Ro5‐4864, a ligand of the mitochondrial translocator protein, protects against heart failure in mice via regulation of the p62‐Keap1‐Nrf2 axis

Heart failure

Heart failure (HF) is typically characterized by a reduction in systolic function (fractional shortening or ejection fraction), ventricular dilation, cardiomyocyte loss and fibrosis (Segura et al., 2014). On a cellular level, HF is characterized by alterations in myocyte metabolism, calcium handling, oxidative stress and inflammation (Tsutsui et al., 2011). Understanding this complex cellular response in HF may lead to targeted therapeutics which can prevent or reverse pathological remodelling in the failing heart.

TSPO

The 18‐kDa mitochondrial translocator protein (TSPO) is a five‐transmembrane domain protein expressed in many tissues including the heart (Morin et al., 2016; Thai et al., 2018). Located on the outer mitochondrial membrane, TSPO regulates critical mitochondrial functions, such as respiration, the mitochondrial membrane potential and calcium homeostasis often through interactions with the voltage‐dependent anion channel (VDAC) (Gatliff et al., 2014). Several biologic processes such as apoptosis, cell proliferation and cholesterol transport are also regulated in part by TSPO (Campanella & Turkheimer, 2015). We have previously shown that cardiac‐specific conditional knockout of TSPO largely prevented HF development induced by transverse aortic constriction (TAC) (Thai et al., 2018). Mechanistically, we demonstrated that cardiac‐specific ablation of TSPO preserved mitochondrial function by restoring mitochondrial calcium uptake and ATP production, presumably through TSPO's interaction with VDAC (Thai et al., 2018). In addition, TSPO ablation reduced oxidative stress and restored cellular redox balance via normalization of mitophagy (Thai et al., 2018). Specifically, we demonstrated that increase in TSPO expression in TAC‐induced HF was associated with increased accumulation of Parkin‐SQSTM1/p62 aggregates in mitochondria. This effect was disconnected from microtubule‐associated protein 1 light chain 3 (LC3)‐mediated autophagosome formation, and recycling of dysfunctional mitochondria via mitophagy/autophagy. Thus, cardiac TSPO knockout restored halted mitophagy and recycling of damaged mitochondria, leading to improvement in mitochondrial function.

Oxidative stress

Oxidative stress is characterized by excess reactive oxygen species (ROS) production relative to the cell's antioxidant defences (Tsutsui et al., 2011). ROS are unstable free radical molecules that readily react with other molecules in a cell, causing cellular dysfunction, protein and lipid peroxidation and DNA damage (Munzel et al., 2017). This irreversible damage contributes to various cardiovascular conditions, including heart failure (Munzel et al., 2017). In cardiomyocytes excess ROS activates mitogen‐activated protein kinase (MAPK) and transforming growth factor (TGF)‐β pathways, promoting myocardial hypertrophy, apoptosis and necrosis (Munzel et al., 2017). Additionally ROS‐induced alterations in calcium handling exacerbate arrhythmogenesis and cardiac remodelling (De Nicolo et al., 2023; Dedkova et al., 2013). Therefore ameliorating elevated ROS and its pro‐inflammatory downstream effects may limit HF progression. Supporting this we have previously demonstrated that the inhibition of TSPO with another TSPO ligand, PK11195, protected cardiomyocytes against oxidative stress and cell death during ischemia‐reperfusion injury (Seidlmayer et al., 2021).

A critical endogenous mechanism to counteract ROS and limit the inflammatory response is via activation of the nuclear factor Erythroid 2‐related transcription factor (Nrf2) (Ngo & Duennwald, 2022; Salinas et al., 2025). Under basal conditions Nrf2 is ubiquitinated by the Keap1‐Cul3‐E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation (Villeneuve et al., 2010). Upon increased oxidative stress the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in the Kelch‐like ECH‐associated protein 1 (Keap1), resulting in the activation of Nrf2 and its translocation to the nucleus where it activates the expression of genes such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase‐1 (HO‐1), superoxide dismutase 2 (SOD2) and others which play a vital role in cellular protection.

Decreasing the activity of Nrf2‐inhibiting proteins such as Keap1 via competitive binding of p62 was shown to provide an anti‐inflammatory effect by activating Nrf2, resulting in the expression of several proteins, including the glutathione system, that detoxify ROS and inhibit pro‐inflammatory cascades (Mo et al., 2025; Salinas et al., 2025; Zhang et al., 2024). Indeed it was shown that p62/SQSTM1 upregulates NQO1 transcription level by Keap1 recycling via autophagy (Liao et al., 2019). As our prior study (Thai et al., 2018) revealed that elevated TSPO expression in HF was associated with impaired mitophagy and p62/SQSTM1 function, we hypothesized that decreasing TSPO activity with the selective ligand such as Ro5‐4864 (Ro5) could remove the inhibitory effect of Keap1, thereby stabilizing or activating the Nrf2 signalling cascade with resultant cardioprotection.

Ro5‐4864 limits TSPO‐mediated inflammation

Administration of Ro5‐4864 (Ro5), a selective ligand of TSPO, has been demonstrated to suppress inflammatory signalling in mast cells, including reductions in pro‐inflammatory cytokines (Lee et al., 2016). We hypothesized that Ro5 could similarly limit inflammation and oxidative stress in the failing heart, such as in HF. Given that inflammation and mitochondrial dysfunction play central roles in HF pathology, Ro5 intervention may attenuate downstream effects of myocardial inflammation, including fibrosis and pyroptotic cell death, ultimately preserving cardiac function.

Based on these observations we postulated that the administration of Ro5 in a murine model of TAC‐induced HF would recapitulate the cardioprotective effects we previously demonstrated in TSPO knockout mice (Thai et al., 2018). We further hypothesized that this benefit would be driven by p62‐mediated Keap‐1 recycling and subsequent Nrf2 activation leading to decreased oxidative stress and preservation of cardiac function after 8 weeks of aortic constriction. To define mechanisms we administered Ro5 daily for 8 weeks post‐TAC and performed echocardiography. Heart tissue lysates from mice with and without TAC, treated with saline or Ro5, were collected for proteomic analysis of tissue, liquid chromatography‐mass spectrometry (LC/MS) and western blotting. Mechanistic insight was complemented by in vitro cardiomyocyte experiments evaluating ROS modulation by Ro5 under oxidative stress conditions.

Ethical approval

These experiments were conducted with the approval of the University of California Davis IACUC protocol # 21 242. All procedures followed standards expressed in the Guide for the Care and Use of Laboratory Animals, 8th edition. Animals were fed ad libitum. For the surgical procedure (TAC or sham), animals were anesthetized using intraperitoneal ketamine 50–100 mg/kg and xylazine 5–10 mg/kg for intubation. Isoflurane was used to maintain a surgical plane of anaesthesia determined by the absence of rear foot reflexes. Post‐operative analgesia was provided using buprenorphine i.p. at the end of surgery and twice daily for 48–72 h post‐operatively. After 8 weeks animals were killed by removal of the heart after the administration of ketamine 100 mg/kg i.p. and xylazine 5 mg/kg i.p.

Model

C57BL/6J mice were obtained from the Jackson Laboratory (Sacramento, CA, USA) and randomized to undergo TAC or sham surgery. For TAC, the aorta was surgically constricted as previously described (deAlmeida et al., 2010; Thai et al., 2018). A 5‐mm thoracotomy was made, the transverse aorta was isolated and a suture was looped around a blunt 27‐gauge needle, yielding a transverse aortic diameter of approximately 0.4 mm. The sham procedure was identical except that the aorta was not ligated.

Administration of Ro5‐4864

Ro5‐4864 (Ro5) (Sigma Aldrich, St. Louis, MO, USA) was administered by ip at a dose of 0.1 mg/kg daily diluted in phosphate‐buffered saline starting at day 1 after surgery. Four groups of animals were studied, including sham and TAC mice either administered Ro5 in the concentration above or saline. These groups were denoted as Sham‐Saline, Sham‐Ro5, TAC‐Saline and TAC‐Ro5.

Echocardiography

Echocardiography was performed on day 1 of injection and day 56 using a VisualSonics Vevo 2100 echocardiography machine (FujiFilm VisualSonics, Bothell, WA, USA) and an MS 550D probe (22–55 MHz). Systolic function was measured in conscious animals by obtaining 2‐dimensional cross‐sectional images at the level of the papillary muscles and analysed as previously described (Salinas et al., 2025, 2024; Thai et al., 2018, 2021). These images were used to calculate cardiac structure and function.

Western blotting

After 8 weeks of treatment, whole hearts were removed, homogenized (Tissue Tearor, Biospec Products, Bartlesville, OK, USA) and lysed in Pierce RIPA Lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was collected after centrifugation at 13 000 rpm for 20 min at 4°C. The lysis protein concentration was quantified via bicinchoninic acid (BCA) assay, and samples were prepared for western blot analyses. Samples were loaded into Protein Gels (Bio‐Rad Laboratories, Hercules, CA, USA) and run at 85 volts for 1.5 h. The gels were transferred using a Bio‐Rad's Trans‐Blot Turbo Transfer System. The resulting membrane was blocked in 3% milk for 1 h. All antibodies were diluted in 3% milk. For analysing of Nrf2 signalling, we used anti‐p62 (Abcam, Cambridge, United Kingdom), anti‐Nrf2 (Abcam) and anti‐Keap1 (Sigma‐Aldrich) antibodies all at a 1:1000 dilution. To assess fibrosis we used anti‐periostin (Abcam) antibody at a 1:1000 dilution. Secondary HRP antibodies (Abcam) were diluted 1:2000. Housekeeping antibody anti‐GAPDH (MilliporeSigma, Burlington, MA, USA) and its corresponding HRP secondary (Abcam) were prepared at a 1:10 000 dilution. For NQO1 detection we used primary antibodies from Abcam (1:1000) and α‐tubulin from Cell Signalling at a 1:5000 dilution. NQO1 signal was normalized to the intensity of α‐tubulin (used instead of GAPDH as the MW of NQO1 and GAPDH are close). Primary antibodies were incubated overnight at 4°C, whereas secondary HRP antibodies were incubated for 1 h at room temperature. Western blot membranes were imaged with ImageQuant LAS 4000 (Fujifilm, College Station, TX, USA).

Proteomics (LC/MS)

Left ventricular tissue pieces of 0.3 cm diameter were cut from each heart, placed into 1 mL of solubilization buffer of 5% SDS, 50 mm triethyl ammonium bicarbonate, complete protease inhibitor cocktail (Roche, Basel, Switzerland), pH 7.5, all inside MagnaLyser tubes that contain ceramic beads (Roche). They were homogenized using a MagnaLyser instrument (Roche) to achieve full tissue mechanical disruption. Homogenates were subjected to proteolysis by using suspension‐trap (ProtiFi) devices. All operations of reduction, alkylation, tryptic digestion and peptide clean‐up were carried out within these devices. The eluted tryptic peptides were dried in a vacuum centrifuge and re‐constituted in 0.1% trifluoroacetic acid. LC/MS: peptides were injected onto an LC/MS system. They were first trapped on a Thermo PepMap trap for desalting, sent for separation onto a PepSep analytical column with dimensions: 100 µm × 25 cm chromatography done with a Dionex Ultimate 3000 nUPLC at 600 nL/min. The peptides were directly eluted over 90 min into a Tribrid Lumos mass spectrometer (Thermo Fisher) using a gradient mix of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The samples were run in DIA mode; mass spectra were acquired using a collision energy of 35, M1 resolution of 120 K and MS2 30k, using overlapped isolation windows of 47 Da in the m/z range 360–1080 m/z, 3 s cycle time.

Protein data analysis

Data‐independent acquisition (DIA) proteomics were analyzed using Spectronaut 15 (Biognosys, Schlieren, Switzerland) using the direct DIA workflow with the default settings. Briefly, trypsin was set as the digestion enzyme, allowing two missed cleavages. Fixed modification was set for carbamidomethyl, and variable modifications were set to protein N‐term acetylation and methionine oxidation. A minimum of two peptides per protein group were required for quantification. Differential expression was calculated in Spectronaut 15 using its default settings using combined MS1 precursor and MS2 transition data according to Huang et al. (Huang et al., 2020). Quantitation was normalized to the local total ion chromatogram. Proteins identified with fewer than one unique peptide were excluded from the assay. Statistical comparison of relative protein changes was performed with paired t‐tests. The significance level was P‐value less than 0.05%, and log2 ratio more than 0.58. Both DIA analysis results were filtered within Spectronaut by a 1% FDR on peptide and protein level using a target‐decoy approach, which corresponds to a Q value <0.01. The list of quantified proteins was uploaded to SimpliFi (ProtiFi, LLC), an online platform for statistical analysis and visualization of omics data. SimpliFi uses unbiased, nonparametric statistical methods and runs on GPUs for proteomics quantitative data processing. The database used was Mus muscularis FASTA, downloaded from Uniprot, UP000000589. The heatmap was generated using the Heatmapper web server (RRID:SCR_01 6974).

In vitro oxidative stress assay

To isolate cardiomyocytes the mouse heart was excised, and the aorta was cannulated. The heart was retrogradely perfused using a Langendorff perfusion system and digested with collagenase (Judd et al., 2016; Thai et al., 2018, 2021). After further digestion via mechanical dissociation, the isolated cardiomyocytes were purified and plated onto laminin‐coated 24‐well plates (Judd et al., 2016; Kwon et al., 2003). To mimic the in vivo experimental conditions, cardiomyocytes were treated with either 2.5 µg Ro5 per well, 25 µM H₂O₂ or both for 2.5 h to induce oxidative stress (Kwon et al., 2003). To determine mitochondrial superoxide levels mean fluorescence intensity in live cells was quantified using 500 nm MitoSOX Mitochondrial Superoxide Indicator (Thermo Fisher) (Dedkova & Blatter, 2012; Dedkova et al., 2013). To validate that the H₂O₂ exposure sufficiently induced ROS production, and to define the effect of Ro5 on cellular ROS, DCF (dichlorodihydrofluorescein diacetate) was used (Dedkova & Blatter, 2012; Dedkova et al., 2013). Isolated murine cardiomyocytes were incubated with 10 µM DCF for 1 h at 37°C. Cells were washed twice with PBS. Images were taken before and after 30 min of exposure to 100 µM H₂O₂. Western blot, MitoSOX and DCF images were quantified with ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA,) using the gels and multiple ROI features, respectively.

Statistics

All data are expressed as mean ± SD. Statistical significance of differences between experimental groups was determined using two‐way ANOVA, followed by Tukey post‐test when appropriate. A value of P <0.05 was considered statistically significant. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Treatment with Ro‐5 protects the heart against TAC‐induced HF

As expected after 8 weeks of TAC, TAC‐Saline hearts exhibited classic signs of HF, including a reduction in fractional shortening (24 ± 7%) compared to baseline 55 ± 3%, P < 0.0001, (Fig. 1A and  B ), increased left ventricular (LV) size manifest by a significant increase in LV mass (145 ± 42 mg vs. 67 ± 12 mg, P < 0.0001) (Fig. 1C ) and an increase in LV end‐diastolic volume (LVEDV) (68 ± 30 µL vs. 45 ± 10 µL, P < 0.05) (Fig. 1D ). In contrast TAC‐Ro5 hearts exhibited less functional impairment from TAC with fractional shortening in TAC‐Ro5 animals significantly higher than that seen in TAC‐Saline animals (41 ± 10% in TAC‐Ro5 vs. 24 ± 7% in TAC‐Saline, P < 0.001). Ro5‐4864 had no effect on fractional shortening in sham controls (55 ± 2% vs. baseline 55 ± 3%, P = 0.89). Similarly the TAC‐Ro5 hearts did not have significant changes in LVEDV, LVESV, LV mass or cardiac output when compared to either Sham‐Saline or Sham‐Ro5 hearts. Ro5 administration alone in the absence of TAC (Sham‐Ro5) did not have any effect on functional parameters when compared to saline treated animals. Thus, the administration of Ro5‐4864 largely protected animals from the functional consequences of TAC‐induced HF.

Ro‐5 decreases TSPO expression and markers of HF and cardiac fibrosis

The four experimental groups showed marked differences in the proteome as illustrated in the heat map in Fig. 2A . Although the greatest qualitative difference in protein expression occurred between the TAC‐Ro5 and TAC‐saline animals, it was noted that treatment with Ro5 in saline‐treated animals also resulted in small but notable changes in the proteome. For example there was a notable increase in the expression of Pnpt1, Art3, Ndufb6, and Gstk1. Pnpt1, or Polyribonucleotide Nucleotidyltransferase 1, is a gene that encodes a protein involved in mitochondrial RNA processing and degradation. Ndufb6 is the gene which encodes NADH:ubiquinoneoxidoreductase subunit B6 of the mitochondrial complex I located at the mitochondrial inner membrane. Gstk1 gene encodes Glutathione S‐transferase kappa 1 (GSTK1) enzyme that is which belongs to the superfamily of enzymes known as glutathione S‐transferase (GST), which plays a major role in cellular detoxification.

Volcano plots of the proteome in TAC‐saline and TAC‐Ro5 animals (Fig. 2B ) showed a general down‐regulation of proteins in TAC‐Ro5 compared with TAC‐saline animals; specifically TSPO expression was upregulated in TAC‐Saline, while being downregulated in TAC‐Ro5 animals. Consistent with the previously described response to HF (Thai et al., 2018), expression of TSPO increased fourfold with TAC (32 338 AU ± 6604 vs. 8013 AU ± 2272 baseline, P < 0.01), while Ro5 limited this increase to 2.2 fold in TAC mice (17 701 AU ± 5994). As a result TSPO expression was significantly lower (P < 0.01) in the TAC‐Ro5 than the TAC‐Saline animals (Fig. 2C ). These findings indicate that Ro5 treatment and its downstream effects limit the increase in TSPO expression in response to TAC, a finding similar to that seen in the TSPO KO mice.

Proteomic analysis revealed that Ro5 treatment significantly attenuated markers of heart failure and fibrosis, in contrast to the significant elevations in TAC‐Saline hearts as previously reported (Thai et al., 2018). As seen in Fig. 3A , natriuretic peptide A (Patel et al., 2005) was significantly increased in the TAC‐Saline mice vs. Sham‐Saline (52 485 AU ± 37 334 vs. 1135 AU ± 597 baseline, P < 0.005) while its expression was significantly decreased in TAC‐Ro5 (2895 AU ± 734, P < 0.05) as compared to TAC‐Saline. Fibrosis in TAC‐Saline hearts was evident by the increase in collagen alpha‐1 (XII) (Neff & Bradshaw, 2021) (TAC‐saline: 7393 AU ± 4232 vs. Sham‐Saline: 943 AU ± 516, P <0.005), with Ro5 treatment decreasing collagen alpha‐1 (XII) expression in TAC hearts (TAC‐Ro5: 1254 AU ± 546 vs. TAC‐saline: 7393 AU ± 4232, P < 0.005) (Fig. 3B ). In addition, periostin, another marker of fibrosis in the heart (Qiao et al., 2024), was significantly elevated in TAC‐Saline hearts (TAC‐Saline 0.604 ± 0.3531 vs. Sham‐Saline 0.0065 ± 0.007, P <0.0005) and decreased in TAC‐Ro5 animals compared to TAC‐Saline animals (0.0817 ± 0.1654 vs. 0.604 AU ± 0.3531, P < 0.005), as evident from western blot data (Fig. 3C and  D ).

Periostin, a 90‐kDa non‐structural matricellular protein, is typically undetectable in the healthy heart as shown in Fig. 3C and  D ; however a pathological insult such as TAC or myocardial infarction stimulates periostin production by the activated fibroblasts, which secrete it to the extracellular matrix (Gil et al., 2022). Mechanical stress and inflammation after TAC surgery triggers the production of inflammatory cytokines IL‐4,13 and TGF‐β which work through transcription factors like twist and c‐fos to promote periostin expression and extracellular matrix remodelling. Based on these facts we hypothesized that Ro5 could slow down heart failure and cardiac remodelling via its effects on inflammation.

Effect of Ro5 on immune system and inflammation

Analysis of the proteomic pathway revealed that Ro5 caused a significant downregulation in proteins involved in oxidative stress and inflammation in TAC mice compared to saline‐treated TAC. Figure 4 shows the distribution of proteins that were affected by Ro5 in the setting of TAC, with the –log P value related to the magnitude of change and the size of the circle indicating the number of proteins with significant changes. As can be seen proteins involved in neutrophil degranulation, immune system and innate immune system were significantly downregulated (–logP 7 to 8) in Ro5‐treated TAC animals compared to other proteins (–logP < 4.5). Overall these data suggest that pathological insult such as TAC was inducing severe cardiac remodelling and fibroblast activation, most likely due to increased inflammation and oxidative stress.

Effect of Ro5 on oxidative stress

Markers of oxidative stress decreased in the TAC‐Ro5 hearts compared to the TAC‐Saline hearts. As seen in Fig. 5, TAC‐Saline hearts, when compared to baseline (Sham‐Saline), had significantly elevated levels of glutathione reductase (46 370 AU ± 5433 vs. 16 806 AU ± 4819, P < 0.0001), glutathione synthetase (89 037 AU ± 8141 vs. 26 267 AU ± 20 905, P < 0.0001), glutathione peroxidase 1 (202 669 AU ± 46 415 vs. 128 334 AU ± 13 932, P < 0.01) and thioredoxin reductase 1 (66 251 AU ± 14 886 vs. 39 157 AU ± 5021, P < 0.005) (Fig. 5A–D ), signifying increased levels of inflammation with TAC (Bachhawat et al., 2020). Treatment with Ro5 in the setting of TAC significantly limited the increase in these proteins: glutathione reductase (29 206 AU ± 2823 vs. 46 370 AU ± 5433, P < 0.005), glutathione synthetase (61 596 AU ± 7283 vs. 89 037 AU ± 8141, P < 0.05), glutathione peroxidase 1 (122 590 AU ± 18 150 vs. 202 669 AU ± 46 415, P < 0.005) and thioredoxin reductase 1 (43 731 AU ± 7723 vs. 66 251 AU ± 14 886, P < 0.05) (Fig. 5A–D ); however, levels of glutathione reductase and glutathione synthetase remained higher than Sham‐Saline hearts. Quantification of specific proteins related to neutrophil degranulation and the immune system showed significant reductions in immunoglobulin heavy constant gamma 2B (TAC‐Saline:1 405 229 AU ± 652 808 vs. TAC‐Ro5: 347 882 AU ± 232 814, P < 0.05) and immunoglobulin kappa variable 5–39 (25 609 AU ± 7164 TAC‐Saline vs. 10 993 AU ± 10 198 TAC‐Ro5, P < 0.001) (Fig. 5E , F ) in the TAC‐Ro5 hearts. These significant changes imply that Ro5 decreased the immune response, inflammation and oxidative stress after TAC.

The role of Nrf2 in inflammation

Given the evidence that Ro5 decreased inflammation under conditions of TAC, mechanisms for this effect were explored, with specific attention to Nrf2, its suppressor Keap1 and p62, a competitive inhibitor of Keap1 (Kageyama et al., 2018). Nrf2 is a transcription factor that reduces oxidative stress and NLRP3 inflammasome activation via activation of ROS detoxifying proteins (Chen & Maltagliati, 2018). Consistent with a beneficial effect of Ro5 in this model, Nrf2 significantly increased with Ro5 administration both in sham (0.8241 AU ± 0.09 vs. 0.26 AU ± 0.05 baseline, 315%, P < 0.05) and TAC mice (1.47 AU ± 0.23 vs. 0.6176± 0.22, 239%, P < 0.005) (Fig. 6A and  C ). Nrf2 expression was also elevated in TAC‐Saline when compared to Sham‐Saline hearts, but this increase was not statistically significant. Ro5 treatment during TAC significantly lowered the expression of p62 (0.683 ± 0.118 in in TAC‐Ro5 vs. 1.176± 0.202 in TAC‐Saline, –42%, P < 0.001), with concurrent significant reductions in Keap1 (0.392 ± 0.182 in TAC‐Ro5 vs. 0.737± 0.136 in TAC‐Saline, –47%, P < 0.05) and increased expression of the antioxidant defence protein NAD(P)H quinone oxidoreductase 1 (NQO1: 1.485 ± 0.089 in TAC‐Ro5 vs. 1.030 ± 0.179 in TAC‐Saline, +44%, P < 0.005) (Fig. 6).

We previously showed that elevated TSPO expression in HF was associated with impaired mitophagy and P62/SQSTM1 accumulation in the heart (Thai et al., 2018), which was improved by TSPO KO. In this study we demonstrated that decreasing TSPO activity with the selective ligand Ro5‐4864 (Ro5) removed the inhibitory effect of Keap1 via its effect on p62, stabilizing or activating the Nrf2 signalling cascade leading to cardioprotective effects. The interconnection of p62 and TSPO, as proteins involved in cellular processes such as autophagy and mitochondrial function, suggests that Ro5, a ligand of TSPO, could decrease p62 expression secondary to enhanced mitophagy and restoration of autophagic flux (Gatliff et al., 2014; Thai et al., 2018).

Effect of Ro5 on mitochondrial ROS in vitro

The inflammatory model presented above was further supported by in vitro experiments in which murine cardiomyocytes were exposed to ROS‐inducing concentrations of H₂O₂ under control and Ro5 conditions. The antioxidant effect of Ro5 was demonstrated by a significant reduction in mitochondrial superoxide production as measured by MitoSOX staining (Fig. 7A ). There was a significant increase in mitochondrial superoxide after H₂O₂ treatment (13.61 AU ± 0.82 vs. 11.73 AU ± 0.48 baseline, P < 0.0001). However co‐treatment of Ro5 and H₂O₂ significantly decreased superoxide production (12.24 AU ± 0.75, P < 0.0001), closely matching in vivo observations. To examine cellular H₂O₂ production, DCF was employed with fluorescent measurements made after 30 min of H₂O₂ exposure. As seen in Fig. 7B DCF fluorescence was significantly lower in Ro5‐treated cardiomyocytes (H₂O₂+Ro5: 19.06 AU ± 7.54 vs. H₂O₂ alone: 32.18 AU ± 12.83, P < 0.005). In agreement with these data we found that the expression of superoxide dismutase 2 (SOD2) significantly decreased in TAC‐Saline hearts (TAC‐Saline: 1 990 515 AU ± 95 411 vs. Sham‐Saline: 2 413 494 AU ± 195 787, P <0.005), and partially restored by Ro5 treatment in TAC hearts (TAC‐Ro5: 2 308 670 AU ± 207 821, P <0.05). Together these experiments suggest that Ro5 lowers mitochondrial and total cellular ROS, and restores SOD2 expression presumably via Nrf2 signalling pathway activation.

Ro5 protects against TAC‐mediated heart failure

The present study demonstrates that Ro5, a ligand of TSPO, mitigates the structural and functional consequences of pressure overload in a murine model of HF. Ro5 preserved fractional shortening in TAC mice. Confirming this, the protection proteins associated with heart failure and myocardial fibrosis [natriuretic peptide A and collagen‐alpha‐1 (VII)] were significantly lower in the TAC‐Ro5 animals versus the TAC‐Saline animals. These functional and structural benefits were accompanied by profound changes in oxidative stress.

Ro5‐4864 limits TSPO‐mediated inflammation through TSPO inhibition and Nrf2 activation

The 18 kDa mitochondrial TSPO is critically involved in numerous biological processes and is generally viewed as a stress responder with its expression upregulated with myocardial ischemia and HF (Morin et al., 2016; Thai et al., 2018). TSPO, in association with VDAC, influences the regulation of mitochondrial respiration, mitochondrial membrane potential, mitophagy, apoptosis and elevation of ROS in normal and disease states (Gatliff et al., 2014). Ca²⁺ enters the mitochondria via the VDAC and serves as a critical mediator of energy production (Thai et al., 2018). When the heart is under stress, such as during HF, TSPO expression increases, leading to a decrease in mitochondrial Ca²⁺ influx (Thai et al., 2018). Additionally TSPO can form a complex with VDACs and adenine nucleotide transporters (ANTs) to open the mitochondrial permeability transition pore (mPTP), which allows for the movement of <1.5 kDa molecules through the inner‐mitochondrial membrane (Lee et al., 2020; Morin et al., 2016). Once the mPTP opens, oxidative phosphorylation and ATP synthesis become uncoupled, leading to energy depletion, loss of mitochondrial membrane potential, production of mROS and apoptosis (Lee et al., 2020; Morin et al., 2016). As shown in Fig. 4, Ro5 significantly blunted the increase in TSPO seen with TAC. These findings align with our previous TSPO KO study (Thai et al., 2018), reinforcing the therapeutic potential of targeting TSPO.

Ro5 has numerous impacts on cellular functions such as steroidogenesis, inflammation and TCA cycle activity, either through its binding with TSPO or possibly off‐target effects (Xiao et al., 2010; Yousefi et al., 2013). At the cellular level Ro5 has been shown to increase mitochondrial steroidogenesis (Veenman et al., 2007), suppress pro‐inflammatory mast cell effector function (Yousefi et al., 2013) and increase mitochondrial complex I and III activity (Xiao et al., 2010). On a cardiac organ level Ro5 has been shown to limit the hypertrophic response to isoprenaline infusion (Jaiswal et al., 2010), reduce infarct size (Obame et al., 2007) and decrease reperfusion arrhythmias and contractile impairment (Brown et al., 2008). In the current experiments Ro5 recapitulated the beneficial functional and cellular effects previously seen with TSPO KO (Thai et al., 2018). Specifically, Ro5 preserved LV systolic function in the setting of TAC, concurrent with a reduction in markers of HF and fibrosis.

Mechanistically, we demonstrate that Ro5 exerted a marked effect on the inflammatory response in HF. Data from LC/MS experiments showed that using Ro5 during TAC limited the increase in proteins involved in glutathione metabolism (Fig. 4), consistent with lower oxidative stress as well as lower production of immunoglobulins (Kaya et al., 2012). It is likely that these changes in glutathione metabolism reflect a response to lower oxidative stress rather than a primary effect of Ro5. Concurrent experiments in cardiac myocytes exposed to the pro‐oxidant H₂O₂ demonstrated that Ro5 decreased markers of oxidative stress. Taken together these experiments provide strong evidence that Ro5 results in lower oxidative stress.

Ro5‐mediated Nrf2 activation, an antioxidant defence mechanism

This anti‐inflammatory profile was tightly coupled with increased transcription factor Nrf2 (Fig. 5). Nrf2 is a critical regulator of antioxidant and cytoprotective defence mechanisms, by directly binding to the promoter regions of antioxidant response elements (Zhang et al., 2013). Whereas Nrf2 is decreased in heart failure (Tian et al., 2022), an increase in Nrf2 has been associated with a lower inflammatory response in multiple models of injury and inflammation (Zhang et al., 2013). Specifically, the activation of Nrf2 provided a novel mechanism to protect the murine heart against pathological cardiac hypertrophy and heart failure by suppressing oxidative stress (Li et al., 2009). In that study Nrf2 knockout mice developed hypertrophy, fibrosis and heart failure in the setting of TAC.

In our study we examined the effects of Ro5‐4864 on Nrf2 and its key regulatory proteins, p62 and Kelch‐like ECH‐associated protein 1 (Keap1). Under normal conditions Keap1 acts as a negative regulator of Nrf2 by promoting its ubiquitination and subsequent proteasomal degradation. Conversely p62 activates Nrf2 signalling through two mechanisms: (1) by sequestering Keap1, thereby preventing Keap1 from binding and degrading Nrf2, and (2) by facilitating the autophagic degradation of Keap1 within autophagosomes, further reducing Keap1's inhibitory influence on Nrf2. During TAC we observed significant increases in both p62 and Keap1, indicating impaired autophagic flux (Fig. 5). This observation aligns with our previous findings in a rabbit model of heart failure induced by combined pressure and volume overload, where mitochondrial quality control was disrupted (Thai et al., 2019). In that model heart failure was associated with significant accumulation of Parkin and p62 in mitochondria and a corresponding reduction in the LC3‐II/LC3‐I ratio and autophagosome formation. In the present study Ro5‐4864 treatment during TAC significantly reduced p62 expression, accompanied by decreases in KEAP1 levels and increased expression of the Nrf2 target protein NQO1, consistent with the activation of the Nrf2 antioxidant defence pathway (Fig. 5).

P62 is a protein that acts as a scaffold and adaptor in autophagy, facilitating the removal of damaged or unnecessary cellular components. TSPO is a protein primarily found in the outer mitochondrial membrane and is involved in various cellular processes, including cholesterol transport and mitophagy. We have previously shown that conditional knockout of TSPO in this model of TAC increased mitophagy and was associated with lower oxidative stress (Thai et al, 2018). The interconnection of p62 and TSPO, as proteins involved in cellular processes such as autophagy and mitochondrial function, suggests that Ro5, a ligand of TSPO, could decrease P62 expression secondary to enhanced mitophagy and restoration of autophagic flux. Similar discoveries were reported in models of neurodegeneration and neuropathic pain (Fairley et al., 2024; Hao et al., 2022), where TSPO ligands such as Ro5‐4864 and XBD173 have been shown to enhance mitophagy and restore autophagic flux. In neuropathic pain model (Hao et al., 2022), Ro5‐4864 increased the LC3‐II/LC3‐I ratio and decreased p62 protein accumulation, indicating a more efficient autophagic process. Interestingly recent work has shown that retention of KEAP1 within phase‐separated p62 bodies can drive liver injury under autophagy‐deficient conditions (Takada et al., 2025), highlighting the critical role of the p62–Keap1–Nrf2 axis in multiple pathological contexts.

Limitations

There are several limitations to this study. Although the TAC model of HF is commonly used to test interventions, and these experiments demonstrated a robust protective effect of Ro5‐4864, it is unknown whether this effect is demonstrable in other forms of HF such as that induced by chemotherapeutic agents (e.g. doxorubicin), toxins (e.g. methamphetamine) or ischemic heart disease. Additionally whether this effect extends to larger animal models, including human, is unknown. Although these data support a model of Ro5‐induced upregulation of P62, resulting in lower Keap1, increased Nrf2 and lower oxidative stress, other models can also be considered. For example Ro5 has been shown to modulate mitochondrial function and reduce oxidative damage, potentially by influencing mitochondrial permeability transition and reducing reactive oxygen species production.

The measurement techniques in this study have limits in their utility. Although protein expression provides some insight, the amount of protein in a tissue sample does not reflect its possible compartmentation (nuclear vs. cytosolic in the instance of Nrf2) or phosphorylation status (He et al., 2020).

With respect to protein electrophoresis, the specificity of the antibody as well as the use of an appropriate control can impair the accuracy of the results. Finally the number of animals in each group limited the statistical strength of the analysis, potentially resulting in a Type II error in some measurements.