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Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

  1. Ying Ann Chiao  Is a corresponding author
  2. Huiliang Zhang
  3. Mariya Sweetwyne
  4. Jeremy Whitson
  5. Ying Sonia Ting
  6. Nathan Basisty
  7. Lindsay K Pino
  8. Ellen Quarles
  9. Ngoc-Han Nguyen
  10. Matthew D Campbell
  11. Tong Zhang
  12. Matthew J Gaffrey
  13. Gennifer Merrihew
  14. Lu Wang
  15. Yongping Yue
  16. Dongsheng Duan
  17. Henk L Granzier
  18. Hazel H Szeto
  19. Wei-Jun Qian
  20. David Marcinek
  21. Michael J MacCoss
  22. Peter Rabinovitch  Is a corresponding author
  1. Department of Pathology, University of Washington, United States
  2. Aging and Metabolism Program, Oklahoma Medical Research Foundation, United States
  3. Department of Genome Science, University of Washington, United States
  4. Buck Institute for Research on Aging, United States
  5. Department of Radiology, University of Washington, United States
  6. Biological Sciences Division, Pacific Northwest National Laboratory, United States
  7. Department of Environmental and Occupational Health Sciences, University of Washington, United States
  8. Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, United States
  9. Department of Cellular and Molecular Medicine, University of Arizona, United States
  10. Social Profit Network, United States
Research Article
Cite this article as: eLife 2020;9:e55513 doi: 10.7554/eLife.55513
8 figures, 1 table and 4 additional files

Figures

Figure 1 with 3 supplements
SS-31 treatment reverses cardiac aging phenotypes and improves exercise performance in old mice.

Doppler echocardiography showed that 8-week SS-31 treatment (a) improved diastolic function (increased Ea/Aa) and (b) enhanced myocardial performance (reduced myocardial performance index, MPI) of old male mice. (c) Fractional shortening (FS) was not altered by SS-31 treatment. (a–c) n = 7 male mice/group were analyzed by repeated measure ANOVA with Tukey’s multiple comparison test between time points and Sidak post hoc analysis between treatment groups. (d) 8- week SS-31 treatment reversed the age-related increase in normalized heart weight. n = 10 (for young saline, young SS-31 and old saline) and n = 8 (for old SS-31) male mice. Data were analyzed by one-way ANOVA with SNK post hoc analysis. (e) Treadmill running was impaired (reduced running time) in old control male mice but was rescued by SS-31 treatment. n = 10 (for young saline, young SS-31 and old saline) and n = 9 (for old SS-31) male mice, analyzed by one-way ANOVA with SNK post hoc analysis. (f) The improved diastolic function in old female mice (increased Ea/Aa) after 8 week of SS-31 treatment persisted for 2–4 weeks after cessation of treatment. n = 3 for saline control and n = 7 for SS-31 treatment, analyzed by repeated measure ANOVA with Tukey’s multiple comparison test between time points and Sidak post hoc analysis between treatment groups.

Figure 1—figure supplement 1
Individual trajectories of SS-31 responses in cardiac function of male mice.

8-week SS-31 treatment improved diastolic function and myocardial performance over time. Linear regression analyses treating each mouse in a group as a separate point revealed increased Ea/Aa (a) over time in old SS-31 treated (R = 0.45, p<0.001) but not old control mice (p=0.23) and reduced MPI (b) in old SS-31 treated (R = 0.30, p=0.011) but not old control mice (p=0.98). (c) FS remained unchanged in old SS-31 treated (p=0.26) and old control mice (p=0.42) during the 8-week treatment. N = 7 mice per group.

Figure 1—figure supplement 2
Individual trajectories of SS-31 responses in exercise performance of male mice.

Treadmill running time trended to decline in majority of old saline control mice (p=0.11) but not in old SS-31 treated mice (p=0.48); n = 10 for old control and n = 9 for old SS-31, analyzed by Wilcoxon signed-rank test with pairing of 0 week and 8 week data.

Figure 1—figure supplement 3
Individual trajectories of SS-31 responses in cardiac function of female mice.

8-week SS-31 treatment improved diastolic function in old female mice (p<0.001 between 0 week and 8 week by paired T-test); this improvement persisted 2 weeks after cessation of treatment (p=0.006 between 0 week and 10 week by paired T-test). At 4 weeks after treatment cessation, some mice displayed declined Ea/Aa, but overall, Ea/Aa was not significantly different from baseline (p=0.38 between 0 week and 12 week by paired T-test); n = 3 for saline control and n = 7 for SS-31 treatment.

Figure 2 with 1 supplement
SS-31 treatment reduces ROS production and improves respiration in cardiomyocytes.

(a) SS-31 treated cardiomyocytes showed reduced mitochondrial superoxide, indicated by reduced MitoSox signal (normalized to mitochondrial content by the ratio to MitoTracker Green), compared to old controls. *p<0.05 vs old saline; n = 67 cells from three female mice for old saline and n = 71 cells from three female mice for old SS-31, compared by unpaired T-test. (b) SS-31 treated cardiomyocytes showed reduced hydrogen peroxide, indicated by reduced mitoPY1 signal (normalized to mitochondrial content using MitoTracker Deep Red), compared to old controls. *p<0.05 vs old saline; n = 31 cells from three female mice for old saline and n = 29 cells from three female mice for old SS-31, compared by unpaired T-test. Images for MitoSox and MitoPY1 measurements can found in Figure 2—source data 1 and Figure 2—source data 2. (c) Averaged traces of oxygen consumption rate (OCR, + / - SEM) of isolated cardiomyocytes from young, old, and old SS-31 treated male and female mice measured by the Seahorse XF Cell Mito Stress Test. Cardiomyocytes from old mice exhibited increased basal respiration (d) and proton leak (e) compared to that of young mice, and these age-related increases were reversed in cardiomyocytes from 8-week SS-31 treated old mice. (f) Old cardiomyocytes exhibited reduced respiratory control ratio (RCR) compared to young cardiomyocytes and this decrease was partially restored by 8-week SS-31 treatment. (d–f) *p<0.05 vs. young saline; #p<0.05 vs. old saline; n = 16 wells from four mice for young, n = 29 wells from six mice for old and n = 35 wells from six mice for old SS-31, analyzed by one-way ANOVA with SNK post hoc analysis.

Figure 2—figure supplement 1
SS-31 treatment increases mitochondrial membrane potential in aged cardiomyocytes.

*p<0.05 vs old saline. n = 19 (Control) and 18 (SS-31) cells were analyzed by unpaired T-test.

SS-31 treatment does not alter expression of subunits of oxidative phosphorylation complexes.

Immunoblotting using anti-OXPHOS antibody detected no differences in expression levels of OXPHOS subunits (NDUFB8, SDHB, UQCRC2, MTCO1, and ATP5A) in hearts of old male mice treated with SS-31 for 8 weeks. Only transient changes in NDUFB8 levels were detected at 1 and 2 weeks after SS-31 treatment. *p<0.05 vs Control; +p<0.05 vs 1-week SS-31; #p<0.05 vs 2-week SS-31 treatment; n = 6 for Control, n = 7 for 1 week and 2 week and n = 5 for 8 week, analyzed by one-way ANOVA with SNK post hoc analysis.

SS-31 treatment reduces protein oxidation and senescence in old hearts.

(a) A histogram of the distribution of changes in glutathionylation levels in peptides from old control and old SS-31 treated hearts; n = 3 female mice per group, analyzed as described in the Materials and method section. (b) Increased levels of protein carbonylation were detected in hearts of old control mice, but not old SS-31 treated mice, when compared to young control mice; n = 5 female mice per group, analyzed by one-way ANOVA with SNK post hoc analysis. (c–d) IHC staining of cellular senescence markers, p16 (c) and p19 (d), detected reduced p16 and p19 positive nuclei in old SS-31 treated heart compared to old control hearts; n = 5 male mice per group, analyzed by unpaired T-test. Images for p16 and p19 staining can be found in Figure 4—source data 1 and Figure 4—source data 2.

Figure 5 with 2 supplements
SS-31 treatment partially restores age-related proteomic remodeling.

A heatmap of z-scores the 88 proteins that were significantly altered by both aging (q < 0.05 for old control vs. young control) and SS-31 treatment (q < 0.05 for old SS-31 vs. old control); n = 9, 10, and eight male mice for young control, old control and old SS-31, respectively, analyzed as described in the method section. We computed the z-scores of the average log2 abundance values for each of the three groups, where we adjusted the data, by protein, to have a mean of zero and a standard deviation of 1. The heatmap was generated using the ComplexHeatmap (v.1.20.0) R package (Gu et al., 2016), where both the sample groups and the proteins were clustered via the hclust function with the ‘complete’ agglomeration method. Distance matrix for clustering were computed using ‘Euclidean’ distance. The resulting heatmap presents the proteins in rows and sample groups in columns, both of which were grouped according to the clustering results. Row labels on the right are the UniProt ID_Gene Name of each protein. The identities and fold changes of all protein identified are listed in Supplementary file 3.

Figure 5—figure supplement 1
SS-31 treatment induces modest changes in metabolome that partially attenuates the age-related changes.

A heat map of the relative levels of the 18 metabolites that were significantly (FDR < 0.05) different among treatment groups; n = 10 (for YCL, YSS, OCL) and n = 8 (for OSS) were male mice were analyzed as described in the method section.

Figure 5—figure supplement 2
A network of metabolite set enrichment for the 11 metabolites that were significantly (p<0.05, by Tukey’s HSD) altered by aging.
SS-31 rescues the age-related hypo-phosphorylation of MyBP-C.

(a) Old murine hearts displayed reduced levels of MyBP-C phosphorylation at Ser282, which is normalized by SS-31 treatment. (b–c) Aging and SS-31 treatment did not alter phosphorylation of cTnI at Ser23/24 (b) and Ser150 (c) in hearts For panel a-c, n = 5, 6, and five male mice for young control, old control and old SS-31, respectively, analyzed by one-way ANOVA Dunnett’s post hoc analysis for panel a-c. (d) Titin isoform ratio (N2BA/N2B ratio) did not change with SS-31 treatment; n = 8 and 6 female mice were used for old control and old SS-31, respectively, and were compared by unpaired T-test.

The cardiac benefit of SS-31 treatment is not additive to that of mCAT expression but the two interventions differentially regulate myofilament protein phosphorylation.

(a) Diastolic function (Ea/Aa) improved at both 8 and 12 weeks after AAV9-mCAT administration. n = 3 (saline) and n = 5 (AAV-mCAT) female mice were analyzed by repeated measure ANOVA with Tukey’s multiple comparison test between time points and Sidak post hoc analysis between treatment groups. (b) 8-week SS-31 improved diastolic function in old WT but did not further improve the function of old mCAT mice; n = 5 (for SS-WT and SS-mCAT) and n = 6 (for Saline-WT) mixed-sex mice were analyzed by repeated measure ANOVA with Tukey’s multiple comparison test between time points. (c) Late-life mCAT expression reduced Ser282 phosphorylation of MyBP-C. (d–e) Late-life mCAT expression increased phosphorylation of cTnI at Ser23/24 (d) and Ser150 (e). For panel c-e, n = 3 and 5 female mice were used for saline and AAV-mCAT, respectively, and were analyzed by unpaired T-test.

Schematic outline of results and interpretation.

While mCAT and SS-31 both inhibit electron transport chain produced ROS, they do so by different mechanisms. Both inhibit a ROS-mediated vicious cycle (ROS induced mtDNA and protein damage leads to greater ROS generation; striped arrows) and ROS-Induced redox signaling. However, by promoting electron transport, preventing proton leakage and augmenting ATP production, SS-31 also improves mitochondrial energetics. By improving mitochondrial energetics and reducing pathologic redox signaling, SS-31 promotes phosphorylation of cMyBP-C to enhance myofilament relaxation kinetics, while mCAT expression does so through promoting phosphorylation of cTnI.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (M. musculus; male and female)C57BL/6JNational Institute of Aging Charles River colonyRRID:IMSR_JAX:000664
Genetic reagent (M. musculus)mCATRabinovitch Lab;
PMID:15879174
RRID:IMSR_JAX:016197;
Tg(CAG-OTC/CAT)4033Prab
Now available at The Jackson Lab
Transfected construct (M. musculus)AAV9-mCATDuan Lab,
PMID:19690612
AV.RSV.MCATAdeno-associated virus construct to transfect and express mCAT transgene
AntibodyAnti-OXPHOS (Rabbit polyclonal)Abcamab110413
RRID:AB_2629281
(1:500)
AntibodyAnti-Troponin I (Rabbit polyclonal)Cell Signaling Technology#4002
RRID:AB_2206278
(1:1000)
AntibodyAnti-pSer23/24-Troponin I (Rabbit polyclonal)Cell Signaling Technology#4004
RRID:AB_2206275
(1:1000)
AntibodyAnti-pSer150-Troponin I (Rabbit polyclonal)ThermoFisherPA5-35410
RRID:AB_2552720
(1:1000)
AntibodyAnti- cMyBP-C (Mouse monoclonal)Santa Cruz SC-137237SC-137237
RRID:AB_2148327
(1:1000)
AntibodyAnti-pSer282-cMyBP-C (Rabbit polyclonal)EnzoALX-215–057 R050
RRID:AB_2050502
(1:2000)
Antibodyanti-p19 (Rabbit polyclonal)LSBioLS-C49180
RRID:AB_1192824
(1:300)
Antibodyanti-p16 (Rabbit polyclonal)Abcamab211542(1:300)
AntibodyDonkey anti-Rabbit Secondary Antibody, HRPThermoFisherA16035
RRID:AB_2534709
(1:10000)
AntibodyGoat anti-Mouse Secondary Antibody, HRPThermoFisherA16072
RRID:AB_2534745
(1:10000)
Peptide, recombinant proteinSS-31 peptide (Elamipretide)Stealth BioTherapeutics3 µg/g body weight/day
Commercial assay or kitOxiSelect protein carbonyl ELISA kitCell BiolabsSTA-310
Commercial assay or kitImmPRESS-VR Anti-Rabbit IgG HRP Polymer Detection KitVector LaboratoriesMP-6401–15
Commercial assay or kitSeahorse XF Cell Mito Stress Test KitAligent/Seahorse Bioscience103015–100
Commercial assay or kitMitoSOX RedThermoFisherM36008
Commercial assay or kitMitoPY1Fisher Scientific/Tocris Bioscience44-281-0
Commercial assay or kitMitoTracker GreenThermoFisherM7514
Commercial assay or kitMitoTracker Deep RedThermoFisherM22426
Commercial assay or kitJC-1 DyeThermoFisherT3168
Commercial assay or kitBCA protein assayThermo Scientific23225
Commercial assay or kitPierce Reversible Protein Stain Kit for PVDF MembranesThermo Scientific24585
Commercial assay or kitSuperSignal West
Pico PLUS
Chemiluminescent Substrate
Thermo Scientific34580
Software, algorithmGraphpad PrismGraphpadRRID:SCR_002798
Software, algorithmAlphaView SoftwareProteinSimple
Software, algorithmMetaboanalyst 4.0www.metaboanalyst.ca;
PMID:29762782
RRID:SCR_015539
Software, algorithmTopographMacCoss Lab software; PMID:22865922
Software, algorithmComplexHeatmaphttps://github.com/jokergoo/ComplexHeatmap
PMID:27207943
RRID:SCR_017270

Additional files

Supplementary file 1

A dataset of relative abundances of all metabolites measured.

https://cdn.elifesciences.org/articles/55513/elife-55513-supp1-v2.csv
Supplementary file 2

A table of all metabolites that showed significant differences in one or more comparisons.

https://cdn.elifesciences.org/articles/55513/elife-55513-supp2-v2.xlsx
Supplementary file 3

A dataset of identities, fold changes and statistics of all proteins identified in the proteomic analysis.

https://cdn.elifesciences.org/articles/55513/elife-55513-supp3-v2.csv
Transparent reporting form
https://cdn.elifesciences.org/articles/55513/elife-55513-transrepform-v2.pdf

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