Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Dec 18;5(12):e1573.
doi: 10.1038/cddis.2014.526.

Defective sarcoplasmic reticulum-mitochondria calcium exchange in aged mouse myocardium

C Fernandez-Sanz  1 , M Ruiz-Meana  1 , E Miro-Casas  1 , E Nuñez  2 , J Castellano  1 , M Loureiro  2 , I Barba  1 , M Poncelas  1 , A Rodriguez-Sinovas  1 , J Vázquez  2 , D Garcia-Dorado  1
Affiliations

Defective sarcoplasmic reticulum-mitochondria calcium exchange in aged mouse myocardium

C Fernandez-Sanz et al. Cell Death Dis. .

Abstract

Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria-sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5-6 months). Mitochondrial membrane potential and resting O2 consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O2 consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca2+, we investigated the effect of age on mitochondrial Ca2+ uptake. Although no age-dependent differences were found in Ca2+ uptake kinetics in isolated mitochondria, mitochondrial Ca2+ uptake secondary to SR Ca2+ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca2+ handling. Age-dependent alterations in SR Ca2+ transfer to mitochondria and in Ca2+ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR-mitochondria communication underlies inefficient interorganelle Ca2+ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effect of advanced age on (a) β-galactosidase, (b and c) lipofuscin autofluorescence, (d and f, green color) lysosome vesicles, (e and f, red color) mitochondrial pool labeling in intact mouse cardiomyocytes, (g) citrate synthase (CS) activity in isolated heart mitochondria and (h) mitochondrial yield determined as mitochondrial protein with respect to total cardiac protein in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from mouse hearts. Mean±S.E.M. from n=14–25 replicates (5–10 hearts)
Figure 2
Figure 2
(a) ATP/PCr in intact hearts from young and old mice, quantified by NMR spectroscopy (left panel; n=3 hearts). Mitochondrial membrane potential (JC-1 ratio fluorescence) in intact cardiac myocytes from young and old mouse hearts under resting contidions and after induction of maximal mitochondrial depolarization with DNP (right panel). (n=14–26 cardiomyocytes per group, five hearts). (b) Resting O2 consumption (state-2) and ADP-stimulated O2 consumption (state-3) in subsarcolemmal and interfibrillar mitochondria from young (<6 months) and old (>20 months) mouse hearts, mediated by substrates of complexes 1–4, normalized by citrate synthase (CS) activity. RCR, respiratory control rate for each respiratory complex (state-3/state-2). Mean±S.E.M. from n=14–28 replicates (5–10 hearts)
Figure 3
Figure 3
(a) Effect of aging on Ca2+ transient amplitude (panel-1), and SR Ca2+ release/uptake kinetics in field-stimulated (1 Hz) fluo-4 loaded cardiac myocytes from young and old mouse hearts (panels-2 to 5) and in total SR Ca2+ content after caffeine stimulation (panel-6). *, P<0.05 with respect to young, n=9–12 cardiomyocytes per group (six hearts). (b) Spontaneous spark frequency, amplitude, rate and diffusion in quiescent fluo-4 loaded cardiomyocytes from young and old mouse hearts. FWHM, full width at half maximum (μm). **, P<0.001 with respect to young. Data represent mean±S.E.M. from 1600 sparks (34 cardiomyocytes, four hearts)
Figure 3
Figure 3
(a) Effect of aging on Ca2+ transient amplitude (panel-1), and SR Ca2+ release/uptake kinetics in field-stimulated (1 Hz) fluo-4 loaded cardiac myocytes from young and old mouse hearts (panels-2 to 5) and in total SR Ca2+ content after caffeine stimulation (panel-6). *, P<0.05 with respect to young, n=9–12 cardiomyocytes per group (six hearts). (b) Spontaneous spark frequency, amplitude, rate and diffusion in quiescent fluo-4 loaded cardiomyocytes from young and old mouse hearts. FWHM, full width at half maximum (μm). **, P<0.001 with respect to young. Data represent mean±S.E.M. from 1600 sparks (34 cardiomyocytes, four hearts)
Figure 4
Figure 4
(a) Mitochondrial Ca2+ uptake throughout time in response to SR Ca2+ release (10 mmol/l caffeine, arrow) in digitonin-permeabilized rhod-2 loaded cardiac myocytes from old and young mouse hearts. N=8–11 cardiomyocytes per group (n=5 hearts). (b) Maximal mitochondrial Ca2+ uptake (rhod-2) normalized by maximal SR Ca2+ release (fluo-4) in young and old permeabilized mouse cardiomyocytes. Addition of 10 μmol/l Ru360 (a specific blocker of the mitochondrial Ca2+ uniporter) prevented caffeine-induced mitochondrial Ca2+ uptake in both groups of ages. *, P<0.05 with respect to young. n=7–11 cardiomyocytes per group (five hearts). (c) Absence of age-dependent differences in the in vitro mitochondrial Ca2+ uptake kinetics (CG5N fluorescence), when exposing isolated subsarcolemmal (SSM) and interfibrillar (IFM) cardiac mitochondria to an external Ca2+ pulse of 30 μmol/l (arrow). Addition of 10 μmol/l Ru360 prevented mitochondrial Ca2+ uptake. Mean±S.E.M. of four replicates per group (six hearts). (d) NAD(P)H/NAD(P)+ ratio 2 min after electrical stimulation at 1 Hz and 5 Hz to induce high contractile activity in intact cardiomyocytes from young and old mouse hearts (left panel), and kinetics of the NAD(P)H consumption in its reduced form, expressed with respect to total cell NADPH, during 5 Hz stimulation (right panel). Data represent mean±S.E.M. (10–19 cardiomyocytes per group, nine hearts). (e) Left panel: total glutathione (GSHtot) and oxidized glutathione levels (GSSG) in myocardial tissue of young and old mice. The inset shows the fraction of oxidized glutathione with respect to total one; Right panel: cytosolic and mitochondrial ROS production 2 min after electrical stimulation at 5 Hz to induce high contractile activity in intact cardiomyoctes from young and old mouse hearts, as quantified by DCF and MitoSox fluorescence. The inset shows the kinetics of short-term mitochondrial ROS production during pacing (arrow points the onset of 5 Hz stimulation)
Figure 5
Figure 5
(a) Confocal fluorescent images of young and old mouse cardiomyocyte simultaneously labeled with anti-RyR (red), anti-VDAC (green) and Hoechst (blue) for visualization of SR, mitochondria and nuclei, respectively. (b) Effect of aging on RyR–VDAC spatial interaction, as quantified by Mander's coefficient (m1) analysis, expressed as the percentage of RyR – with respect to total RyR fluorescence – that overlaps with VDAC (left panel); in the right panel, total RyR and VDAC fluorescence. Mean±S.E.M. from 4 to 6 cardiomyocytes per group (four hearts). (c) Confocal fluorescent images of the RyR–VDAC interaction in different individual cardiomyocytes isolated from young and old mouse hearts, detected by proximity ligation assay (PLA). Positive cross-reactivity – reflecting an intermolecular distance of <40nm – is shown in red, nuclei are depicted in blue (Hoechst). (d) Aging was associated with a significant reduction in cell fluorescence resulting from RyR–VDAC cross-reactivity (left panel) and in the number of amplification spots (right panel), as quantified by PLA assay. Mean±S.E.M. of 1071–3250 blobs per group (15 cardiomyocytes, two hearts). (e) Western blot representative bands and quantification of the expression of proteins involved in SR and mitochondria Ca2+ transport and interorganelle communication in whole-heart homogenate, microsomal and mitochondrial fractions from young and old mice. Each protein of interest was normalized by the corresponding protein of reference as follows: in total homogenates, VDAC1/Grp75, Mfn2/GAPDH and Grp75/GAPDH; in microsomal fraction, Mfn2/VDAC1, VDAC1/Grp75, Grp75/GADPH and RyR/Grp75; in mitochondria, VDAC1/Grp75, Grp75/ANT1/2 and Mfn2/ANT1/2. Mean±S.E.M. of n=8 replicates from four hearts/group)
Figure 6
Figure 6
Effect of aging on: (a) quantitative redox proteomics using GELSILOX of proteins from mitochondria-associated membranes (MAMs; left) and of the Cys-containing peptides detected in mitochondrial VDAC proteins (right). The left graph represents the percentage of Cys-containing peptides identified in oxidized (red) or reduced (blue) forms. The numbers in the right table represent the standardized variable at the peptide level as in b; negative values indicate an increase (in green) and positive values a decrease (in red) in the peptides containing Cys in oxidized form (Ox) or in reduced form (Red) in mitochondrial VDAC proteins in hearts from old mice. (b) The abundance of mitochondrial respiratory complexes (1–5) in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Data are shown as cumulative distributions of the standardized variable at the protein level (i.e., corrected log2 ratios of proteins expressed in units of standard deviation) for all oxidative phosphorylation proteins. The black sigmoid is the theoretical null hypothesis distribution; a displacement toward the left indicates an increase in protein concentration. All the categories follow very closely the null hypothesis distribution, indicating that aging does not affect the abundance of mitochondrial respiratory proteins. (c) Alterations in the abundance of oxidized (red) and reduced (blue) Cys-containing peptides in SSM and IFM from young and old mice hearts. Peptides containing Cys residues in different oxidation states were quantified using the GELSILOX method. The sigmoid curves represent the cumulative distribution of the standardized variable at the peptide level (i.e. corrected log2-ratios of peptides expressed in units of S.D.), for all peptides containing either oxidized or reduced Cys sites that belong to proteins from OxPhos complexes
Figure 7
Figure 7
Effect of 0.75 μmol/l colchicine on: (a) RyR–VDAC spatial interaction quantified by Mander's coefficient (m1) analysis, indicating a reduction of the percentage of RyR (with respect to total RyR) that overlaps with VDAC in young cardiac myocytes, without effect in old cardiomyoyctes (left panel); and RyR–VDAC positive cross-reactivity detected by proximity ligation assay (PLA; right panel; n=4–6). (b) The amplitude of Ca2+ transients in fluo-4 loaded cardiomyocytes submitted to field-stimulation (left panel), and mitochondrial Ca2+ uptake in response to SR Ca2+ release in digitonin-permeabilized cardiac myocytes (right panel). Ru360 10 μmol/l was used to specifically inhibit mitochondrial Ca2+ uniporter (n=6–11). (c) In vitro mitochondrial Ca2+ uptake (CG5N fluorescence) in isolated subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria from young mouse hearts with or without colchicine (yellow and black), and from old mouse hearts (red), exposed to an external 30 μmol/l Ca2+ pulse (arrow; top panels), complex 11-mediated O2 consumption (state-2) and ADP-stimulated O2 consumption (state-3) in SSM from young mouse hearts, normalized by citrate synthase activity, in the absence (control) or in the presence of colchicine (bottom panel). Mean±S.E.M. of four replicates per group (six hearts)
Figure 8
Figure 8
Schematic representation of the proposed mechanism by which aging induces SR–mitochondria disruption in cardiomyocytes
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. 1Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G et al. Executive summary: heart disease and stroke statistics--2010 update: a report from the American Heart Association. Circulation 2010; 121: 948–954. - PubMed
    1. 2Boengler K, Schulz R, Heusch G. Loss of cardioprotection with ageing. Cardiovasc Res 2009; 83: 247–261. - PubMed
    1. 3Strait JB, Lakatta EG. Aging-associated cardiovascular changes and their relationship to heart failure. Heart Fail Clin 2012; 8: 143–164. - PMC - PubMed
    1. 4North BJ, Sinclair DA. The intersection between aging and cardiovascular disease. Circ Res 2012; 110: 1097–1108. - PMC - PubMed
    1. 5Terentyev D, Gyorke I, Belevych AE, Terentyeva R, Sridhar A, Nishijima Y et al. Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ leak in chronic heart failure. Circ Res 2008; 103: 1466–1472. - PMC - PubMed

Publication types