MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype

Abstract

Rationale: Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This cancer-like phenotype is promoted by increased cytosolic calcium ([Ca²⁺]_cyto), aerobic glycolysis, and mitochondrial fission.

Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAH's cancer-like phenotype.

Methods: PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore-forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid-mediated up-regulation, as well as through modulation of the upstream microRNAs (miRs) miR-138 and miR-25. In vivo, nebulized anti-miRs were administered to rats with monocrotaline-induced PAH.

Measurements and main results: Impaired MCUC function, resulting from down-regulation of MCU and up-regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAH's pathogenesis. MCUC dysfunction decreases intramitochondrial calcium ([Ca²⁺]_mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca²⁺]_cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca²⁺]_cyto, raising [Ca²⁺]_mito, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR-138) down-regulate MCU directly and also by decreasing MCU's transcriptional regulator cAMP response element-binding protein 1. Nebulized anti-miRs against miR-25 and miR-138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model.

Conclusions: These results highlight miR-mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted.

Keywords: cAMP response element–binding protein; microRNA-25- and -138-5p; mitochondrial calcium uptake protein 1; pyruvate dehydrogenase.

Figure 1.

Reduced expression of the mitochondrial calcium uniporter (MCU) and decreased intramitochondrial calcium in pulmonary artery smooth muscle cells (PASMCs) in human and experimental pulmonary arterial hypertension (PAH). (A) Representative images and mean values for intramitochondrial calcium in control subject and patients with PAH measured using a calcium-sensitive fluorescent Förster resonance energy transfer (FRET) probe (a cyan fluorescent protein–calmodulin–yellow fluorescent protein construct) by confocal microscopy (n = 10; **P < 0.01). The FRET ratio (528/485 nm) depends on the probe’s calmodulin conformation, which is determined by the amount of bound calcium and shown with white reflecting higher intramitochondrial calcium concentration than red, as described (56). Ratio bar is from 0 to 2.5, scale bar = 5 μm. (B) Cytosolic calcium, measured using FURA-2, is increased in PAH PASMCs (n = 25; **P < 0.01). The amount of calcium within the sarcoplasmic reticulum (the pool released by cyclopiazonic acid [CPA]) is reduced in PAH versus control PASMCs (n = 25; **P < 0.01). (C) Quantitative reverse-transcription polymerase chain reaction measurement of mRNA expression for each of the MCU complex’s components. Only the expression of MCU mRNA is altered in human PAH PASMCs (n = 6–7; **P < 0.01). (D) Representative immunoblots and densitometric quantification demonstrating decreased MCU protein expression in human PAH versus control PASMCs (n = 4–7; **P < 0.01). (E) Confocal (left) and stimulated emission depletion (STED) super-resolution (right) images of the mitochondrial network of control (top) and PAH (bottom) human PASMCs. In normal patients, STED images reveal that MCU (green) is localized in uniform discrete bands spanning the inner mitochondrial membrane into the matrix, where NADH dehydrogenase (complex I, red) is located. Scale bar = 2 μm for confocal images and 0.5 μm for STED images. (F) Representative immunohistochemistry of human control versus PAH lungs (of n = 10 per group) demonstrating increased expression of MCU protein (brown) in the media and endothelium of pulmonary arteries (representative of >10 patients/group; see additional images in Figures E1A and E1B). The increased medial thickness (arrows) is visible in the small pulmonary arteries of patients with PAH. Scale bar = 100 μm. Representative immunoblots and densitometric quantification showing (G) decreased expression of MCU and its transcriptional regular cAMP response element–binding protein 1 (CREB1) in lungs from rats with monocrotaline (MCT)-PAH versus control rats (n = 5/group; **P < 0.01) and (H) increased expression of mitochondrial calcium uptake protein 1 (MICU1), a negative regulator of MCU, in human PAH PASMCs (n = 5–7; *P < 0.05). EMRE = essential MCU regulator.

Figure 2.

Decreased mitochondrial calcium uniporter (MCU) expression in pulmonary arterial hypertension (PAH) causes a reciprocal increase in cytosolic calcium concentration and mitochondrial fission. Cytosolic calcium was measured in human pulmonary artery smooth muscle cells (PASMCs) using fluo-3 and FURA Red (3 μM for 30 min) and expressed as the ratio of 530/640 nm emission measured at 488 nm. (A) Representative and mean data showing cytosolic calcium concentrations are elevated in normal PASMCs when MCU expression is reduced by exposure to small interfering RNA targeting MCU (si-MCU) (10 nM for 72 h), partially recapitulating the PAH phenotype (n = 3 individual cell lines per group, with a total of 6–17 cell images per line; *P < 0.05, **P < 0.01). (B) si-MCU reduces intramitochondrial calcium, demonstrating the role MCU expression plays in establishing a reciprocal relationship between mitochondrial and cytosolic calcium concentration (n = 3 control human PASMC lines, six discrete cell measurements per line; *P < 0.05). (C) Cytosolic calcium is higher in PAH versus control PASMCs. Overexpressing MCU by plasmid transfection (1 ng DNA/2 ml for 72 h) lowered the baseline cytosolic calcium level in PAH PASMCs to near normal control levels (A). In addition, MCU transfection greatly augmented the intramitochondrial calcium pool (which is reflected by the significantly greater increase in cytosolic calcium), which occurs in response to the mitochondrial uncoupling agent, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (1 μM) (n = 3 individual cell lines per group, with a total of 6–17 cell images per line; *P < 0.05, **P < 0.01). (D–G) PASMCs were transfected with photoactivatable, mitochondrial-targeted green fluorescent protein (Mito-PA-GFP) and then loaded with a potentiometric dye, tetramethylrhodamine (10 nM at 37°C for 30 min). Mitochondrial networking was evaluated with photoactivating Mito-PA-GFP as described in the Methods section in the online supplement. Mitochondrial networking factor (MNF), a metric that is proportionate to the degree of fusion, is calculated as the number of green pixels outside the white photoactivation boxes measured in the first image after photoactivation (5 s) divided by the number of green pixels within the activation area, as previously described (4, 5). Scale bar = 5 μm. Representative images (D and E) and mean ± SEM data (F) show a fused mitochondrial network (high MNF) in normal human PASMCs versus a fragmented network in identically imaged PAH PASMCs (low MNF). Inhibiting MCU expression in normal PASMCs (using si-MCU) recapitulates the PAH phenotype. Conversely, MCU overexpression in PAH PASMCs (using MCU plasmid transfection) restores the network fusion (n = 3 individuals per group, with experiments on each individual cell line repeated five to seven times; *P < 0.05; **P < 0.01). (G) The MCUC inhibitor ruthenium red (RuR; 10 μM for 48 h) mimics the effects of si-MCU, leading to fragmentation (reduced MNF). RuR had no additional effects on the already fragmented mitochondrial network of PAH PASMCs (n = 3 individuals per group, with experiments on each individual cell line repeated three to five times; **P < 0.01). [Ca²⁺]_cyto = cytosolic calcium concentration; [Ca²⁺]_mito = intramitochondrial calcium concentration; Con = control; si-control = scrambled siRNA control.

Figure 3.

Mitochondrial calcium uniporter (MCU) gene transfer restores pyruvate dehydrogenase (PDH) activity and improves the coupling of glycolysis to glucose oxidation in pulmonary arterial hypertension (PAH) pulmonary artery smooth muscle cells (PASMCs). (A–I) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as markers of mitochondrial and glycolytic flux were measured in PASMCs using the Seahorse XF^e24 Extracellular Flux Analyzer (Seahorse XFe24, Agilent Technologies Canada Inc., Mississauga, ON, Canada), as previously described (8). All measurements were made on n = 25,000–50,000 cells/well and five wells per cell line (sample). Each experimental group consisted of two to three patient cell lines (samples). All data were normalized to total protein per well before analysis. (A) OCR (left) is higher and ECAR (right) is lower in naive normal PASMCs compared with PAH PASMCs. (B) Glycolytic activity is higher in naive PAH PASMCs compared with normal. (C) Naive normal PASMCs have a higher OCR/ECAR ratio than PAH PASMCs (****P < 0.0001). (D) Silencing MCU using small interfering RNA targeting MCU (si-MCU) reduced OCR (left) and increased ECAR (right) in normal PASMCs. (E) Silencing MCU increased glycolytic activity in normal PASMCs. (F) si-MCU decreases the OCR/ECAR ratio in normal PASMCs (****P < 0.0001). (G) OCR is markedly reduced in PAH PASMCs with and without MCU overexpression (MCU ox) (vs. control PASMCs; note smaller scale compared with A and D). Overexpression of MCU (via transfection of MCU plasmid) has little effect on OCR (left) but disproportionately lowers ECAR (right). (H) Glycolytic activity is decreased in PAH PASMCs with MCU overexpression compared with control PAH. (I) MCU overexpression increases the OCR/ECAR ratio in PAH PASMCs (****P < 0.0001). (J) Representative images and mean data (n = 4/group; *P < 0.05) showing that PDH activity is greater in control PASMCs than in PAH PASMCs and that in both cell types PDH activity is inhibited by the MCU complex inhibitor ruthenium red (RuR; 10 μM) or Si-MCU. PDH activity was measured in homogenized PASMCs using a PDH activity dipstick in which the intensity of the nitroblue tetrazolium band (arrows) is proportionate to enzyme activity (ab109882; MitoSciences-Abcam, Eugene, OR). (K) Lactate production is increased in normal PASMCs by inhibiting MCU expression (using si-MCU) and is reduced in PAH PASMCs by overexpressing MCU (by plasmid transfection) (n = 4; *P < 0.05). Data in panels A, B, C, D, and H are mean ± SEM. 2-DG = 2-deoxyglucose; Anti A/Rote = antimycin A and rotenone; FCCP = carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; NC = negative control small interfering RNA.

Figure 4.

Pulmonary artery smooth muscle cell (PASMC) migration, proliferation, and apoptosis are regulated by the mitochondrial calcium uniporter (MCU). (A and B) PASMC migration (basal or stimulated by platelet-derived growth factor [PDGF]) was measured using the iCELLigence system, an impedance-based system of measuring cell number and cell movement (ACEA Biosciences, San Diego, CA) wherein higher slope indicates faster migration. Ruthenium red (RuR; 10 μM for 6 h) increased PASMC migration in control (A) and pulmonary arterial hypertension (PAH) (B) PASMCs. The increase was additive to that achieved with PDGF in PAH PASMCs (*P < 0.05; n = 3/group). (C and D) Rates of PASMC proliferation are faster in PAH versus control PASMCs. RuR (10 μM for 37–41 h) and small interfering RNA targeting MCU (si-MCU) increase rates of cell proliferation in both control and PAH PASMCs (**P < 0.01; n = 5/group). Proliferation was measured using a Click-iTEdU kit (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. (E) Transfection with the MCU plasmid reduces rates of cell proliferation in both control and PAH PASMCs. Values are mean ± SEM; **P < 0.01; n = 5/group. (F) Inhibition of the negative regulator of the mitochondrial calcium uniporter complex, mitochondrial calcium uptake protein 1 (MICU1), using si-MICU1, reduces cell proliferation in PAH PASMCs (**P < 0.01; n = 4/group). (G) Transfection with the MCU plasmid increases baseline apoptosis in PAH PASMCs cultured in serum (*P < 0.05; n = 4/group).

Figure 5.

Down-regulation of mitochondrial calcium uniporter (MCU) results from increased expression of microRNA (miR)-138 but is reinforced by miR-25 and reduced expression of cAMP response element–binding protein 1 (CREB1). The nomenclature of miRs is in evolution, and because the sequence of rat and human miRs is the same, we use the more generic names, miR-138 and miR-25. However, all anti-miRs were constructed against the human miR sequences for hsa-miR-138-5p and hsa-miR-25-3p. (A) Expression of miR-138 is increased in pulmonary arterial hypertension (PAH) pulmonary artery smooth muscle cells (PASMCs) (*P < 0.01; n = 9–12/group). Note the trend for increased expression of miR-25 (P = NS). (B) HEK293A cells were transfected with a luciferase reporter bearing the 3′-UTR of the MCU gene, as well as a miR control, (A) miR-25 or (B) miR-138. Both miR-25 and miR-138 were found to repress the activity of luciferase reporter, indicating their binding to their target sites in the MCU 3′-UTR (*P < 0.05; n = 6). (C and D) Anti–miR-138 and anti–miR-25 restore expression of (C) MCU and (D) CREB1 mRNA (top) and protein (bottom) (*P < 0.01; n = 3/group). (E) CREB small interfering RNA (si-CREB) does not significantly increase expression of miR-25 or miR-138 (*P < 0.05; ***P < 0.001; n = 3/group). (F) si-CREB1 transfection reduces the expression of CREB1 in normal PASMCs (immunoblot and aggregate data) (*P < 0.05; **P < 0.01; n = 4/group). This leads to a reduction in the expression of MCU protein, thereby confirming the relevance of the observed down-regulation of CREB1 in PAH to the associated reduction in MCU expression in PAH (Figure 6B). (G) Quantitative reverse-transcription polymerase chain reaction (top) and immunoblot and aggregate data showing that CREB1 and phospho-CREB1 (serine-133; Ser-133p-CREB) expression are decreased in PAH versus control PASMCs (*P < 0.05; **P < 0.01; n = 4–6/group). (H) Representative images and mean data showing that expression of activated CREB1, defined as CREB IF (green) in the PASMC nucleus, is increased in PAH (middle column). Scale bars = 10 μm (top) and 50 μm (bottom). CREB1 intensity is expressed in arbitrary fluorescent units (AFU). In addition, a higher percentage of PAH PASMCs versus control PASMCs have detectable CREB1 immunofluorescence in the nucleus (colocalized with 4′,6-diamidino-2-phenylindole [DAPI]) (*P < 0.05; n = 3 lines per group; discrete three to four measurements per line). The negative control had no primary antibody. p-ATF-1 = activating transcription factor-1; si-control = scrambled siRNA control; UTR = untranslated region.

Figure 6.

Anti-microRNA (miR) therapy regresses pulmonary arterial hypertension (PAH) in vivo. (A) Protocol for the administration of the anti-miRs in rats with established PAH. Ten days after injection of monocrotaline (MCT; 60 mg/kg subcutaneous; n = 10 rats/group) rats were anesthetized and nebulized biweekly for 2 weeks with either negative control (5 nmol in 50 μl, mirVana miRNA inhibitor negative control#1, Ambion), anti–miR-25, or anti–miR-138 (5 nmol in 50 μl, mirVana miRNA inhibitor, Ambion). (B) Compared with control rats (n = 9), MCT caused severe PAH with elevations of total pulmonary vascular resistance (TPR) and right ventricular (RV) hypertrophy, evident as an increase in Fulton index, and RV failure, evident as an increase in end diastolic pressure (EDP). Anti–miR-138 was more effective than anti–miR-25 in increasing cardiac output (CO; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). PAP = pulmonary arterial pressure; RVSP = right ventricle systolic pressure. (C) Summary data and (D) representative images demonstrating that the regression of PAH was accompanied by a reduction in the medial thickness of small pulmonary arteries (****P < 0.0001). Scale bar = 25 μm. (E) Representative immunofluorescence images of mitochondrial calcium uniporter (MCU) expression (red) in pulmonary arterioles (α-smooth muscle actin [αSMA], green) from MCT challenged rats, with and without treatment with nebulized anti–miR-25 or anti–miR-138. Nuclei are stained in blue. (F) Schematic illustrating the proposed model in which increases in miR-138, reinforced by increases in other miRs including miR-25, suppress both cAMP response element–binding protein 1 (CREB1) and the MCU itself. miR-138 directly inhibits expression of MCU and reinforces this effect indirectly by down-regulating CREB1. The loss of MCU expression, exacerbated by increased expression of mitochondrial calcium uptake protein 1 (MICU1), reduces the function of the MCU complex. This simultaneously overloads cytosolic calcium while depriving the mitochondria of the calcium. The former triggers pulmonary artery smooth muscle cell migration and proliferation (and vasoconstriction), whereas the latter affects the mitochondrion, inhibiting pyruvate dehydrogenase and promoting a shift to uncoupled glycolysis (the Warburg phenomenon), further driving cell proliferation and apoptosis resistance. Changes in intracellular calcium homeostasis also promote mitochondrial fission by activating Drp1, a known driver of cell proliferation (4). There are several major pathways for calcium extrusion from the matrix that were not studied, notably NCLX (recently identified Na⁺–Ca²⁺ exchanger [20]) and the H⁺–Ca²⁺ exchanger (21). It is possible that changes in efflux mechanisms might also occur in PAH. DAPI = 4′,6-diamidino-2-phenylindole [DAPI]; Drp1 = dynamin related protein 1; IP3 = inositol 1,4,5-trisphosphate receptor; LVS = left ventricle + septum; VDAC = voltage-dependent anion channel.