ATPase Inhibitory Factor-1 Disrupts Mitochondrial Ca2+ Handling and Promotes Pathological Cardiac Hypertrophy through CaMKIIδ - PubMed (original) (raw)

. 2021 Apr 23;22(9):4427.

doi: 10.3390/ijms22094427.

Pablo I Sánchez-Aguilera 1, Nils Bomer 1, Shigeki Miyamoto 2, Harmen G Booij 1, Paula Giraldo 1, Silke U Oberdorf-Maass 1, Kirsten T Nijholt 1, Salva R Yurista 1, Hendrik Milting 3, Peter van der Meer 1, Rudolf A de Boer 1, Joan Heller Brown 2, Herman W H Sillje 1, B Daan Westenbrink 1

Affiliations

ATPase Inhibitory Factor-1 Disrupts Mitochondrial Ca2+ Handling and Promotes Pathological Cardiac Hypertrophy through CaMKIIδ

Mario G Pavez-Giani et al. Int J Mol Sci. 2021.

Abstract

ATPase inhibitory factor-1 (IF1) preserves cellular ATP under conditions of respiratory collapse, yet the function of IF1 under normal respiring conditions is unresolved. We tested the hypothesis that IF1 promotes mitochondrial dysfunction and pathological cardiomyocyte hypertrophy in the context of heart failure (HF). Methods and results: Cardiac expression of IF1 was increased in mice and in humans with HF, downstream of neurohumoral signaling pathways and in patterns that resembled the fetal-like gene program. Adenoviral expression of wild-type IF1 in primary cardiomyocytes resulted in pathological hypertrophy and metabolic remodeling as evidenced by enhanced mitochondrial oxidative stress, reduced mitochondrial respiratory capacity, and the augmentation of extramitochondrial glycolysis. Similar perturbations were observed with an IF1 mutant incapable of binding to ATP synthase (E55A mutation), an indication that these effects occurred independent of binding to ATP synthase. Instead, IF1 promoted mitochondrial fragmentation and compromised mitochondrial Ca2+ handling, which resulted in sarcoplasmic reticulum Ca2+ overloading. The effects of IF1 on Ca2+ handling were associated with the cytosolic activation of calcium-calmodulin kinase II (CaMKII) and inhibition of CaMKII or co-expression of catalytically dead CaMKIIδC was sufficient to prevent IF1 induced pathological hypertrophy. Conclusions: IF1 represents a novel member of the fetal-like gene program that contributes to mitochondrial dysfunction and pathological cardiac remodeling in HF. Furthermore, we present evidence for a novel, ATP-synthase-independent, role for IF1 in mitochondrial Ca2+ handling and mitochondrial-to-nuclear crosstalk involving CaMKII.

Keywords: CaMKII; calcium handling; cardiomyocyte hypertrophy; heart failure; mitochondria.

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Conflict of interest statement

M.P.G.: N.B., H.G.B., P.G., S.M.O., K.T.N., S.M., S.R.Y., P.v.d.M., J.H.B., H.H.W.S. and B.D.W. do not report conflicts of interest relative to this report. The UMCG, which employs N.B., H.G.B., P.G., S.O.M., K.T.N., S.R.Y., P.v.d.M., H.H.W.S. and B.D.W. has received research grants and/or fees from Abbott, AstraZeneca, Bristol-Myers Squibb, Novartis, Novo Nordisk, and Roche. R.A.d.B. received personal fees from Abbott, AstraZeneca, Novartis.

Figures

Figure 1

Figure 1

Expression of ATPase inhibitory factor-1 (IF1) is increased in heart failure, decreases mitochondrial respiratory capacity, and promotes glycolysis in cardiomyocytes. (A) IF1 mRNA levels in mice with heart failure induced by cardiomyocyte autonomous expression of Gαq and wild-type (WT) littermate controls (n = 4), and mice with heart failure induced by transverse aortic constriction (TAC) (n = 6) or myocardial infarction (MI) (n = 6) surgeries and sham-operated controls (SHAM). (B) mRNA expression of IF1 in cardiac lysates from patients with end-stage heart failure and normal control hearts (n = 11). (C) IF1 mRNA expression in neonatal rat ventricular myocytes (NRVMs) treated with insulin-like growth factor-1 (IGF-1, 10 nM), isoproterenol (ISO, 100 nM) or vehicle (Ctl) for 48 h (n = 4). (D) Neonatal rat ventricular myocytes (NRVMs) were infected with an adenoviral vector expressing human IF1 (ad-IF1-WT) or a control virus expressing green fluorescent protein (GFP) (ad-CTL) for 48 h. The line graph depicts changes in the oxygen consumption rate (OCR) of NRVMs after serial treatments with oligomycin (oligo), FCCP, and rot + AA assessed with the Seahorse system. The graph represents five independent experiments. (E) Bar graph depicting differences in basal respiration, ATP-linked respiration, maximal respiration, and mitochondrial spare capacity (n = 5). Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01 by parametric test _t_-test. (F) Changes in extracellular acidification rate (ECAR) in cells infected with ad-IF1-WT or ad-CTL after serial additions of glucose, oligomycin, and 2-deoxyglucose (2-DG, n = 4). (G) Bar graph depicting differences in glycolysis in NRVMs treated as in F (n = 4). (H) mRNA levels of lactate dehydrogenase (LDH) and pyruvate kinase (PRK) assayed with RT-qPCR (n = 4). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. SHAM/Ctrl/ad-CTL using the Mann–Whitney U test or _T_-test where appropriate.

Figure 2

Figure 2

IF1 overexpression promotes mitochondrial reactive oxygen species (ROS) production and cardiomyocyte hypertrophy. NRVMs were infected with an adenoviral vector expressing the human IF1 (ad-IF1-WT) or a control virus (ad-CTL) for 48 h. All bar graphs depict the results of at least three independent experiments. (A) Basal mitochondrial ROS emissions detected with the mitochondrial ROS sensor MitoSOX® (n = 4). (B) mRNA expression of the mitochondrial ROS-sensitive genes NADPH oxidase 2 (NOX2), nuclear receptor factor-2 (NRF2), and heat shock protein-60 (HSP60) (n = 4). (C) Semi-log-run polymerase chain reaction was performed using specific primers for mitochondrial DNA fragments in the mitochondrial D-Loop region to determine oxidative damage to mitochondrial DNA (n = 4). (D) Changes in the protein levels of mitochondrial respiratory chain complexes in cells infected with ad-IF1-WT and ad-CTL detected by Western blot. Different complex levels were normalized for mitochondrial heat shock protein-70 (mtHSP70) levels (n = 4). (E) Typical example of immunofluorescent staining of NRVMs cultured with a fluorescent-labeled anti-α-actinin (red, left panel) and (right panel) average cardiomyocyte surface area of α-actinin-positive cells (n = 5). (F) mRNA expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01 vs. ad-CTL by nonparametric Mann–Whitney U test using the Mann–Whitney U test or _T_-test where appropriate.

Figure 3

Figure 3

An IF1 mutant incapable of binding to ATP synthase induces similar degrees of mitochondrial stress and cardiomyocyte hypertrophy. NRVMs were infected with an adenovirus expressing an IF1 mutant harboring an E55A substitution that renders the protein unable to interact with ATP synthase or control virus (ad-CTL) for 48 h. (A) Changes in the oxygen consumption rate (OCR) of NRVMs after serial treatments with oligomycin (oligo), FCCP, and rot + AA assessed with the Seahorse system (n = 5). (B) Bar graph depicting differences in basal respiration, ATP-linked respiration, maximal respiration, and mitochondrial spare capacity (n = 5). (C) Changes in the extracellular acidification rate (ECAR) in cells infected with ad-IF1-E55A or ad-CTL after serial additions of glucose, oligomycin, and 2-deoxyglucose (2-DG, n = 4). (D) Bar graph depicting differences in glycolysis in NRVMs treated as in F (n = 4). (E) mRNA levels of lactate dehydrogenase (LDH) and pyruvate kinase (PRK). (F) Basal mitochondrial ROS levels assayed with MitoSOX® (n = 4). (G) mRNA levels of NOX2, NRF2, and HSP60. (H) Bar graph depicting differences in cardiomyocyte cross-sectional area (n = 5). Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01 vs. ad-CTL using the Mann–Whitney U test or _T_-test where appropriate.

Figure 4

Figure 4

IF1 induces mitochondrial fission by promoting dynamin-related protein 1 (DRP1) translocation. NRVMs were infected with adenovirus ad-IF1-WT or empty vector (ad-CTL) for 48 h. (A) Representative Z-stack image of neonatal rat ventricular myocyte stained with MitoTracker® Deep Red (green) and anti-α-actinin (red) using confocal microscopy. (B) 3D imaging reconstruction was employed for mitochondrial network analysis (30–36 Z-stacks slices were recorded per cell). Representative example of confocal stack acquisition followed by 3D reconstruction after image deconvolution of cells stained as in A. (C) Bar graphs depicting differences in mitochondrial number per cell (left bar graph) and fission index (right bar graph). Both ratios were calculated from 3D multislice reconstruction values. Fission index estimation was measured using the number of mitochondrial normalized for mitochondrial volume per cell (ad-CTL, n = 17 cells and ad-IF1-WT, n = 18 cells). (D) Dynamin-related protein 1 (DRP1), cytochrome c oxidase subunit 4 (COXIV), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein level in mitochondrial and cytosolic fractions of NRVMs infected with IF1-WT and ad-CTL and (left panel). (E) DRP1 protein levels normalized for COXIV in the mitochondrial fraction (n = 4). (F) PTEN-induced kinase 1 (PINK1) and mitofusin-2 (MFN2) protein levels normalized for GAPDH. (G) Bar graphs depict densitometric analysis from protein levels normalized using immunoblot. Data are presented as mean ± SEM. ** p < 0.01, and *** p < 0.001 vs. ad-CTL using the Mann–Whitney U test or _T_-test where appropriate.

Figure 5

Figure 5

IF1 reduces mitochondrial calcium, promotes sarcoplasmic reticulum (SR) calcium overload, and activates calcium–calmodulin kinase II (CAMKII) signaling. NRVMs were infected with adenovirus ad-IF1-WT and ad-CTL for 48 h. (A) Bar graph depicting differences in basal mitochondrial Ca2+ detected by with Rhod-2 AM (n = 4). (B) Time lapse of mitochondrial Ca2+ release measurements using Fluo-4 AM before and after addition of FCCP (n = 5). (C) Differences in the area under the curve of Fluo-4 AM fluorescence after the addition of FCCP (n = 5). (D) Cytosolic Ca2+ measurements using Fura-2 AM in cells infected with ad-IF1-WT or ad-CTL using an epifluorescence microscope (n = 4). (E) Representative experiment of potassium chloride (KCL)-induced sarcoplasmic reticulum (SR) Ca2+ release (n = 3). (F) Bar graph depicting differences in the area under the curve of Fura-2 AM 340/380 fluorescence ratio after the addition of KCL (n = 3). (G) Bar graphs depicting mRNA levels of different calcium regulatory genes assessed by qPCR; voltage-dependent anion channel 1 (VDAC1); mitochondrial calcium uniporter (MCU); essential MCU regulatory element (EMRE),; dominant-negative pore-forming subunit of the MCU ß (MCUB); mitochondrial calcium uptake protein 1 and 2 (MICU1 and MICU2 respectively); MCU regulator 1 (MCUR1); mitochondrial sodium calcium exchanger (NCLX), (n = 4). Data are presented as mean ± SEM. * p < 0.05 and ** p < 0.01 vs. ad-CTL using the Mann–Whitney U test or _T_-test where appropriate.

Figure 6

Figure 6

IF1-induced cardiomyocyte hypertrophy is dependent upon CaMKIIδ activation. NRVMs were infected with adenovirus ad-IF1-WT and ad-CTL for 48 h. (A) Representative immunoblot depicting phosphorylation of CAMKII (threonine 286) and CaMKII-dependent phosphorylation of phospholamban (p-PLN, thr17) and adenosine monophosphate-activated protein kinase (p-AMPK, thr172), normalized for the total protein (left panel). (B) Bar graphs depicting differences in phosphorylation between experimental groups (n = 7). (C) NRVMs were co-infected with adenovirus expressing IF1-WT or ad-CTL in the presence or absence of a catalytically dead mutant of CAMKIIδC (ad-dnCAMKII) for 48 h. Representative immunoblots from whole-cell lysate using an antibody specific for p-PLN (thr17), total PLN, CAMKII (PAN), and GAPDH (n = 4). (D) Maximal mitochondria respiration through oxygen consumption rate (OCR) recorded using Mito Stress Test Seahorse assay. (E) Bar graph depicting basal mitochondrial ROS levels assayed with MitoSOX® (n = 4). (F) Typical example of immunofluorescent staining of cultured NRVMs with a fluorescent-labeled anti-α-actinin (red, left panel) and (right panel) average cardiomyocyte surface area of α-actinin-positive cells (scale bar represents 50 µm, n = 4). (G) ANP mRNA level normalized for 36B4 (n = 4). * Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. ad-CTL using the Mann–Whitney U test (A,B) or the Kruskal–Wallis test (D–G).

Figure 7

Figure 7

Schematic representation of the proposed mechanism responsible for IF1-mediated cardiomyocyte hypertrophy. Under conditions of respiratory collapse, ATPase inhibitory factor-1 (IF1) prevents maladaptive ATP hydrolysis by ATP synthase (complex V). The function of IF1 under normal respiring conditions remains poorly described. We present evidence for a novel, ATP-synthase-independent, role for IF1 in mitochondrial calcium handling and mitochondrial-to-nuclear crosstalk involving calcium–calmodulin kinase II (CaMKII). Specifically, we found that IF1 induced mitochondrial oxidative stress, promoted mitochondrial fission, and compromised mitochondrial calcium handling in primary cardiomyocytes. These perturbations resulted in sarcoplasmic reticulum calcium overload and the activation of CaMKII-mediated pathological cardiomyocyte hypertrophy. ETC, electron transport chain, ROS, reactive oxygen species, SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; PLN, phospholamban; SR, sarcoplasmic reticulum.

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