Mitochondrial Calcium Uniporter Regulates Metabolic Remodeling and Smooth Muscle Cell Proliferation in Type 2 Diabetes

Abstract

Background: Excessive proliferation of vascular smooth muscle cells (VSMCs) is a consequence of type 2 diabetes (T2D) that increases the risk for atherosclerosis and restenosis after angioplasty. Here, we sought to determine whether and how mitochondrial dysfunction in T2D drives VSMC proliferation with a focus on increased reactive oxygen species and intracellular [Ca²⁺] that both drive cell proliferation, occur in T2D, and are regulated by the mitochondrial Ca²⁺ uniporter (MCU).

Methods: Using a mouse model of T2D, we performed in vivo phenotyping after mechanical injury and established the mechanisms of excessive proliferation in cultured VSMCs. The T2D model was induced by high-fat diet and low-dose streptozotocin in both wild type mice and mice with the VSMC-specific inhibition of the mtCaMKII (mitochondrial Ca²⁺/calmodulin-dependent kinase IImtCaMKII), a regulator of Ca²⁺ entry via the MCU.

Results: In VSMCs from T2D model mice, MCU inhibition reduced both in vivo neointima formation after mechanical injury, as well as in vitro proliferation of cultured VSMCs. Further, in VSMCs from T2D mice, the composition of the MCU complex and MCU activity were altered with loss of MICU1 (mitochondrial calcium uptake 1). In addition, VSMC mitochondrial reactive oxygen species was elevated and mitochondrial respiration blunted. The increase in cytosolic reactive oxygen species induced activation of G6PD (glucose-6-phosphate dehydrogenase), a key enzyme of the pentose phosphate pathway. However, inhibiting MCU or MICU1 overexpression on VSMCs from T2D mice decreased intracellular reactive oxygen species, preserved mitochondrial respiration and ATP production, decreased activity of G6PD, and normalized cell proliferation. These data suggest the MCU complex controls a T2D-induced metabolic switch that promotes cell proliferation.

Conclusions: Collectively, these data indicate that MCU complex remodeling in T2D drives neointimal restenosis, suggesting MCU as a therapeutic target.

Keywords: diabetes; metabolism; mitochondria; neointima; vascular smooth muscle cells.

Figure 1. Inhibition of MCU by mtCaMKIIN prevents neointimal hyperplasia after vascular injury.

A, Blood glucose levels over time after loading with glucose (2.0 g/kg i.v.), in NG mice and T2D mice of the WT and mtCaMKIIN genotypes (n=5–10). B and C, Levels of (B) cholesterol (n=10–20 mice per condition) and (C) insulin (n=3–6 mice per condition) in serum from NG and T2D mice of the WT and mtCaMKIIN genotypes. D, Verhoeff‐Van Gieson staining of carotid arteries 21 days after endothelial denudation. The yellow line demarcates the internal elastic lamina. E, Neointimal area at 100 μm from the bifurcation of the common carotid artery (n=8 mice per condition). F, Number of nuclei and (G) percentage of PCNA‐positive cells in the neointima at the same level as in (E; n=6 mice per condition). Statistical analyses were performed using 2‐way ANOVA (A), Kruskal–Wallis test (B and C), and Mann–Whitney test (E–G). mtCaMKIIN indicates mitochondrial calcium and calmodulin‐dependent protein kinase II inhibiting peptide; NG, normoglycemic; PCNA, proliferating cell nuclear antigen; T2D, type 2 diabetes; and WT, wild type.

Figure 2. T2D‐induced MCU1 deficiency triggers phosphorylation of MCU at Serine 92.

A, Representative immunoblots assessing phosphorylation (ie, activation) of CaMKII (p‐CaMKII, Thr287) and MCU (p‐MCU, Ser92) in mitochondrial fractions of VSMCs from NG and T2D WT mice. B and C, Quantification of immunoblots for (B) p‐CaMKII normalized (n=9) to CaMKII and (C) p‐MCU normalized to MCU (n=6) as in (A). D, Representative immunoblots assessing the expression of MICU1 and MCU in mitochondrial fractions from cultured VSMCs isolated from NG and T2D WT mice. E, Quantification of immunoblots for MICU1 normalized to COX IV (n=5) as in (D). F and G, Blue‐native PAGE (F) and SDS PAGE (G) of mitochondrial fractions of VSMCs isolated from NG and T2D WT mice, immunoblotted for MICU1 and MCU (F), or MCU and COX IV (G; n=3). H, Representative images of coronary artery sections from autopsies of patients who were normoglycemic and patients with T2D, immunostained for MICU1 and (SMC‐actin). Scale bars: 500 μm (4×) and 100 μm (insert, 20×; n=2). I, Representative immunoblots showing p‐MCU (Ser92) in mitochondrial fractions of VSMCs from T2D WT mice transduced with adenoviruses expressing MICU1, mtCaMKIIN, or control. J, Quantification of immunoblots for p‐MCU normalized to MCU (n=3) as in (I). Statistical analyses were performed Mann–Whitney test (B, C, E) and Kruskal–Wallis for (J). CaMKII indicates calcium and calmodulin‐dependent protein kinase II; MCU, mitochondrial calcium uniporter; MICU1, mitochondrial calcium uptake 1; mtCaMKIIN, mitochondrial calcium and calmodulin‐dependent protein kinase II inhibiting peptide; NG, normoglycemic; Ser92, Serine 92; SMC‐actin, smooth muscle actin; T2D, type 2 diabetes; VSMC, vascular smooth muscle cell; and WT, wild type.

Figure 3. MCU phosphorylation at Ser92 regulates proliferation, mitochondrial calcium uptake, and mitochondrial ROS in T2D VSMCs.

A, Cell counts of VSMCs isolated from WT NG mice, electroporated with WT or S92D MCU and counted after 72 hours in culture with or without PDGF (20 ng/mL). Data are expressed as fold change over levels at 0 hours (n=10). B, Cell counts of VSMCs isolated from NG and T2D WT mice counted after 72 hours in culture with or without PDGF (20 ng/mL). Data are expressed as fold change over levels at 0 hours (n=10). C, Cell counts of VSMCs isolated from T2D WT mice, transduced with empty or MICU1 adenoviruses and counted after 72 hours in culture with or without PDGF (20 ng/mL). Data are expressed as fold change over levels at 0 hours (n=5). D, Cell counts of VSMCs isolated from T2D WT mice, transduced with empty or mtCaMKIIN adenoviruses and counted after 72 hours in culture with or without PDGF (20 ng/mL). Data are expressed as fold change over levels at 0 hours (n=6). E through H, Mitochondrial Ca²⁺ uptake over time (as measured by mtPericam) in cultured, permeabilized VSMCs from NG (E) and T2D WT mice (F), and VSMCs from T2D WT mice overexpressing MICU1 (G) or mtCaMKIIN (H) with CaCl₂ added at 0.1, 1, and 3 μmol/L by adenoviral transduction for 72 hours (n=4). Statistical analyses were performed using Kruskal–Wallis test for (A through D). MCU indicates mitochondrial calcium uniporter; MICU1, mitochondrial calcium uptake 1; mtCaMKIIN, mitochondrial calcium and calmodulin‐dependent protein kinase II inhibiting peptide; NG, normoglycemic; PDGF, platelet‐derived growth factor; ROS, reactive oxygen species; Ser92, Serine 92; T2D, type 2 diabetes; VSMC, vascular smooth muscle cell; and WT, wild type.

Figure 4. Increased mitochondrial and cytosolic ROS are driven by MCU complex activity in T2D VSMCs.

A and B, Representative images of mitochondrial ROS levels as measured by roGFP targeted to the mitochondrial matrix in cultured VSMCs from NG and T2D WT mice, and vascular smooth muscle cells (VSMCs) from T2D mice overexpressing mtCaMKIIN or MICU1 by adenoviral transduction for 72 hours. B, Quantification of mitochondrial ROS levels as measured by roGFP targeted to the mitochondrial matrix (n=6). C and D, Cytosolic ROS levels as measured by untargeted roGFP in cultured VSMCs from NG and T2D WT mice, and VSMCs from T2D mice overexpressing mtCaMKIIN or MICU1 by adenoviral transduction for 72 hours (C). D, Quantification of cytosolic ROS levels as measured by roGFP (n=8). E and F, Immunoblots for 4‐HNE modified proteins in cytosolic and mitochondrial fractions of mouse aortas dissected from WT NG and T2D and T2D mtCaMKIIN mice (E). (F) Quantification of 4‐HNE normalized to GAPDH for cytosolic fractions and to TOM20 for mitochondrial fractions (n=6). G through I, Cell counts of VSMCs isolated from WT NG (G), T2D (H), and T2D mtCaMKIIN (I) mice counted after 72 hours in culture with or without PDGF (20 ng/mL), with application of ROS scavengers—cytosolic (TEMPO, 1 mmol/L) and mitochondrial (10 μmol/L). Data are expressed as fold change over numbers at 0 hours (n=6–8). Statistical analyses were performed using Kruskal–Wallis test for (B), (D), (F–I). 4‐HNE indicates 4‐hydroxynonenal; MICU1, mitochondrial calcium uptake 1; mtCaMKIIN, mitochondrial calcium and calmodulin‐dependent protein kinase II inhibiting peptide; mtTEMPO, mitochondrial TEMPO; NG, normoglycemic; PDGF, platelet‐derived growth factor; roGFP, reduction–oxidation sensitive green fluorescent protein; ROS, reactive oxygen species; T2D, type 2 diabetes; VSMC, vascular smooth muscle cell; and WT, wild type.

Figure 5. Mitochondrial respiration (OCR) but not glycolysis (ECAR) is impaired by T2D and restored by MCU inhibition.

A, ETC complex I activity in mitochondrial fraction of WT NG and T2D VSMCs, as well as in T2D mtCaMKIIN samples as determined by the decrease in the rate of absorbance at 340 nm with and without rotenone incubation for 10 minutes. B, Quantification of activity of ETC complex I, plotted as the difference between absorbance curve slopes with and without rotenone (as in A), (n=5). C, OCR (from the Seahorse assay—a mitochondrial stress test) in VSMCs isolated from WT NG, WT T2D, and mtCaMKIIN T2D mice, with and without PDGF treatment (20 ng/mL; n=7). D, ECAR (from the Seahorse assay), (n=7). E, Quantification of basal (C) OCR (for WT NG and T2D VSMCs), with and without PDGF (n=7; as in (C)). F, Quantification of ATP‐linked OCR as a subtraction of OCR under oligomycin from basal OCR. (n=7; as in C). G, Quantification of ECAR from Seahorse experiments under oligomycin treatment for T2D VSMCs isolated from WT and mtCaMKIIN mice, with and without PDGF (n=8; as in D). H, Mitochondrial ATP levels in VSMCs from NG or T2D mice transduced with empty, mtCaMKIIN, or MICU1 adenoviruses (n=7–8). I, Aconitase 2 (ACO2) activity in VSMCs from NG or T2D mice transduced with empty or mtCaMKIIN adenoviruses or treated with 10 μmol/L mtTempo for 48 hours as a control (n=6–7). Statistical analyses were performed using Kruskal–Wallis test for (B), (E–I). ECAR indicates extracellular acidification rate; ETC, electron transport chain; MCU, mitochondrial calcium uniporter; MICU1, mitochondrial calcium uptake 1; mtCaMKIIN, mitochondrial calcium and calmodulin‐dependent protein kinase II inhibiting peptide; NADPH, nicotinamide adenine dinucleotide phosphate; NG, normoglycemic; OCR, oxygen consumption rate; PDGF, platelet‐derived growth factor; T2D, type 2 diabetes; VSMC, vascular smooth muscle cell; and WT, wild type.

Figure 6. In T2D VSMCs, increased NADPH production and ATP generation through the PPP drive VSMC proliferation and are dependent on MCU complex activity.

A, Representative traces of an NADPH‐linked luminescence assay, with or without the PPP inhibitor 6‐aminonicotinamide (6‐AN, 50 μmol/L) in lysates from NG and T2D WT VSMCs infected with empty, mtCaMKIIN, or MICU1 adenoviruses. B, Quantification of NADPH levels in (A; n=5–6). C, Cytosolic ATP levels in cells from (A), assessed with measuring the ratio of GFP/RFP fluorescence after adenoviral infection with ATP sensor (n=10). D, Cell counts of VSMCs from (A) counted after 72 hours in culture with or without PDGF (20 ng/mL). Data are expressed as fold change over numbers at 0 hours (n=5–8). Statistical analyses were performed using Mann–Whitney (B and C) and Kruskal–W Wallis test (D). GFP indicates green fluorescent protein; MCU, mitochondrial calcium uniporter; MICU1, mitochondrial calcium uptake 1; mtCaMKIIN, mitochondrial calcium and calmodulin‐dependent protein kinase II inhibiting peptide; NADPH, nicotinamide adenine dinucleotide phosphate; NG, normoglycemic; OCR, oxygen consumption rate; PDGF, platelet‐derived growth factor; PPP, pentose phosphate pathway; RFP, red fluorescent protein; T2D, type 2 diabetes; VSMC, vascular smooth muscle cell; and WT, wild type.

Figure 7. In T2D VSMCs, increased NADPH production and proliferation through the PPP rely on MCU‐dependent mtROS production.

A, Representative traces of an NADPH‐linked luminescence assay, with or without mtTempo (10 μmol/L for 48 hours) in lysates from NG and T2D WT VSMCs infected with empty or mtCaMKIIN adenoviruses. B, Quantification of NADPH levels in (A; n=6). Statistical analyses were performed using Mann–Whitney test mtCaMKIIN indicates mitochondrial calcium and calmodulin‐dependent protein kinase II inhibiting peptide; mtROS, mitochondrial reactive oxygen species; mtTempo, mitochondrial TEMPO; NADPH, nicotinamide adenine dinucleotide phosphate; NG, normoglycemic; PPP, pentose phosphate pathway; T2D, type 2 diabetes; VSMC, vascular smooth muscle cell; and WT, wild type.