C) NCX1 protein levels measured by immunoblotting in whole heart lysates from miR-214 KO mice compared with WT at baseline and after IR (24 hours and 7 days). Quantification was normalized to tubulin as a loading control and then compared with WT. Data are representative of 2 independent experiments. Mean ± SEM; n = 3. *P < 0.04, **P < 0.01.

Figure 5. miR-214 regulation of Ca²⁺ signaling and cell death genes.

(A) Predicted miR-214 binding sites in the 3′-UTR of Bim, CamkIId, and Ppif mRNA. Bim contains 4 predicted sites, while CamkIId and Ppif each contain one. Site position relative to beginning of the 3′-UTR is indicated above each panel. Seed and target sequences are highlighted in red and base pairing between miR-214 and target site marked by vertical lines. (B and C) Target protein levels measured by immunoblotting in whole heart lysates from miR-214 KO mice compared with control at baseline (B) and after IR (7 days) (C). Quantification is normalized to indicated loading control and then compared with WT. Data are representative of 2 independent experiments. Mean ± SEM; n = 3. *P < 0.05, **P < 0.01. (D) Ability of miR-214 to directly repress the activity of luciferase reporter constructs that contain the 3′-UTR for CamkIId and Ppif as indicated. Transfection with or without miR-214 expression plasmid is indicated. Luciferase activity was normalized to β-galactosidase activity and compared with empty vector measurements. Luciferase assays were performed in triplicate and are representative of 2–3 independent experiments. Data are mean ± SEM. *P < 0.05.

Figure 6. miR-214 is required to maintain efficient Ca²⁺ handling.

(A) Representative Ca²⁺ traces obtained from isolated WT or miR-214 KO cardiomyocytes. Myocytes were imaged in the presence of 1.8 mM or 5.0 mM extracellular Ca²⁺. n = 15–20 cells, with 3–5 animals per group for all experiments. (B) Quantitative analyses of intracellular Ca²⁺ levels shown as corrected average peak Ca²⁺ transients in WT and miR-214 KO cardiomyocytes in 1.8 mM or 5.0 mM extracellular Ca²⁺ (top). Ca²⁺ transient decay shown as τ (seconds) comparing WT and KO at 1.8 mM and 5.0 mM extracellular Ca²⁺ (bottom). All data are mean ± SEM; ***P < 0.0001.

Figure 7. miR-214 protection of cardiomyocytes and regulation of target genes in vitro.

(A) RNA isolated from neonatal rat cardiomyocytes transfected with antimiR-214 or 15-mer control (100 nM) was analyzed by miR-214–specific RT-PCR and qPCR to assess levels of miR-214 suppression. **P < 0.01. (B and C) TUNEL staining of neonatal rat cardiomyocytes transfected with control or antimiR-214 and then subjected to in vitro simulation of IR. (B) Representative images of total nuclei (blue) and apoptotic nuclei (red) from normoxic and both IR conditions are shown for control and antimiR-214 transfected samples. Scale bar: 100 μm. (C) Quantification of percent TUNEL-positive nuclei. Samples were assayed in triplicate, and results are representative of 2–3 independent experiments. Data are mean ± SEM; **P < 0.01. (D) Levels of miR-214 target mRNAs in cardiomyocytes transfected with antimiR-214 or control antimiR as indicated. Data were normalized to ribosomal 18S and expressed relative to control. Samples were run in triplicate, and results are representative of 2 independent experiments. All data are mean ± SEM; *P = 0.02, **P < 0.01.

Figure 8. Model demonstrating miR-214 cardioprotection against Ca²⁺ overload injury and cell death.

Ischemic injury leads to Ca²⁺ overload, causing a switch to reverse mode NCX1 activity in cardiomyocytes that enhances Ca²⁺ overload and leads to cell death via downstream effectors of Ca²⁺ signaling. miR-214, also induced by ischemic injury, protects the myocyte from damage by attenuating NCX1 levels to prevent excessive Ca²⁺ influx into the cytoplasm. Additional protection by miR-214 occurs through suppression of the Ca²⁺ effector kinase CaMKII and the cell death mediators CypD and BIM. In the absence of miR-214 expression in the heart, higher levels of reverse mode NCX1 and Ca²⁺ effectors further perpetuate Ca²⁺ overload and cell death during IR, resulting in greater impairment of cardiac function.