Cardiomyocyte-specific PCSK9 deficiency compromises mitochondrial bioenergetics and heart function

Abstract

Aims: Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9-/- mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells.

Methods and results: Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9-/- mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9-/- hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9-/- mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9-/- mice. Circulating lipid levels were unchanged in CM-Pcsk9-/- mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9-/- mice had an increased number of mitochondria-endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism.

Conclusion: PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Keywords: Cardiac dysfunction; Cardiomyocyte; Metabolic inflexibility; Mitochondria; Pro-protein convertase subtilisin-kexin type 9 (PCSK9).

Graphical Abstract

Model of the effects of Pcsk9 deletion in the heart. We propose that cardiac PCSK9 maintains the normal composition of cholesterol and phospholipids in the mitochondrial membrane by modulating their transport through mitochondria-associated ER membrane contacts (MAMs). Reduced cardiac Pcsk9 expression alters the lipid composition of mitochondrial membranes and cristae morphology, leading to impaired assembly and activity of mitochondrial respiratory complexes (especially ATP synthase), and thereby decreased OXPHOS and ATP production, and increased ROS production. These impairments indicate metabolic inflexibility characterized by reduced mitochondrial oxidative metabolism and insufficient compensatory energy production by glycolytic pathways, making the heart less efficient in generating energy to support demand. PCSK9 cardiac deficiency leads to progressive DCM, impaired heart function/structure, and compromised energy production. TCA, tricarboxylic acid cycle

Figure 1

Characterization of CM-Pcsk9^−/− mice. (A) Schematic of conditional knockout of Pcsk9. Exons 2 and 3 were flanked with loxP sites. Cre-mediated recombination led to a Pcsk9Δ allele. Half arrows indicate the P1–P3 primers used for genotyping and model validation. (B and C) Analysis of genomic DNA from (B) isolated primary cardiomyocytes and cardiac fibroblasts and (C) the indicated tissues from CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice with PCR primers targeting the Pcsk9loxP allele, the Pcsk9Δ allele, and αMHC-Cre. (D and E) Quantification of Pcsk9 mRNA expression in (D) primary cardiomyocytes (n = 4) and (E) the indicated tissues (n = 6–7) from CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice, determined with a probe covering exons 3 and 4. (F) Representative immunoblots and quantification of PCSK9 protein levels in isolated primary cardiomyocytes from CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice hearts (n = 4). Vinculin was the loading control. Values are mean ± SEM. P values are shown in the figure vs. CM-Pcsk9^+/+ by two-tailed t-test.

Figure 2

CM-Pcsk9^−/− mice have heart failure and die prematurely. (A) Body weight and (B) photographs of hearts (left) and heart weight/tibia length (HW/TL) (right) of 10- and 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 7–10). Scale, 5 mm. (C) Representative images of cardiomyocytes stained with fluorescent wheat germ agglutinin (WGA) (red) and phalloidin (green) (left) and quantification (right) of cross-sectional area (CSA) of cardiomyocytes from 10- and 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 7–10). Scale, 25 µm. (D) Relative frequency of cross-sectional area of cardiomyocytes from 10- and 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (≥1600 cells; n = 7–10). (E) Representative Sirius Red-stained heart sections from 10- and 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (left) and quantification of fibrosis (right) (n = 8–10). Scale, 1 mm or 25 µm. (F) Lactate dehydrogenase (LDH) release from cardiomyocytes isolated from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 9). (G) Representative transmission electron microscope images (left) and quantification (right) of sarcomere, I-band and A-band length from LV tissue of 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (≥472 sarcomeres; n = 3). Scale, 1 µm. (H) Lung weight/tibia length (LW/TL) of 10- and 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 7–10). (I) Cardiac function during dobutamine stress in 10- and 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 10; see also Supplementary material online, Table S2). (J) Kaplan–Meier survival curve of wild-type mice expressing αMHC-Cre (WT/αMHC-Cre), CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 7–11). Values are mean ± SEM. P values are shown vs. CM-Pcsk9^+/+ by two-tailed t-test (F, G, I, J) or two-way ANOVA (A, B, C, E, H).

Figure 3

Cardiac mitochondrial metabolism is impaired in CM-Pcsk9^−/− mice. (A) Significantly up- and down-regulated Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genome pathways related to metabolic pathways in RNA-seq data from hearts from 28-week-old CM-Pcsk9^−/− vs. CM-Pcsk9^+/+ mice (n = 5). RNA-seq statistics were retrieved from gene set statistics from PIANO analysis. FDR, false-discovery rate. (B) Variation plots of genes (mRNA) related to cardiac metabolism in hearts of 28-week-old CM-Pcsk9^−/− vs. CM-Pcsk9^+/+ mice (n = 8–10). (C) Left: Variation plots of proteins related to cardiac metabolism in cardiomyocytes from 28-week-old CM-Pcsk9^−/− vs. CM-Pcsk9^+/+ mice (n = 12). Right: representative immunoblots. β-actin was the loading control. (D) Schematic metabolic network showing alterations in genes and proteins in 28-week-old CM-Pcsk9^−/− vs. CM-Pcsk9^+/+ mouse hearts. Proteins (in boxes) and mRNA (in italics) that were significantly modulated are coloured. Unexplored proteins are indicated in grey. (E) Total acyl- and free carnitine content and their ratio in cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 11). (F) Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and their ratio in isolated cardiomyocytes from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 8). UFA, total unsaturated fatty acids. Data are mean ± SEM. P values are shown vs. CM-Pcsk9^+/+ by two-tailed t-test.

Figure 4

Impairment of mitochondrial but not glycolytic function in CM-Pcsk9^−/− cardiomyocytes. OCR and ECAR were determined using the Seahorse flux analyser in primary cardiomyocytes isolated from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice and normalized to total cellular protein content for each well (12 wells from n = 9). (A and B) OCR (left), quantification of respiratory parameters (middle), and energetic maps obtained by plotting ECAR and OCR values at baseline and under FCCP-induced stress (right) of CM-Pcsk9^+/+ and CM-Pcsk9^−/− cardiomyocytes using pyruvate/glucose (A) or palmitate (B) as the substrate. OCR was measured under basal conditions and after addition of oligomycin (1 µM) to inhibit ATP synthase, FCCP (1 µM) to uncouple oxidative phosphorylation, and antimycin A (2 µM) and rotenone (2 µM) to gauge non-mitochondrial respiration. (C) Left: measurement of OCR-related fatty acid oxidation (FAO) in cardiomyocytes pre-treated with etomoxir (CPT1B inhibitor, 100 μM, 15 min) before palmitate addition (150 μM) at time 0. Right: quantification of total (endogenous + exogenous) fatty acid oxidation at baseline and under FCCP-induced stress (maximal). See Supplementary material online, Figure S3A. (D) ECAR (left) and quantification of glycolytic parameters (right) in glucose-deprived cardiomyocytes after addition of glucose (10 mM) to fuel glycolysis and OXPHOS, oligomycin (2 μM) to inhibit ATP synthase, and 2-deoxyglucose (2-DG) (100 mM) to inhibit glucose catabolism. (E) Glycolytic proton efflux rate and quantification of glycolysis in cardiomyocytes after addition of 0.5 μM rotenone/antimycin A and 2-deoxyglucose (2-DG) (100 mM). (F) Intracellular and extracellular lactate levels in primary cardiomyocytes isolated from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 6). Lactate concentrations were measured under basal conditions and after addition of 2 µM rotenone/antimycin and 2-deoxyglucose (2-DG) (100 mM), and normalized to total protein content. Data are mean ± SEM. P values are shown vs. CM-Pcsk9^+/+ by two-tailed t-test (A–E) and two-way ANOVA (F).

Figure 5

Mitochondrial OXPHOS complexes are altered in CM-Pcsk9^−/− mice. (A) Representative immunoblots (after SDS electrophoresis) with antibodies against subunits of mitochondrial ETC complexes: NDUFB8 [complex (Cx) I], SDHA (Cx II), UQCRC2 (Cx III), COXIV (Cx IV) and ATP5A (Cx V) (left) and quantification (right) in 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− cardiomyocytes (n = 12). β-actin served as loading control. (B) Representative immunoblots (after blue native polyacrylamide gel electrophoresis) (top) and quantification (bottom) of supercomplexes (SCs), individual ETC complexes, and Complex V oligomers/monomers in cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 8). (C) Representative images (top) and quantification (bottom) of in-gel activity of SCs, individual ETC complexes and Complex V oligomers/monomers in cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 9). Activity was normalized to total mitochondrial protein (Coomassie gel) and to that of CM-Pcsk9^+/+. (D–F) OCR was determined using the Seahorse flux analyser in mitochondria isolated from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice hearts and normalized to total mitochondrial protein content for each well (12 wells from n = 9). (D) Measurement (left) and quantification (right) of OCR determined by electron flow assay using pyruvate/malate/FCCP (10 mM/5 mM/4 µM) to drive Complex I-dependent respiration in an uncoupled state, rotenone (2 µM) to inhibit Complex I, succinate (5 mM) to drive Complex II-dependent respiration, antimycin A (4 µM) to inhibit Complex III, and ascorbate/TMPD (10 mM/0.1 mM) to drive Complex IV-dependent respiration in cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice. (E) Quantification of State III and State IIIu respiration and (F) respiratory control ratio (State III/State IV) determined in a coupling assay in cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice. Pyruvate/malate (10 mM/5 mM) and succinate/rotenone (5 mM/2 µM) were used to drive Complex I- and Complex II-dependent respiration, respectively. Respiration was initiated by adding ADP (5 mM, State III) and stopped by adding oligomycin (2.5 µM, State IV). FCCP (4 µM) dissipated mitochondrial membrane potential and initiated respiration (State IIIu). See Supplementary material online, Figure S4B and C. (G) Mitochondrial levels of reactive oxygen species in cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 6), determined with Amplex Red assay at baseline or under stress induced by FCCP (4 µM). Antimycin A (4 µM) was used as a positive control of massive ROS production. (H) Mitochondrial levels of reactive oxygen species in 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− cardiomyocytes (n = 9), determined with MitoSOX Red probe. Values are mean ± SEM. P values are shown vs. CM-Pcsk9^+/+ by two-tailed t-test.

Figure 6

Lipid composition, proximity with ER and morphology of mitochondria are altered in CM-Pcsk9^−/− mice. (A) Cardiolipin and total phospholipid levels, (B) PE levels and composition, (C) PC levels and composition, and (D) free cholesterol levels in membranes of cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 11). (E) Quantification of mitochondrial membrane potential (ΔΨm) at baseline or under stress induced by FCCP (1 µM; 10 min; positive control of ΔΨm disruption) in isolated cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 7–9). (F) Pseudocolour images showing the Laurdan dye generalized polarization (GP) index at each pixel position and average GP index from 10 to 15 images in isolated cardiac mitochondria from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 3). Red: rigid; blue: fluid. Scale, 10 µm. (G) Representative TEM images (top) and quantification of distance between mitochondria and ER (bottom, left panel) and of the percentage of mitochondria having contact (<30 nm) with the ER (bottom, right panel) in 28-week-old CM-Pcsk9^−/− vs. CM-Pcsk9^+/+ hearts (≥100 junctions from ≥12 images; n = 3). Scale, 100 nm. (H) Representative TEM images of mitochondrial ultrastructure and quantification of (I) mitochondrial number and mitochondrial area per cardiac area (≥1480 mitochondria for >67 images) and (J–M) individual mitochondrial area/perimeter and mitochondrial shape descriptors (≥782 mitochondria for >67 images) in LV tissue from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 3). AU, arbitrary units. Scale, 1 µm. (N) Representative TEM images and percentage of indicated mitochondrial features (≥1480 mitochondria from >67 images) in LV tissue from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 3). Scale, 100 nm. (O) Cristae density (length per mitochondrial area) (30 mitochondria) and (P) quantification (left) and distribution (right) of cristae width (≥300 cristae) in LV tissue from 28-week-old CM-Pcsk9^+/+ and CM-Pcsk9^−/− mice (n = 3). Scale, 250 nm. Data are mean ± SEM. P values are shown in the figure vs. CM-Pcsk9^+/+ by two-tailed t-test (A–D, F, G, I–M, O, P) or two-way ANOVA (E).

Figure 7

Acute Pcsk9 silencing causes a shift to glycolytic metabolism in cardiomyocytes and compromises ability to meet increased energy demand. H9c2 cardiomyocytes treated with siRNA against Pcsk9 (siPCSK9) or scrambled control (siCT) for 24 h were used for all experiments. (A) Representative gels (left) and quantification (right) of in-gel activity of ETC complexes in mitochondria from Pcsk9-deficient H9c2 cardiomyocytes (n = 4–6). Activity was normalized to total mitochondria protein (Coomassie gel) and to cells treated with scrambled control. (B) mRNA expression of Acadl, Hadh, Acaa2, Pdha1, and Pdk4 in Pcsk9-deficient H9c2 cardiomyocytes and in cells treated with scrambled control (n = 4). (C–H) OCRs and ECARs were determined using the Seahorse flux analyser and ATP production was determined using the Seahorse XF real-time ATP rate assay in H9c2 cardiomyocytes treated with siPCSK9 or scrambled control; values were normalized to total cellular protein content for each well (12 wells from 4 to 6 independent experiments). (C) OCRs and quantification of respiratory and ATP production parameters in H9c2 cardiomyocytes using pyruvate/glucose as substrate, oligomycin (1 µM) to inhibit ATP synthase, FCCP (5 µM) to uncouple oxidative phosphorylation, and antimycin A (1 µM) and rotenone (1 µM) to gauge non-mitochondrial respiration. (D) Left: OCR-related fatty acid oxidation (FAO) in H9c2 cardiomyocytes pre-treated with etomoxir (40 μM, 15 min) before addition of palmitate (150 μM) at time 0. Right: Quantification of total (endogenous + exogenous) fatty acid oxidation at baseline and under FCCP-induced stress. (E) ECARs and quantification of glycolytic parameters in glucose-deprived H9c2 cardiomyocytes after addition of glucose (10 mM) to fuel glycolysis and OXPHOS, oligomycin (2 μM) to inhibit ATP synthase, and 2-deoxyglucose (2-DG) (50 mM) to inhibit glucose catabolism. (F) Metabolic flux analysis showing quantification of mitochondrial (mitoATP) and glycolytic (glycoATP) ATP production in H9c2 cardiomyocytes. (G) ATP rate index indicating the changes in metabolic phenotype, calculated from data in (F). An increase in this index indicates a more oxidative/less glycolytic phenotype. (H) Energetic map of mitoATP vs. glycoATP of H9c2 cardiomyocytes using carbohydrates (glucose) or fatty acids (palmitate) as substrate [from (F)]. Values are mean ± SEM. P values are shown vs. CM-Pcsk9^+/+ by t-test (A–C, E–G) and two-way ANOVA (D).

Figure 8

Cardiomyocyte-secreted PCSK9 does not reverse mitochondrial dysfunction induced by Pcsk9 silencing. (A) Representative confocal images (left) and quantification (right) of the subcellular localization of PCSK9-GFP (green) and markers for Golgi apparatus (Gm130), endoplasmic reticulum (KDEL) and mitochondria (ATP5A) (all red) in H9c2 cardiomyocytes transfected with PCSK9-GFP or empty control (CT-GFP) for 24 h. Co-localization was analysed on a minimum of 5 fields of view (6710 μm) each containing approximately 9–15 cells from 3 independent experiments. Scale, 20 and 5 µm. (B) Immunoblot of PCSK9 in H9c2 cardiomyocytes transfected with PCSK9-GFP or CT-GFP for 24 h. Intracellular and secreted PCSK9 was detected using anti-GFP in cell lysates and media (at a concentration proportional to the amount of cell lysate deposit). GAPDH was used as a loading and cells control (n = 4). (C and D) Conditioned media from H9c2 cardiomyocytes transfected with PCSK9-GFP or CT-GFP for 24 h was used to treat H9c2 cardiomyocytes previously transfected with siRNA against Pcsk9 (siPCSK9) or scrambled control (siCT) for 24 h. After 24 h incubation, OCRs were determined using the Seahorse flux analyser in the media-treated H9c2 cardiomyocytes. Values were normalized to total cellular protein content for each well (12 wells from 6 to 8 independent experiments). (C) Experimental scheme. (D) OCRs and quantification of respiratory and ATP production parameters in H9c2 cardiomyocytes using pyruvate/glucose as substrate, oligomycin (1 µM) to inhibit ATP synthase, FCCP (5 µM) to uncouple oxidative phosphorylation, and antimycin A (1 µM) and rotenone (1 µM) to gauge non-mitochondrial respiration. Values are mean ± SEM. P values are shown vs. indicated condition by one-way ANOVA followed by Tukey’s post-test.