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. 2020 Dec 15;9:e60827. doi: 10.7554/eLife.60827

Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte

Huiliang Zhang 1, Nathan N Alder 2, Wang Wang 1,3, Hazel Szeto 4, David J Marcinek 5, Peter S Rabinovitch 1,✉
Editors: Holly van Remmen6, Jessica K Tyler7
PMCID: PMC7738186  PMID: 33319746

Abstract

Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. Age-related heart impairment is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes. Most importantly, the tetra-peptide SS-31 prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore opening, rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, and leads to substantial reversal of diastolic dysfunction. Our results uncover the excessive proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 can reverse this clinically important complication of cardiac aging.

Research organism: Mouse, Rat

Introduction

Mitochondria are both the primary source of organismal energy and the major source of cellular reactive oxygen species (ROS) and oxidative stress during aging (Dai et al., 2014). Aged cardiac mitochondria are functionally changed in redox balance and are deficient in ATP production (Lesnefsky et al., 2016). Numerous reported studies have focused on redox stress and ROS production in aging (Dai et al., 2014). However, in its simplistic form, the free radical theory of aging has become severely challenged (Pérez et al., 2009).

While more attention has been placed on mitochondrial electron leak and consequent free radical generation, proton leak is a highly significant aspect of mitochondrial energetics, as it accounts for more than 20% of oxygen consumption in the liver (Brand, 2005) and 35–50% of that in muscle in the resting state (Rolfe and Brand, 1996). There are two types of proton leak in the mitochondria: (1) constitutive, basal proton leak, and (2) inducible, regulated proton leak, including that mediated by uncoupling proteins (UCPs) (Divakaruni and Brand, 2011). In skeletal muscle, a majority of basal proton conductance has been attributed to adenine nucleotide translocase (ANT) (Brand et al., 2005). Although aging-related increased mitochondrial proton leak was detected in the mouse heart, kidneys, and liver by indirect measurement of oxygen consumption in isolated mitochondria (Harper et al., 1998; Serviddio et al., 2007), direct evidence of functional impact remains to be further investigated. Moreover, the exact site and underlying mechanisms responsible for aging-related mitochondrial proton leak are unclear.

SS-31 (elamipretide), a tetrapeptide (D-Arg-2′,6′-dimethyltyrosine-Lys-Phe-NH2), binds to cardiolipin-containing membranes (Birk et al., 2013) and improves cristae curvature (Szeto, 2014). Prevention of cytochrome c peroxidase activity and release has been proposed as its major basis of activity (Szeto, 2014; Szeto and Birk, 2014). SS-31 is highly effective in increasing resistance to a broad range of diseases, including heart ischemia reperfusion injury (Cho et al., 2007; Szeto, 2008), heart failure (Dai et al., 2013), neurodegenerative disease (Yang et al., 2009), and metabolic syndrome (Anderson et al., 2009). In aged mice, SS-31 ameliorates kidney glomerulopathy (Sweetwyne et al., 2017) and brain oxidative stress (Hao et al., 2017) and has shown beneficial effects on skeletal muscle performance (Siegel et al., 2013). We have recently shown that administration of SS-31 to 24-month-old mice for 8 weeks reverses the age-related decline in diastolic function, increasing the E/A from just above 1.0 to 1.22, restoring this parameter 35% toward that of young (5-month-old) mice (Chiao et al., 2020). However, how SS-31 benefits and protects aged cardiac cells remains unclear.

In this report, we investigated the effect and underlying mechanism of action of SS-31 on aged cardiomyocytes, especially on the mitochondrial proton leak. Using the naturally aged rodent model we provided direct evidence of increased proton leak as the primary energetic change in aged mitochondria. We further show that the inner membrane protein ANT1 mediates the augmented proton entry in the old mitochondria. Most significantly, we demonstrate that SS-31 acutely prevents the excessive mitochondrial proton entry and rejuvenates mitochondrial function through direct association with ANT1 and stabilization of the ATP synthasome.

Results

SS-31 alleviates the excessive mitochondrial proton leak in old cardiomyocytes

To examine whether SS-31 restores aging mitochondrial function, we applied the Seahorse mitochondrial stress assay to intact primary cardiomyocytes in a non-working state (not paced for contraction). The Seahorse assay revealed higher mitochondrial basal respiration in cells from old mice than that in young mouse cells (Figure 1A,C); however, the maximal respiratory rate was not significantly different (Figure 1A,D). The increased basal respiration was attributable to a higher proton leak in old cardiomyocytes (164 ± 16 in 24 month vs 82 ± 12 in young, pmol/min/800 cells, n = 7–14, p<0.01) (Figure 1A,B). Although, SS-31 has only a minor and non-significant effect on young cardiomyocytes (Figure 1—figure supplement 1), acute in vitro treatment of isolated old cardiomyocytes with SS-31 (100 nM, 1 μM, or 10 μM for 2 hr), caused reduced mitochondrial proton leak (Figure 1A,B and Figure 1—figure supplement 2), shifting their respiratory pattern closer to that of young cells. These results indicate that SS-31 directly protects aging cardiac energetics through rapid rejuvenation of mitochondrial respiration in cardiomyocytes, and in particular, by reducing proton leak.

Figure 1. SS-31 alleviates the excessive mitochondrial proton leak of cardiomyocytes from 24 mo old mice.

(A) Representative Seahorse assay traces of cardiomyocytes isolated from untreated young and old mice, then exposed or not to 100 nM SS-31 for 2 hr in vitro. Aging-increased basal respiration (C), which was attributable to the augmentation of proton leak (B), but did not affect maximal respiration (D). ANT1 inhibitors Bongkrekic acid (BKA, 10 μM) and carboxyatractyloside (CAT, 20 μM) 2 hr treatment decreased the proton leak (B) and basal respiration (C) but also decreased maximal respiration (D) in old cardiomyocytes. N = 5–14 mice in each group; one-way ANOVA followed by Fisher’s LSD test. *p<0.05, **p<0.01 vs young; #p<0.05, ##p<0.01 vs old controls.

Figure 1.

Figure 1—figure supplement 1. SS-31 has a minor effect on mitochondrial respiration in the young cardiomyocytes.

Figure 1—figure supplement 1.

None of the differences are significant. N = 5 mice in each group. Student’s t-test was applied to determine the statistical significance.
Figure 1—figure supplement 2. SS-31 reaches its inhibitory effect on proton leak suppression at low concentrations.

Figure 1—figure supplement 2.

SS-31 decreased the mitochondrial proton leak. N = 6–14 mice in each group. **p<0.01 vs young, #p<0.05 vs old controls. One-way ANOVA with Fisher’s LSD test was applied.
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SS-31 restores the resistance to external pH gradient stress in mitochondria of old cardiomyocytes

The evaluation of mitochondrial proton leak by Seahorse assay is indirect, as it is based on the oxygen consumption rate. Thus, to directly investigate the reduction of mitochondrial proton leak in old cardiomyocytes by SS-31, we expressed the protein mt-cpYFP, a mitochondrial matrix-targeted pH indicator (Demaurex and Schwarzländer, 2016; Schwarzländer et al., 2012; Wang et al., 2016a; Wei-LaPierre et al., 2013) in the rat cardiomyocytes (Figure 2—figure supplement 1). Taking advantage of the pH sensitive character of mt-cpYFP, we developed a novel protocol to evaluate mitochondrial proton leak by exposing mitochondria to a pH gradient stress in saponin permeabilized, mt-cpYFP expressing cardiomyocytes (Figure 2A,B). The drop in mt-cpYFP 488/405 ratio is due to proton leak through the mitochondrial inner membrane into the mitochondrial matrix. To evaluate the physical properties of the mitochondrial inner membrane in the absence of mitochondrial activity, we permeabilized the cardiomyocytes in a buffer that contained no substrates, ATP, or ADP. We found that aging reduced cardiomyocyte mitochondrial resistance to a proton gradient stress (Figure 2A,B). More importantly, we found 10 μM SS-31 treatment in vitro restored cardiomyocyte mitochondrial inner membrane resistance to the pH gradient stress in the aged cardiomyocytes (Figure 2A,B). SS-31 treatment largely prevented the decline in matrix pH of old cells after the external pH was reduced to 6.9 (Figure 2—figure supplement 2) and 5.3 (Figure 2B,C) and slowed the rate of cpYFP 488/405 change after pH 6.9 (Figure 2—figure supplement 3, Figure 2D). At pH 4.5 SS-31 continued to enhance resistance to proton permeability in the treated old cells. This is unlikely to represent a biological benefit at this nonphysiologic pH, but does indicate the substantial change in physical properties of the inner membrane after interaction with SS-31 (Figure 2B). To further evaluate the kinetics of SS-31 effect on mitochondrial proton permeability, we analyzed cpYFP fluorescence ratios at various times after exposure of the saponin treated cardiomyocytes to 10 μM SS-31. SS-31 protection on the mitochondrial matrix proton entry became significant and near maximal after 7–10 min of SS-31 treatment (Figure 2E). We examined the dose effect of SS-31 on proton permeability and found near-maximal effects at 100 nM SS-31 (Figure 2F). In summary, this is the first direct evidence that aging increases mitochondrial inner membrane proton permeability in aged cardiomyocytes and that SS-31 protects mitochondria from this proton leak.

Figure 2. SS-31 restores the resistance of cardiomyocytes from old rat to proton entry into the mitochondrial matrix during external pH gradient stress.

(A) Typical image of the effects of pH gradient stress on permeabilized rat cardiomyocyte mt-cpYFP fluorescence. Upper panel, young, middle panel, old, lower panel, old+SS-31 (10 μM, 3 days) visualized after exposure of the cells to pH 7.5 and, later, to pH 5.3. The excitation is 488 nm and collection is at 505–730 nm. (B) Saponin (50 μg/ml) permeabilized cardiomyocytes expressing mt-cpYFP were exposed to progressively lower external pH. Proton permeability of old mitochondria was greater than that of young mitochondria, but preincubation of old cells with 10 μM SS-31 for 3 days enhanced the mitochondrial inner membrane resistance to the pH stress. The traces were averaged from 4 to 19 experiments. The arrows indicate the changes of pH. (C) Quantitation of the SS-31 treatment effect on the mitochondrial matrix cpYFP ratio at pH 5.3. The data are from 7 to 8 min after the pH was adjusted to 5.3. The mean value of the normalized 488/405 ratio in old group is 0.59 with a SD ±0.10 and the range is from 0.46 to 0.77. N = 5–19 rats in each group, analyzed by one-way ANOVA with Dunnett’s test. (D) SS-31 decreased the rate of cpYFP 488/405 ratio drop at pH 6.9. N = 4–10 rats in each group. The rate is calculated as indicated in Figure 2—figure supplement 3. Student’s t-test was applied to determine the statistical significance. The time dependence (N = 3–4 rats in each group) (E) and dose dependence (N = 3–14 rats in each group) (F) of SS-31 protection of mitochondrial resistance to pH gradient stress are shown and analyzed by one-way ANOVA with Fisher’s LSD test. After cardiomyocyte permeabilization, 10 μM SS-31 was added for the times shown in (E) or at the doses shown in (F) for 30 min, followed by pH stress. *p<0.05, **p<0.01 vs Young; #p<0.05, ##p<0.01 vs Old.

Figure 2.

Figure 2—figure supplement 1. pH calibration of mt-cpYFP in adult cardiac myocytes with Nigericin.

Figure 2—figure supplement 1.

10 μM Nigericin was added to the mitochondrial permeabilization buffer. (A) shows the pH calibration of 488 nm and 405 nm, the ratio of 488/405 (normalized to that of the value at pH = 7.5) as the pH is gradually lowered. The ratio of 488/405 reaches the lowest point at pH 6.0, followed by a small increase starting at pH 5.5, consistent with the known pH response of this dye (Wei-LaPierre et al., 2013). (B) shows the emission spectra of mt-cpYFP pH 8.0, 6.9, and 5.3 when excited at 488 nm (C) shows the emission spectra of mt-cpYFP pH 8.0, 6.9, and 5.3 when excited at 405 nm. The 488 nm excitation is sensitive to the pH change but the 405 nm excitation is much less pH dependent.
Figure 2—figure supplement 2. SS31 restores the resistance to external pH gradient stress in the old cardiomyocytes at pH 6.9.

Figure 2—figure supplement 2.

N = 5–18 in each group. One-way ANOVA with Dunnett’s test was applied. **p<0.01 vs young, #p<0.05 vs old controls.
Figure 2—figure supplement 3. Method of quantitation of the slope of mt-cpYFP fluorescence ratio change after permeabilized cells are exposed to external pH 6.9.

Figure 2—figure supplement 3.

The trace is from old rat cardiomyocytes.
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ANT1 inhibitors restore resistance to proton leak of mitochondria in old cardiomyocytes

In search of the source of the uncoupled mitochondrial proton leak in the aged cells, we examined possible involvement of proton leakage through ATPase and mitochondrial UCPs. As expected, the ATPase inhibitor Oligomycin A failed to inhibit the proton leak in pH challenged permeabilized aged cells (Figure 3A,B). Levels of UCP2, which is the dominant isoform of UCPs in the heart, do not change with age in hearts (Figure 3—figure supplement 1). Genipin, an inhibitor of UCP2, showed no effect on the proton leak in permeabilized aged cells (Figure 3A,B). These results suggest that ATPase and UCP2 may not be the source of the excess proton leak in the aged hearts.

Figure 3. ANT1 inhibitors restore resistance to proton leak of mitochondria in old rat cardiomyocytes.

ANT1 inhibitors 10 μM Bongkrekic Acid (BKA) and 20 μM carboxyatractyloside (CAT), but not 50 μM Genipin (UCP2 inhibitor) or 1 μM oligomycin A (OA, ATPase inhibitor), protected the mitochondrial matrix from decreased pH after exposure to external pH 5.3 (N = 4–19 rats in each group) (A) and reduced the rate of 488/405 decline after exposure to pH 6.9 (N = 4–10 rats in each group) (B). BKA, CAT, Genipin, or OA were added immediately after the mitochondria permeabilization. One-way ANOVA with Fisher’s LSD test was applied. *p<0.05, **p<0.01 vs Young, #p<0.05, ##p<0.01 vs Old.

Figure 3.

Figure 3—figure supplement 1. Aging effect on ANT1 and UCP2 cardiac protein abundance.

Figure 3—figure supplement 1.

Aging slightly, but significantly decreases ANT1 levels in the mouse heart. Western blot of ANT1 (A, C) and UCP2 (B, D). N = 6 mice in each group. Student’s t-test was applied to determine the statistical significance. *p<0.05 vs young. The total protein loading control was stained with MemCode Reversible Protein Stain Kit for Polyvinylidene difluoride Membranes (Pierce, Rockford, IL). The identity of the band as UCP2 in B was confirmed using a recombinant UCP2 protein standard (Origene, CAT#: TP761109), as shown in E.
Figure 3—figure supplement 2. Mitochondrial proton entry in buffer without phosphate.

Figure 3—figure supplement 2.

Mitochondrial pH stress on permeabilized rat cardiomyocytes in a buffer that did not contain phosphate.
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Recently, the inner membrane protein ANT1 (also called AAC) was identified as the major site of proton leak in mitochondria of multiple tissues (Bertholet et al., 2019), and was shown to contribute to the majority of the proton leak in muscle cells (Brand et al., 2005). Treatment of old cardiomyocytes with either the ANT1 inhibitor bongkrekic acid (BKA) (Ruprecht et al., 2019) or carboxyatractyloside (CAT) (Pebay-Peyroula et al., 2003) completely suppressed the excess proton leak in the Seahorse assay, though unlike SS-31, they also decreased the maximal respiratory rate (Figure 1B,D), which is consistent with the effect seen in the ANT triple knockout model (Karch et al., 2019). We treated permeabilized old cardiomyocytes with the ANT1 inhibitors and examined the mt-cpYFP response to an external pH gradient using the protocol described above. BKA suppressed the proton leak in old cardiomyocytes, evidenced by the preserved 488/405 ratio at pH 5.3 and a slower 488/405 ratio decrease at pH 6.9 (Figure 3A,B). Similar inhibition was found with CAT treatment (Figure 3A,B). Taken together, these data implicate ANT1 as the major site of proton leak in aging hearts.

Considering that the inorganic phosphate carrier might be a potential source of the elevated proton entry in old mitochondria, we examined the proton permeability of old mitochondria to pH stress in a buffer that did not contain phosphate. In this buffer, the proton entry characteristics of mitochondria from old cells was indistinguishable from that of mitochondria stressed in the presence of inorganic phosphate (Figure 3—figure supplement 2).

SS-31 attenuates the excessive mitochondrial flash (mitoflash) activity of aged cardiomyocytes, while normalizing membrane potential and ROS

The mitoflash (Feng et al., 2017; Hou et al., 2014; Shen et al., 2014; Wang et al., 2008; Wang et al., 2016b; Zhang et al., 2015) is triggered by nanodomain proton influx into the mitochondrial matrix (Wang et al., 2016c). Thus, we wondered whether the increased proton leak in the old cells triggered excessive mitoflash activity. We evaluated mitoflash activity in isolated young and old rat cardiomyocytes using the indicator mt-cpYFP, as established in the previous studies noted above. The mitochondrial mitoflash activity in the cells from old (26 mo) cardiomyocytes was higher than that of young (five mo) cells (2.8 ± 0.3 in old vs 1.4 ± 0.2,/1000 μm2/100 s in young cells, n = 28–88, p<0.05). Confirming this, we detected an increase in mitoflash activity in Langendorff perfused intact aged hearts from mt-cpYFP transgenic mice (Figure 4—figure supplement 1). 1 hr treatment with SS-31 normalized the mitoflash activity in old cells to the young cell level (Figure 4D). The mitochondrial ANT1 inhibitors BKA and CAT showed super-suppression of the flash activity, reducing this frequency to half of that of young cells (Figure 4D). These data support the notion that proton leak from ANT1 triggers the mitoflash in cardiomyocytes and is responsible for the excess mitoflash activity of old cells. Moreover, the mitochondrial membrane potential, which is generally lower in old cardiomyocytes (Serviddio et al., 2007), is restored to youthful levels by SS-31 treatment (Figure 4E). Also, SS-31 reduced ROS production in the aged cardiomyocytes (Figure 4F). Thus, the reduction of mitochondrial proton leak by SS-31 is accompanied by a more youthful membrane potential and dynamic function (mitoflash), as well as less oxidative stress.

Figure 4. SS-31 attenuates excessive mitoflash activity in aged rat cardiomyocytes.

(A–C) Mitoflash events within the regions shown in the red boxes took place at the times shown by vertical bars during the 100 s scanning time in the representative cardiomyocytes from young (A), old (B) and old+SS-31 (C) rat hearts. (D) The rate of mitoflash activity was increased in old rat cardiomyocytes compared to young, but 1 hr SS-31, Bongkrekic Acid (BKA) (10 μM) and Carboxyatractyloside (CAT) (20 μM) treatments decreased the mitoflash frequency in old cells to or below that of young cells. N = 26–87 cells from 3 to 14 rats. **p<0.05 vs young. ##p<0.05 vs old. (E) JC-1 red to green fluorescence ratio, indicative of mitochondrial membrane potential, in cells from young and old mice and old mouse cardiomyocytes treated with SS-31 (10 μM for 12 hr). N = 84–218 cells from 3 to 4 mice. p=0.06 vs young. ##p<0.01 vs old. (F) Mitochondrial ROS production in mouse cardiomyocytes measured by the fluorescence ratio of MitoSOX (5 μM, excitation 540 nm, emission >560 nm) to Mitotracker green (200 nM, excitation 488 nm, emission 505–530 nm). One-way ANOVA with Dunnett’s test was applied. N = 40–84 cells from 3 to 5 mice. **p<0.05 vs young. ##p<0.05 vs old.

Figure 4.

Figure 4—figure supplement 1. Increased mitochondrial flash activity in the intact perfused mouse aged heart.

Figure 4—figure supplement 1.

(A) Typical images from the young (upper panel) and old (lower panel) mouse hearts. (B) Statistical analysis of mitoflash frequency in the analyzed regions indicated by the red boxes in panel A. Young: N = 18 regions from three mice. Old: N = 16 regions from four mice. Student’s t-test was applied to determine the statistical significance. *p<0.05 vs Young.
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SS-31 reverses increased mPTP opening in aged cardiomyocytes

Due to the close link previously established between the mitoflash and mitochondrial permeability transition pore (mPTP) opening (Hou et al., 2014), we evaluated mPTP activity by the photon-triggered mPTP opening protocol (Figure 5A; Zorov et al., 2000). Consistent with previous reports in isolated mitochondria (Hafner et al., 2010), we found that the time to mPTP opening is decreased in intact old cardiomyocytes (Figure 5B). SS-31 and the ANT1 inhibitor BKA, which stabilizes the ANT1 in the m-state open toward the mitochondrial matrix, both protect the aging-increased mitochondrial mPTP opening rate (Figure 5B), consistent with previous observations that BKA prevents the onset of the permeability transition (Halestrap et al., 1997). The ANT1 inhibitor CAT, which stabilizes ANT1 in the c-state open toward the cytosol, failed to prevent the rapid opening of the mPTP in old cells (Figure 5B), consistent with previous observations that it facilitates mPTP opening (Halestrap et al., 1997). These data indicate that SS-31 decreases mPTP opening in old cardiomyocytes.

Figure 5. SS-31 reverses the increased speed of mitochondrial permeability transition pore (mPTP) opening in aged mouse cardiomyocytes.

Figure 5.

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(A) A typical image shows 1 Hz line-scanning photo-excitation induced mPTP opening in a cardiomyocyte loaded with the mitochondrial membrane potential (Δψm) dye Tetramethylrhodamine methyl ester (TMRM). The sudden decline of TMRM fluorescence with time (rightward) indicates mPTP opening and Δψm loss. (B) 1 hr SS-31 and Bongkrekic Acid (BKA), but not carboxyatractyloside (CAT) treatments protect the photo-excitation induced mPTP opening. Quantification of time to mPTP opening from 418 to 658 mitochondria from 19 to 32 cells isolated from 3 to 4 mice in each group. One-way ANOVA with Dunnett’s test was applied. **p<0.01 vs young, ##p<0.01 vs old.

SS-31 associates directly with ANT1 and the ATP synthasome

To further investigate the mechanism of SS-31 protection on the proton leak, we used biotinylated SS-31 to evaluate whether SS-31 directly interacts with the ANT1 protein. Hearts were disrupted by douncing, after a low speed spin to remove fragments, mitochondria were collected by high speed spin and disrupted in digitonin, to create lipid rafts containing their associated proteins, a protocol commonly used to prepare mitochondrial supercomplexes (Johnson et al., 2013). This preparation was incubated with SS-31-biotin or biotin only, followed by incubation with streptavidin beads. After washing, the bead-bound fraction was eluted with excess SS-31 and analyzed by Western blotting. Biotin-SS-31 pulled down ANT1, and free SS-31 competed with the biotin-SS-31 binding to ANT1 (Figure 6A,B, Figure 6—figure supplement 1). Most notably, both BKA and CAT inhibited binding of biotin-SS-31 to ANT1 (Figure 6A,B). This competition was observed even at BKA and CAT concentrations in the tens of nanomolar range (data not shown), which is consistent with their reported Kd of binding to ANT1 (Vignais et al., 1976). Biotin-SS-31 pulldown of ANT1 was not inhibited by Genipin or Oligomycin A (Figure 6A,B). These data indicate that SS-31 associates closely with the ANT1 protein. Moreover, native gel and ATPase blot analysis showed that SS-31 stabilized the ATP synthasome, of which ANT1 and ATPase are critical members (Ko et al., 2003; Figure 6D,E). However, SS-31 treatment did not produce a detectable increase in mitochondrial complex proteins by Coomassie blue staining (Figure 6C). Taken together, these data suggest that SS-31 interacts directly with ANT1 and stabilizes the ATP synthasome in old cardiomyocytes.

Figure 6. SS-31 interacts with ANT1 and stabilizes the ATP synthasome in the old mouse heart mitochondria.

(A, B) Biotin-SS-31 pulldown shows the association of biotin-SS-31 to ANT1. Free SS-31 competes with this interaction, while Bongkrekic Acid (BKA) and carboxyatractyloside (CAT) inhibit the interaction of biotin-SS-31 with ANT1. Panel A shows a representative Western blot. N = 6 mice in each group. One-way ANOVA with Fisher’s LSD test was applied. **p<0.01 vs Biotin control, #p<0.05, ##p<0.01 vs Biotin-SS31 pulldown. (C) Coomassie blue staining of isolated mitochondria in a native gel. (D) Left panel is the total protein loading control for the Native gel blot. (D, E) Native gel blotting shows that 10 μM SS-31 stabilizes the mitochondrial synthasome (Syn) in isolated mitochondria. The Syn is highlighted in the red box. The Syn and ATPase Dimer (D) and Monomer (M) were labeled using anti-ATP5A. N = 8 mice in each group. One-way ANOVA with Fisher’s LSD test was applied. *p<0.05 vs young, #p<0.05 vs old.

Figure 6.

Figure 6—figure supplement 1. Silver staining of the Biotin-SS-31 pulldown of Figure 6A from the old mouse heart mitochondria.

Figure 6—figure supplement 1.

The great majority of bands present in the input are not present in the biotin-SS-31 pulldown; those that are show suppression by SS-31 competition.
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Discussion

In this report, we have shown direct evidence of increased proton leak in the aged mitochondria as a primary energetic disturbance and that the increased proton entry in old cardiomyocytes likely takes place through ANT1. Moreover, we demonstrated that SS-31 prevents the proton entry to the mitochondrial matrix and rejuvenates mitochondrial function through direct interaction with ANT1 and stabilization of the ATP synthasome. During aging, the pathological augmented and sustained basal proton leak burdens the mitochondrial work load, resulting in a decline in respiratory efficiency. Blocking this pathological proton leak induced by aging benefits the mitochondria and the heart (Figure 7). We suggest that the restoration of aged mitochondrial function that is conferred by SS-31 is directly attributable to this effect. However, the resulting enhancement in diastolic function is likely to require downstream changes, as the functional benefit took up to 8 weeks to reach full effect, and required post-translational modifications of contractile protein elements (Chiao et al., 2020). It is increasingly recognized that mitochondrial function, including redox status and energetics, has far-reaching effects, including epigenetic alterations and post-translational modifications (Olgar et al., 2019).

Figure 7. Schematic of the mechanism of SS-31 protection of proton leak and rejuvenation of mitochondrial function.

Figure 7.

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Due to increased mitochondrial proton leak, the mitochondria work harder to maintain ATP production, and thus the work load is increased in the aged heart.

ANT1 appears to mediate the pathological mitochondrial proton leak in the aged mouse heart. Although an increased mitochondrial proton leak in the aged heart was previously suggested by indirect oxygen consumption measurement (Serviddio et al., 2007), the site of this augmented proton leak in aging mitochondria has remained a puzzle. We directly evaluated the proton leak using the mitochondrial matrix-targeted pH indicator (mt-cpYFP) and provide evidence that implicates ANT1, the ATP/ADP translocator, as responsible for the pathologically increased proton leak in aged cardiomyocytes. This does not necessarily implicate the ADP/ATP translocase mechanism itself in the proton leak, as in the unenergetic state in which we examined the mitochondrial pH resistance, there would be no ADP/ATP transport activity. Our result is, however, supported by a recent report that proton transport is an integral function of ANT1 (Bertholet et al., 2019). Because the ANT1 protein level is not increased in the aged heart (it is, in fact, mildly but significantly decreased, Figure 3—figure supplement 1), the aging-augmented proton leak through ANT1 must be through altered transport activity or conformational change. Both the inhibitors BKA (locking ANT1 in the matrix face state [Ruprecht et al., 2019]) and CAT (locking ANT1 in the cytosol face state [Pebay-Peyroula et al., 2003]) suppressed the proton leak in the aged cardiomyocytes, suggesting that constraining the conformational state in either position, or otherwise blocking the proton pore reduces ANT1 proton translocation.

Most interestingly, for the first time, we showed that a novel drug, SS-31 (elamipretide), now in clinical trials, prevents the augmented mitochondrial proton leak, rejuvenates mitochondrial function, and reverses aging-related cardiac dysfunction. Mechanistically, we found that SS-31 directly interacts with ANT1 and stabilizes formation of the ATP synthasome. This would seem surprising, given the prior belief that SS-31 affects mitochondria via binding to cardiolipin. However, the notion that SS-31 prevents the proton leak by direct interaction at the pore ‘pocket’ of ANT1 is supported by recent observations based on cross-linking ‘interactome’ mass-spectroscopy that showed that SS-31 is in intimate proximity to two lysine amino acid residues in the water filled cavity of the ANT1 protein (Chavez et al., 2020). Moreover, the cross-linking data suggested that this interaction could have structural consequences and may stabilize the m-state of ANT1 (Chavez et al., 2020). Our observation that both BKA and CAT blocked the SS-31 interaction with ANT1 suggests that SS-31 interacts with ANT1 independent of the ANT1 face to m-state (matrix facing) or c-state (cytoplasmic-facing). These results, and prior evidence of the critical role of ANT1 in mitochondrial health and function (Liu and Chen, 2013), warrant further high resolution structural study of the ANT transporter.

It has recently been shown that SS-31 alters surface electrostatic properties of the mitochondrial inner membrane (Mitchell et al., 2019). The consequences of this effect could include alteration of the channel ion gating properties of the ANT, including conformational changes secondary to enhanced supercomplex and ATP synthasome complex stability. SS-31 effects on stabilization of mitochondrial synthasome (Figure 6D,E) could directly contribute to the enhanced efficiency of mitochondrial respiration that is seen in muscle of SS-31 treated old animals (Siegel et al., 2013) and the improvement in performance of humans with primary mitochondrial myopathy (Karaa et al., 2018). As considerable prior literature indicates that SS-31 interacts with cardiolipin, this interaction would also affect the ATP synthasome, as all the components of the ATP synthasome are ‘floating’ in the inner membrane lipid membrane (of which about 20% is cardiolipin). It is possible that this directly affects stability of the synthasome components. Alternatively, the effect could be due to the change in inner membrane electrostatic properties that has recently been shown to result from incorporation of SS-31 into the inner membrane (Mitchell et al., 2019), as this could indirectly stabilize the ATP synthasome.

The restoration of membrane potential by SS-31 in the old mitochondria (Figure 4E) can be attributed to the suppression of proton leak. However, SS-31 also decreased ROS production (Figure 4F) in the aged cardiomyocytes. It is unclear if the reduced ROS production is associated with modification of the ANT1 shown here or through a parallel mechanism. Blocking this pathological proton leak induced by aging will benefit the mitochondria and the heart. This is not in conflict with the ‘uncoupling to survive hypothesis’, which arises from the positive correlations between increase proton leak, reduced ROS, and increased lifespan (Brand, 2000). This reduced ROS production is interpreted as resulting from decreased electromotive force and consequent reduced electron leak during transport through the respiratory chain. However, SS-31 through its interaction with cardiolipin, abundant in the inner membrane, can improve the efficiency of electron transfer, especially by its known interaction with the heme group of cytochrome c (Szeto, 2014; Szeto and Birk, 2014), thereby reducing ROS production, even as the aged mitochondrial membrane potential is increased. Thus, our data support the conclusion that SS-31 interaction with multiple inner membrane proteins enhances the performance of multiple facets of respiratory mechanics.

In summary, our study reveals that the adenine nucleotide transporter is the most likely candidate responsible for the elevated proton leak in old cardiomyocytes and that SS-31 acutely and directly interacts with the transporter, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes. The improved mitochondrial function leads to complex secondary changes to effect enhanced diastolic function in the aged heart. These findings provide a novel insight for better understanding of the mechanisms of cardiac aging and establish the novel concept that decreasing the pathological proton leak in the aging heart restores mitochondrial function, ultimately reversing cardiac dysfunction in aging.

Materials and methods

Key resources table.

Reagent type
(species) or resource		 Designation Source or reference Identifiers Additional
information
Strain, strain background (M. musculus; male and female) C57BL/6J National Institute of Aging Charles River colony RRID:IMSR_JAX:000664 See Materials and method
Strain, strain background (Rattus norvegicus) F344 National Institute of Aging F344 rats See Materials and method
Genetic reagent (M. musculus) mt-cpYFP Wang lab,
PMID:25252178 		See Materials and method
Transfected construct (Adenovirus) mt-cpYFP Wang lab,
PMID:25252178 		Adenovirus to transfect and express mt-cpYFP
Antibody anti- ANT1 Abcam Cat# ab102032 1:3000
Antibody anti-UCP2 Cell signaling technology Cat# 89326S 1:2000
Antibody anti-ATP5a Abcam Cat# ab14748 1:3000
Peptide, recombinant protein SS-31 peptide (Elamipretide) Stealth BioTherapeutics
Commercial assay or kit Seahorse XF Cell Mito Stress Test Kit Aligent/Seahorse Bioscience 103015–100
Commercial assay or kit MitoSOX Red ThermoFisher M36008
Chemical compound, drug Bongkrekic acid Cayman Chemical 19079
Chemical compound, drug Carboxyatractyloside Cayman Chemical 21120
Chemical compound, drug Genipin Sigma G4796
Chemical compound, drug Oligomycin A Sigma 75351
Chemical compound, drug carbonyl cyanide-p-trifluoromethoxyphenylhydrazone Sigma C2920
Chemical compound, drug Antimycin A Sigma A8674
Chemical compound, drug Rotenone Sigma R8875
Commercial assay or kit MitoTracker Green ThermoFisher M7514
Commercial assay or kit JC-1 Dye ThermoFisher T3168
Commercial assay or kit Biotin ThermoFisher B20656
Commercial assay or kit Streptavidin Agarose beads ThermoFisher 20349
Commercial assay or kit Pierce Reversible Protein Stain Kit for PVDF Membranes Thermo Scientific 24585
Commercial assay or kit BCA protein assay Thermo Scientific 23225
Commercial assay or kit SuperSignal West Pico PLUS Chemiluminescent Substrate Thermo Scientific 34580
Software, algorithm Graphpad Prism Graphpad RRID:SCR_002798
Software, algorithm AlphaView Software ProteinSimple
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Animals

All the animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington and conform to the NIH guidelines (Guide for the care and use of laboratory animals). The use of mice and rats have been approved by IACUC under animal protocols 2174–23 and 2654–03. Young (4–6-month-old) and aged (24–26-month-old) C57BL/6 mice (Charles River colony) and F344 rats (young, 5-7-month old; aged, 25–30-month-old) were obtained from the National Institute of Aging Rodent Resource. The mt-cpYFP transgenic C57BL/6 mice were housed until reaching the age described.

Isolation of adult mouse and rat cardiomyocytes

Single ventricular myocytes were enzymatically isolated from mouse and rat hearts as described previously (Zhang et al., 2013; Zhang et al., 2017). The rod-shaped cardiomyocytes were collected by allowing cells to settle down and adhere to laminin coated-24 well Seahorse plates for intact cell oxygen consumption test, or to glass coverslips for confocal imaging.

Seahorse assay

The XF24e Extracellular Flux Analyzer (Seahorse Bioscience) was used for measuring oxygen consumption in intact resting cardiomyocyte, with XF assay medium containing 5 mM glucose and 1 mM pyruvate. Oligomycin A (OA, 2.5 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 μM), and antimycin A (AA, 2.5 μM) plus 1 μM rotenone (Rot) were added in three sequential injections, as reported (Zhang et al., 2017).

Confocal imaging

We used a Zeiss 510 (Zeiss, Germany) or Leica SP8 (Leica, Germany) for confocal imaging at room temperature. The cells were placed in modified Tyrode’s solution (in mM: 138 NaCl, 0.5 KCl, 20 HEPES, 1.2 MgSO4, 1.2 KH2PO4, 5 Glucose, 1 CaCl2, pH 7.4). For mitochondrial flashes, mt-cpYFP expressing cells were exposed to alternating excitation at 405 and 488 nm and emission collected at >505 nm. Time-lapse 2D images were collected at a rate of 1 second per frame. For mitochondrial superoxide quantitation, we used the ratio of MitoSOX Red (5 μM, excited at 540 nm with emission collected at >560 nm) to mitoTracker Green (200 nM, excited at 488 nm and emission collected at 505–530 nm). For mitochondrial membrane potential measurement, JC-1 was excited at 488 nm and emission collected at 510–545 nm and 570–650 nm. For photon triggered mPTP opening, the cells were loaded with 120 nM Tetramethylrhodamine methyl ester (TMRM) and line scanned at 1 Hz as described previously (Zorov et al., 2000).

Cell permeabilization and pH stress

Rat cardiomyocytes were cultured with mt-cpYFP adenovirus (Wang et al., 2016b) for 3 days in M199 medium. After incubation in Ca2+-free Tyrode’s solution for 30 min, the medium was changed to a solution of 100 mM potassium aspartate, 20 mM KCl, 10 mM glutathione, 10 mM KH2PO4, 0.1 mM EGTA, 8% dextran 40,000, pH 7.5, with 50 μg/ml saponin for 30 s and then maintained in saponin-free internal solution (Lukyanenko and Gyorke, 1999). The pH of the solution containing the permeabilized cells was then progressively lowered by addition of HCl in quantities previously titrated to result in pH 7.3, 6.9, 5.3, and 4.5, with 8 min between each step. The permeabilized cells were excited using the same settings as for mt-cpYFP above, but using a time-lapse of 6 seconds per frame. The ratio of emission fluorescence at 488 nm from 405 nm excitation indicated the mitochondrial pH change (Wei-LaPierre et al., 2013) and was normalized to a starting (pH 7.5) arbitrary value of 1.0, so as to normalize differences due to variability of the intensity of laser excitation and emission collection between different experiments. Bongkrekic acid (BKA) 10 μM (Adams et al., 2000), CAT 20 μM (Winter et al., 2016), Genipin 50 μM (Wang et al., 2017), OA were added after cell permeabilization.

Western blots

Heart tissue was lysed with RIPA buffer containing a protease inhibitor cocktail (Chiao et al., 2016). Protein samples were denatured and separated via NuPAGE Bis-Tris gel, and transferred to PVDF membranes. The blots were probed with primary antibodies: ANT1 (Abcam, ab102032, 1:3000), UCP2 (Cell Signaling Technology, 89326S, 1:2000) followed by appropriate secondary antibodies.

Biotin-SS-31 pulldown and blot analysis

Hearts were chunked and dounce homogenized in mitochondrial isolation buffer (MIB, in mM: 300 sucrose, 10 Na-HEPES, 0.200 EDTA, pH 7.4) and centrifuged at 800 g for 10 min. The supernatants were centrifuged at 8000 g for 15 min to purify mitochondria. Digitonin was added to the mitochondria at a ratio of Digitonin: protein = 6:1 to break down the membrane system. Treatment drugs were added 30 min before addition of 10 μM biotin-SS-31 (Biotin-D-Arg-dimethyl Tyr-Lys-Phe-NH2) or biotin control (Thermo, B20656). Streptavidin Agarose beads (Thermo, 20349) were added and incubated for 2 hrs at room temperature. The beads were washed with MIB three times and then eluted by 50 μM SS-31. The eluates were boiled with LDS protein loading buffer (Thermo, NP0008) and loaded on NuPAGE for gel electrophoresis and Western blotting with antibody for ANT1 (Abcam, ab102032, 1:3000). In some experiments, after electrophoresis, gels were silver stained using a Pierce Silver Stain Kit (Thermo, #24612).

Native coomassie blue staining and blotting

Mitochondria from mouse hearts were isolated as described previously (Marcu et al., 2012). Mitochondria (100 µg) were solubilized in 4x NativePAGE Sample Buffer containing 5% digitonin and 5% coomassie blue G-250. The samples were loaded on NativePAGE Novex 3–12% Gel and run at 100 V for 1 hr, then at 300 V for 2 hr. For coomassie blue staining, gels were stained with 0.1% Coomassie Brilliant Blue overnight and destained with destaining solution (H2O: Methanol: Acetic Acid = 5:4:1) 5 times at 20 min intervals. For native blotting, gels were transferred to PVDF membranes at 25 V in 4°C overnight and incubated with ATP5a antibody (Abcam, ab14748, 1:3000), followed by anti-mouse secondary antibody.

Perfused mouse heart confocal imaging

mt-cpYFP transgenic mice were anesthetized with pentobarbital (150 mg/kg). The heart was removed, cannulated via the ascending aorta, and put on a modified perfusion system and in a custom made chamber on the confocal stage as previously reported (Zhang et al., 2018; Zhang et al., 2017). The perfusion was maintained under a constant flow (~2 ml/min) with O2/CO2-bubbled KHB solution (in mM: 118 NaCl, 0.5 EDTA, 10 D-glucose, 5.3 KCl, 1.2 MgCl2, 25 NaHCO3, 0.5 pyruvate, and 2 CaCl2, pH 7.4) at 37°C. To minimize motion artifact during imaging, 10 µM (-)-Blebbistatin (Toronto Research Chemicals) was included. During imaging, the left ventricle was gently pressed to further suppress motion artifact. Mitoflashes were imaged using the procedure described above.

Data statistics

Data are shown as mean ± SEM. For the multiple groups comparisons one-way ANOVA was applied followed with appropriate test. Student’s t-test was used to determine the statistical significance between two group comparisons. p<0.05 was considered statistically significant.

Acknowledgements

We thank Drs. Mariya Sweetwyne, Ying Ann Chiao, Martin Brand, Michael MacCoss and Gaomin Feng for technical support and helpful discussions and the services of the WM Keck Microscopy Center at the University of Washington. SS-31 (elamipretide) was kindly provided by Stealth Biotherapeutics (Newton, MA).

This work was supported by NIA P01AG001751 and R56AG055114 to PSR, HL114760, HL137266 and AHA 18EIA33900041 to WW, and a Glenn Foundation for Medical Research Postdoctoral Fellowship and AHA 19CDA34660311 to HZ.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Peter S Rabinovitch, Email: petersr@uw.edu.

Holly van Remmen, Oklahoma Medical Research Foundation, United States.

Jessica K Tyler, Weill Cornell Medicine, United States.

Funding Information

This paper was supported by the following grants:

  • NIA P01AG001751 to Peter S Rabinovitch.

  • NIA R56AG055114 to Peter S Rabinovitch.

  • NHLBI HL114760 to Wang Wang.

  • NHLBI HL137266 to Wang Wang.

  • AHA 18EIA33900041 to Wang Wang.

  • AHA 19CDA34660311 to Huiliang Zhang.

  • Glenn/AFAR Foundation Medical Research Postdoctoral Fellowship to Huiliang Zhang.

Additional information

Competing interests

No competing interests declared.

is the inventor of SS-31 and founder of Stealth Biotherapeutics.

Author contributions

Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft.

Conceptualization, Resources, Methodology, Writing - review and editing.

Resources, Funding acquisition, Writing - review and editing.

Writing - review and editing.

Resources, Writing - review and editing.

Conceptualization, Resources, Funding acquisition, Writing - original draft, Project administration.

Ethics

Animal experimentation: All the animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington and conform to the NIH guidelines (Guide for the care and use of laboratory animals). The use of mice and rats have been approved by IACUC under animal protocols 2174-23 and 2654-03.

Additional files

Transparent reporting form

Data availability

Data available in Dryad: https://doi.org/10.5061/dryad.fqz612jqs.

The following dataset was generated:

Zhang H, Alder NN, Wang W, Hazel SH, Marcinek DJ, Rabinovitch PS. 2020. Reduction of Elevated Proton Leak Rejuvenates Mitochondria in the Aged Cardiomyocyte. Dryad Digital Repository.

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Decision letter

Editor: Holly van Remmen1
Reviewed by: Benjamin Miller2

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Your work is novel and a clear demonstration of the potential role of mitochondrial proton leak in cardiac aging and dysfunction and the protective effect of SS-31.

Decision letter after peer review:

Thank you for submitting your article "Reduction of Elevated Proton Leak Rejuvenates Mitochondria in the Aged Cardiomyocyte" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Benjamin Miller (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

In this manuscript Zhang et al. examine the effects of the compound SS-31, a compound that has shown intervention potential in other systems, on increased mitochondrial proton leak in cardiac myocytes from aged rats and mice. The data are important contribution to the mechanism of action of SS-31, which shows great promise for cardiac aging and the interaction of SS-31 with ANT1. ANT1 is the major isoform of ANT expressed in the heart, and it has been shown previously to contribute to proton leak in cardiac mitochondria. The authors also analyze the effects of SS-31 on mito-flash and mitochondrial permeability transition pore opening. From the work presented here the authors conclude that proton leak is an important underlying mechanism of cardiac dysfunction in aging and provide evidence for the effect of SS-31 in modulating this effect.

While the work was deemed interesting, however the manuscript should be improved by more clarity in the presentation of the data and their interpretation in general. There are several significant concerns that need to be addressed:

Essential revisions:

1) This work does not appear to be a major advance beyond the author's recent publication in eLife showing that "SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts." They also demonstrated improved diastolic function. (Chiao et al., 2020).

2) Authors cannot claim that they have "identified ANT1 as mediating the increased proton permeability of old cardiomyocytes". ANT2 is also expressed, albeit at lower levels than ANT1, in the heart, and is also inhibited by CATR and BKA (e.g., work from Doug Wallace's lab: PMID: 10974536) – and thus could also contribute to the effects of SS-31. Authors state that Bertholet et al., 2019, found that ANT1 was the major site of proton leak; while this is true, they also found that ANT2 can contribute to leak, especially FA- induced leak. Thus authors cannot claim, as they have in the their summary statement that their "study reveals that ANT1 is responsible for the elevated proton leak in old cardiomyocytes and that SS-31 directly interacts with ANT1, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes." Indeed, data in Figure 1 show that SS-31 inhibition of proton leak is significantly less than the inhibition caused by BKA; BKA also inhibits ANT2.

3) Moreover there are other mechanisms contributing to proton leak that could be relevant here: NNT, UCP3 and the inorganic phosphate carrier. See for example the recent work on NNT and its contributions to leak and ROS (Smith et al. JBC 2020; PMID 32747443). Why have the authors not shown blots of ANT2 and other possible proteins involved? Regarding UCP2 blots, authors should show a positive control (e.g., a recombinant protein) in the western blots to convince readers that this band really is UCP2. The antibodies for UCP2 are very poor, and UCP2 protein expression is extremely low, if detectable at all in the heart.

4) We felt that the authors over-interpreted their mt-cp-YFP results. Authors conducted these analyses in the absence of mitochondrial activity, and permeabilized the cardiomyocytes in a buffer that contained no substrates, ATP, or ADP. If authors had found effects under physiological conditions, this would be meaningful. Moreover, the control of the pH of the mitochondrial matrix is not via mitochondrial proton leak; there are many other ion transport mechanisms in the mitochondrial inner membrane and there are many mechanisms in the matrix itself. Authors need to conduct analyses under physiological conditions in the presence of energy substrates.

5) Authors describe how oligomycin failed to inhibit proton leak, but it is well known that it does not inhibit proton leak. Authors need to revise this section.

6) Authors use the well-known inhibitors of ANT, ie. CAT and BKA in non-permeabilized cells, and it is generally thought that they are not cell permeable (PMID: 26950698). Authors need to demonstrate that the inhibitors are traversing the cell membrane.

7) Seahorse data for the single ventricular myocytes of rats and mice are expressed in units per 800 cells. How did authors assess the number of cells that actually adhered and thus that were actually measured in these assays?

8) Why is MitoSOX normalized to mitochondrial content? Are there differences in mitochondrial content in the different experimental groups (old vs young)?

9) Models are important for interpretation. The initial experiment specifies primary cardiac myocytes whereas subsequent studies indicate cardiac myocytes. I think it is important to know for sure what model was used.

10) There are multiple statements of "rejuvenation" "restoring" "more youthful" etc. In a strict sense, the data compare young to old and to old+SS-31. These groups are similar or different. It is not possible to say that the a given group of cells changed with age and with treatment were made like their young self again as implied by the use of these words.

11) How do the authors justify using t-tests? Within each experiment were multiple comparisons so t-tests do not seem justified.

12) The presentation of the figures bounces around quite a bit and are not in sequence. This makes the study hard to follow. In addition, the figures lack detail such as what the abbreviations are and what the error bars are. Although these seem like minor quibbles, when added up, it made it difficult to assess the data.

13) Why was normalization used in many of the assays? Was there a lot of variability at baseline? And if so, why?

14) What was the justification for carrying out experiments in Figure 2 at pH 5.3? This pH does not seem relevant for general mitochondrial function. Do you see the same results at a pH close to actual mitochondria pH?

15) The Discussion made some conclusions that were maybe a bit too much speculation. For example, the proton leak is identified as pathological, although this was not directly demonstrated in the study. Also, protein conformation was inferred as well.

16) It should be emphasized that the proton leak studied is in myocytes in the resting, non-working state oxidizing endogenous substrates. The lack of a resting defect in OCR in this state is therefore plausible compared to the extensive literature describing key defects in OXPHOS with aging.

17) The myocyte yield of the isolation procedure should be discussed. Are only the "best" aged myocytes obtained?

18) The pH dependence of 405 excitation is of some concern. Also, calibration curves should be shown for pH 5.3 and 6.9; which are critical to the work.

19) The proton leak shown is of interest since state 4 rates in isolated cardiac mitochondria have minimal change with age. This should be discussed and reconciled.

20) The similarity of BKA and CAT to block proton leak is somewhat puzzling since the response of MPTP to the inhibitors is of course different. Is there evidence of MPTP opening in the permeabilized cells used for the measurement of mitochondrial pH with the buffer used as the artificial "cytosol"?

21) The differences in Figure 2A are modest; the "normalized" data in Figure 2B and Figure 3C are more reasonable. How were fluorescence data normalized?

22) The dynamic range and reproducibility of the cp-YFP assay should be described in greater detail. It seems puzzling that some groups apparently required n=19 for valid results.

23) Is there a quality control assessment of mitochondrial inner membrane integrity in the permeabilized cells at low pH? This is critical to the conclusion of the study.

eLife. 2020 Dec 15;9:e60827. doi: 10.7554/eLife.60827.sa2

Author response

Essential revisions:

1) This work does not appear to be a major advance beyond the author's recent publication in eLife showing that "SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts." They also demonstrated improved diastolic function. (Chiao et al., 2020).

We wish to respectfully disagree with this interpretation, as the difference between a functional observation (prior work) and elucidation of mechanism (present work) is large. In the recent publication in eLife we showed that chronic SS-31 8-week osmotic minipump treatment decreased the proton leak in aged mice and improved diastolic function. However, the mechanism for this effect was unclear; in fact, the long-term in vivo treatment could even have been produced by indirect compensatory changes, including proteomic or metabolic remodeling, as opposed to a possible direct effect on the cardiomyocyte. Moreover, the underlying mechanism of the excessive proton leak remained elusive. The present study showed that SS-31 directly and acutely decrease excessive proton leak; by directly evaluating the physical properties of the inner membrane in the absence of any active metabolic process we were able to determine the mechanism of the increased proton leak and how SS-31 prevents it in the aged mitochondria. We specified the novelties in the revision as “SS-31 acutely prevents the excessive mitochondrial proton entry”; “In this report we have shown direct evidence of increased proton leak in the aged mitochondria as a primary energetic disturbance and evidence that the increased proton entry in old cardiomyocytes takes place likely through ANT1. Moreover, we demonstrated that SS-31 prevents the proton entry to the mitochondrial matrix and rejuvenates mitochondrial function through direct interaction with the adenine nucleotide transporter and stabilization of the ATP synthasome.”; and “our study reveals that the adenine nucleotide transporter is the most likely candidate responsible for the elevated proton leak in old cardiomyocytes and that SS-31 acutely and directly interacts with the transporter, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes”.

2) Authors cannot claim that they have "identified ANT1 as mediating the increased proton permeability of old cardiomyocytes". ANT2 is also expressed, albeit at lower levels than ANT1, in the heart, and is also inhibited by CATR and BKA (e.g., work from Doug Wallace's lab: PMID: 10974536) – and thus could also contribute to the effects of SS-31. Authors state that Bertholet et al., 2019, found that ANT1 was the major site of proton leak; while this is true, they also found that ANT2 can contribute to leak, especially FA- induced leak. Thus authors cannot claim, as they have in the their summary statement that their "study reveals that ANT1 is responsible for the elevated proton leak in old cardiomyocytes and that SS-31 directly interacts with ANT1, preventing the proton leak and rejuvenating mitochondrial function in the aged cardiomyocytes." Indeed, data in Figure 1 show that SS-31 inhibition of proton leak is significantly less than the inhibition caused by BKA; BKA also inhibits ANT2.

Together with points raised in item #3 below, we agree that the possibilities of other sites as the proton leak contributor, including ANT2, should be considered. However, of these, the crosslinking interactome data recently published by our collaborators identified only ANT1 as interacting with SS-31 (PMID: 32554501). Thus, we are more confident to say ANT1 is involved in the proton leak in the aged mitochondria. As we agree that we cannot with certainty rule out the others sites of proton entry, we have revised the Abstract “our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes” and the Discussion to state that “our study reveals that the adenine nucleotide transporter is the most likely candidate responsible for the elevated proton leak in old cardiomyocytes”.

3) Moreover there are other mechanisms contributing to proton leak that could be relevant here: NNT, UCP3 and the inorganic phosphate carrier. See for example the recent work on NNT and its contributions to leak and ROS (Smith et al. JBC 2020; PMID 32747443). Why have the authors not shown blots of ANT2 and other possible proteins involved? Regarding UCP2 blots, authors should show a positive control (e.g., a recombinant protein) in the western blots to convince readers that this band really is UCP2. The antibodies for UCP2 are very poor, and UCP2 protein expression is extremely low, if detectable at all in the heart.

Specific to inorganic phosphate carrier, we checked the potential involvement of the mitochondrial phosphate carrier (PiC) in proton entry by applying the pH stress to mitochondria in a buffer without phosphate. The proton entry characteristics of mitochondria from old cells was indistinguishable from that of mitochondria in buffer containing phosphate. This data is added as Figure 3—figure supplement 2, with the statement in the text “Considering that the inorganic phosphate carrier might be a potential source of the elevated proton entry in old mitochondria, we examined the proton permeability of old mitochondria to pH stress in a buffer that did not contain phosphate; in this buffer, the proton entry characteristics of mitochondria from old cells was indistinguishable from that of mitochondria stressed in the presence of inorganic phosphate (Figure 3—figure supplement 2).”

With respect to UCP2, we confirmed the UCP2 WB in the heart samples with a UCP2 standard. The band we detected was confirmed as UCP2. This blot confirms the results now included as Figure 3—figure supplement 1E and referred to in the text. Moreover, our data is consistent with previous work from Sreekumaran Nair lab demonstrated that the lack of age-related change in UCP2 transcript levels (PMID: 11171595, see Figure 3 from that paper).

4) We felt that the authors over-interpreted their mt-cp-YFP results. Authors conducted these analyses in the absence of mitochondrial activity, and permeabilized the cardiomyocytes in a buffer that contained no substrates, ATP, or ADP. If authors had found effects under physiological conditions, this would be meaningful. Moreover, the control of the pH of the mitochondrial matrix is not via mitochondrial proton leak; there are many other ion transport mechanisms in the mitochondrial inner membrane and there are many mechanisms in the matrix itself. Authors need to conduct analyses under physiological conditions in the presence of energy substrates.

We strongly believe that performing the pH stress experiments in the absence of energy substrates is the clearest and most informative protocol. In the presence of energy substrates, the mitochondria will actively pump protons out of the matrix, making it more difficult and complicated to evaluate proton entry. Thus, the buffer without substrates allows us to evaluate the fundamental process of proton entry to the mitochondrial matrix. In this fashion, looking at the physical properties of the inner membrane in the absence of metabolic activity is highly informative and reduces many possible complications. Also, we do not claim that proton leak is the primary method of regulating pH, only that the age-related excessive leak is via ANT and is prevented by SS-31, and that this is correlated with improved behavior of MPTP opening, membrane potential and mitochondrial flashes in aged cardiomyocytes.

5) Authors describe how oligomycin failed to inhibit proton leak, but it is well known that it does not inhibit proton leak. Authors need to revise this section.

Thank you. We changed the description to “As expected, the ATPase inhibitor Oligomycin A failed to inhibit the proton leak”

6) Authors use the well-known inhibitors of ANT, ie. CAT and BKA in non-permeabilized cells, and it is generally thought that they are not cell permeable (PMID: 26950698). Authors need to demonstrate that the inhibitors are traversing the cell membrane.

We believe that this question is based on a misunderstanding – in the assay used in this report the plasma membrane is permeabilized by saponin. However, it may be of interest to point out that in cardiomyocytes both BKA (PMID: 19452617; PMID: 11110776; PMID: 26746144) and CAT (PMID: 26746144; PMID: 26548633) have been shown to be effective in non-permeabilized cells. This is indeed in contrast to the report cited by the reviewer (PMID: 26950698) which found that BKA and CAT had a milder effect on intact T98G cells than permeabilized cells.

7) Seahorse data for the single ventricular myocytes of rats and mice are expressed in units per 800 cells. How did authors assess the number of cells that actually adhered and thus that were actually measured in these assays?

For the cell number determination, we counted the cell numbers using hemocytometer after cell isolation. Only the rod shaped cells were counted as live cells. Usually the starting cell concentration was ~4*104/ml. Then the cells were diluted to 8000 cell/ml and 100ul (containing ~800 cells) were plated in each Seahorse well. We had previously coated the plate wells with laminin (50ug/ml) to allow the cells to adhere to the bottom of the plate. The cells were allowed to attach to the plate for at least 2 hours before changed the culture medium to Seahorse Assay medium without disturbing the cells. In initial experiments Seahorse wells were examined by microscopy to confirm plated numbers and equality between young and old cardiomyocytes. As expected, no differences were seen between young and old cells. This time-consuming secondary confirmation was not continued in later experiments.

8) Why is MitoSOX normalized to mitochondrial content? Are there differences in mitochondrial content in the different experimental groups (old vs young)?

The reason that the ratio of MitoSOX to MitotrackerGreen is analyzed is to reduce the cell to cell variability in analysis, which is primarily due to differences in cell size and dye uptake between cells (cell variation in dye transport being largely the same for the two indicators allows the MitoTracker Green intensity to be used to normalize the changes seen in MitoSOX staining to make them independent of cell size and dye uptake). This ratiometric approach is exceedingly common in flow and image cytometry. There were no significant differences seen in average Mitotracker Green intensity between old vs young cells.

9) Models are important for interpretation. The initial experiment specifies primary cardiac myocytes whereas subsequent studies indicate cardiac myocytes. I think it is important to know for sure what model was used.

We appreciate this reminder. All experiments were performed with primary cardiac myocytes. For pH stress and mitochondrial flash experiments, rat cardiomyocytes were used; mouse heart and cardiomyocytes were used in all other experiments. We have revised the language used in the manuscript to clearly note this accordingly.

10) There are multiple statements of "rejuvenation" "restoring" "more youthful" etc. In a strict sense, the data compare young to old and to old+SS-31. These groups are similar or different. It is not possible to say that the a given group of cells changed with age and with treatment were made like their young self again as implied by the use of these words.

We are grateful for bringing this to our attention. In the revised manuscript we are careful to state only that the phenotypes of the treated cells are closer to that of young cells.

11) How do the authors justify using t-tests? Within each experiment were multiple comparisons so t-tests do not seem justified.

Thank you for bringing this to our attention. We did the t-tests as analysis of differences between the young vs. old and between old vs. a single treatment. In this revision, we applied one-way ANOVA analysis to all multiple group comparisons then followed this with student t-tests to look at pairwise comparisons, allowing readers to see both results. We updated the statistical analysis in the Materials and methods part in this revision.

12) The presentation of the figures bounces around quite a bit and are not in sequence. This makes the study hard to follow. In addition, the figures lack detail such as what the abbreviations are and what the error bars are. Although these seem like minor quibbles, when added up, it made it difficult to assess the data.

Thank you for this observation, which we have now corrected. The Figure 3C which showed the method of 488/405 ratio slope calculation was previously placed after the slope statistical analysis of in Figure 2D, which was hard to follow. We moved Figure 3C to become new Figure 2—figure supplement 3 and mentioned this before the statement of Figure 2D to make it easier to follow. Also, we added the full spellings for the abbreviations. All the error bars are standard errors of the mean and this information is added to the figure legends.

13) Why was normalization used in many of the assays? Was there a lot of variability at baseline? And if so, why?

Thank you for this question. The reasons for each normalization are as following:

For the pH stress assay, we normalized the cpYFP 488/405 ratio to the starting value. This is required due to inter-experimental variation in relative intensities of the two wavelengths due to variability in confocal laser powers, detector settings and efficiency.

For the superoxide evaluation, we normalized the mitoSOX signal to the MitotrackerGreen signal. The advantage of this normalization is described in response to query number 8, above.

For the biotin-SS31 pulldown ANT1 blot data, we normalized bands to the Biotin-SS31 group in each experiment. This eliminated the variation due to differences in sample loading amounts and blotting procedures.

For the ANT1 and UCP2 Western Blot data, we normalized the blotting bands to the total protein content in that lane, as detected by reversible protein stain. This eliminated variation from differences in sample loading amounts.

14) What was the justification for carrying out experiments in Figure 2 at pH 5.3? This pH does not seem relevant for general mitochondrial function. Do you see the same results at a pH close to actual mitochondria pH?

We appreciate this question. The mitochondrial intermembrane pH is around 6.9. While we did see a similar phenotype for proton entry at pH 6.9 (we have added this data as Figure 2—figure supplement 2 in this revised version and have noted this in the text), differences between proton permeability of young vs old inner membranes and between SS-31 treated and untreated were larger and less variable at pH 5.3. We believe that the observations at pH 5.3 are more clearly indicative of physical chemical differences in inner membrane permeability, in spite of this being a non-physiologic pH.

15) The Discussion made some conclusions that were maybe a bit too much speculation. For example, the proton leak is identified as pathological, although this was not directly demonstrated in the study. Also, protein conformation was inferred as well.

We thank the reviewer for this comment and have revised the language used. We have restricted the use of the term pathological to exclusively refer to previous observations of diastolic dysfunction of the aged hearts, which is not a healthy condition.

The conformational states in the ANT pore conferred by BKA and CAT treatment are based on prior work by others: PMID: 30611538 and PMID: 14603310, which are generally well accepted. We have changed the wording to make this reference clearer.

16) It should be emphasized that the proton leak studied is in myocytes in the resting, non-working state oxidizing endogenous substrates. The lack of a resting defect in OCR in this state is therefore plausible compared to the extensive literature describing key defects in OXPHOS with aging.

Thank you for this constructive suggestion. We emphasized the resting and non-working myocyte states in both the Materials and methods and Results sections.

17) The myocyte yield of the isolation procedure should be discussed. Are only the "best" aged myocytes obtained?

Each experiment, only preparations having at least 80% rod shape cells (which means live cardiomyocytes) were used. Thus, while we cannot completely exclude the possibility that there was some selection present, this would have to have been a small effect that could not have appreciably affected the reported results. For the aged myocytes, we did observe that the cells were on average larger, compared with the young cells, as has been previously documented by imaging of heart tissue sections by us and others.

18) The pH dependence of 405 excitation is of some concern. Also, calibration curves should be shown for pH 5.3 and 6.9; which are critical to the work.

Thank you for this suggestion. We added the pH calibration results of both 488 and 405 excitations in Figure 2—figure supplement 1. Also, we replaced the previous pH 4.5 and 6.0 panels with the calibration results at pH 5.3 and 6.9 in Figure 2—figure supplement 1.

19) The proton leak shown is of interest since state 4 rates in isolated cardiac mitochondria have minimal change with age. This should be discussed and reconciled.

An accurate State IV is difficult to achieve since there may be contaminating ATPases in the crude mitochondrial preparation which convert newly formed ATP back to ADP, and thus prevent a true “ADP exhausted” state. Thus, most investigators use oxygen consumption in the presence of oligomycin as a surrogate of state IV. This is the parameter that we have previously shown to be elevated in aged cardiomyocytes and reverse by SS-31 (PMID: 32648542).

20) The similarity of BKA and CAT to block proton leak is somewhat puzzling since the response of MPTP to the inhibitors is of course different. Is there evidence of MPTP opening in the permeabilized cells used for the measurement of mitochondrial pH with the buffer used as the artificial "cytosol"?

The discrepancy of BKA and CAT effect on proton leak and mPTP opening is reasonable, as ANT is only a regulator of the mPTP, while the ATPase itself, especially the "c" ring is thought by many to be the pore of the mPTP. Thus, while we hypothesize that both BKA and CAT change the conformation of the ANT pore in such a way as to inhibit proton entry, only one of these conformational states regulates the mPTP to slow its opening. In fact, this difference is a strong argument against the mPTP pore itself being the source of excessive proton leak in old cardiomyocytes, as CAT, which has no delay on mPTP opening times (Figure 5B), would not be expected to alter proton permeability, which it does.

21) The differences in Figure 2A are modest; the "normalized" data in Figure 2B and Figure 3C are more reasonable. How were fluorescence data normalized?

The differences seen by viewers in the fluorescence image are dependent on the observer’s display settings (for example γ) and may vary among viewers. On our displays and printed copies, the variation in intensities appear consistent with the quantified data, but the reviewer’s comment is an important reason for not relying on the visual image. To quantify the fluorescence intensity we first subtract the signal intensities in the background that does not contain any cell (this is generally low). Then, for each image area that contains a cell (ascertained by threshold settings and standard image segmentation algorithms) the integrated fluorescence value at the 488nm detection channel is divided by the corresponding value at from the 405nm detection channel. We set the ratio of 488/405 at the beginning as one (see query 13 above) and all later values are normalized to this starting value.

22) The dynamic range and reproducibility of the cp-YFP assay should be described in greater detail. It seems puzzling that some groups apparently required n=19 for valid results.

Thank you for the suggestion. We put the cp-YFP assay spectral characteristics at pH 5.3 in the Figure 2—figure supplement 1. The reproducibility of the data comparing old vs. young cells and old SS-31 treated cells at pH 5.3 for example (Figure 2C), yields a mean of 0.59, a SD of ± 0.10 and a range of 0.46 to 0.77 for the old compared to the starting pH 7.5 value. This range is non-overlapping with the mean of either young or old-SS-31 treated cells at pH 5.3. This is added to the Figure 2C legend to give a better understanding of the range and reproducibility of the assay.

The number n=19 arises because we used the results of multiple experiments to validate and confirm the data. It does not require a sample size of 19 to reach a significant difference in the statistical test.

23) Is there a quality control assessment of mitochondrial inner membrane integrity in the permeabilized cells at low pH? This is critical to the conclusion of the study.

As the mt-cpYFP indicator (269 aa, ~28kd) is still held within the mitochondrial matrix, this indicates that at the very least, the inner membrane does not break down enough to let this size molecule escape. Furthermore, and more to the point, while the reviewer is correct in thinking that a generalized breakdown of inner membrane integrity would prevent an informative answer as to the source of proton permeability, the fact that SS-31, BKA and CAT prevent excess proton permeability is a very strong indication that there is not a general loss of inner membrane integrity.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Zhang H, Alder NN, Wang W, Hazel SH, Marcinek DJ, Rabinovitch PS. 2020. Reduction of Elevated Proton Leak Rejuvenates Mitochondria in the Aged Cardiomyocyte. Dryad Digital Repository. [DOI] [PMC free article] [PubMed]

    Supplementary Materials

    Transparent reporting form
    elife-60827-transrepform.docx (252KB, docx)

    Data Availability Statement

    Data available in Dryad: https://doi.org/10.5061/dryad.fqz612jqs.

    The following dataset was generated:

    Zhang H, Alder NN, Wang W, Hazel SH, Marcinek DJ, Rabinovitch PS. 2020. Reduction of Elevated Proton Leak Rejuvenates Mitochondria in the Aged Cardiomyocyte. Dryad Digital Repository.

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

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