2-APQC, a small-molecule activator of Sirtuin-3 (SIRT3), alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis.

Sirtuin 3 (SIRT3) is well known as a conserved nicotinamide adenine dinucleotide+ (NAD+)-dependent deacetylase located in the mitochondria that may regulate oxidative stress, catabolism and ATP production. Accumulating evidence has recently revealed that SIRT3 plays its critical roles in cardiac fibrosis, myocardial fibrosis and even heart failure (HF), through its deacetylation modifications. Accordingly, discovery of SIRT3 activators and elucidating their underlying mechanisms of HF should be urgently needed. Herein, we identified a new small-molecule activator of SIRT3 (named 2-APQC) by the structure-based drug designing strategy. 2-APQC was shown to alleviate isoproterenol (ISO)-induced cardiac hypertrophy and myocardial fibrosis in vitro and in vivo rat models. Importantly, in SIRT3 knockout mice, 2-APQC could not relieve HF, suggesting that 2-APQC is dependent on SIRT3 for its protective role. Mechanically, 2-APQC was found to inhibit the mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K), c-jun N-terminal kinase (JNK) and transforming growth factor-ß (TGF-ß)/ small mother against decapentaplegic 3 (Smad3) pathways to improve ISO-induced cardiac hypertrophy and myocardial fibrosis. Based upon RNA-seq analyses, we demonstrated that SIRT3-pyrroline-5-carboxylate reductase 1 (PYCR1) axis was closely assoiated with HF. By activating PYCR1, 2-APQC was shown to enhance mitochondrial proline metabolism, inhibited reactive oxygen species (ROS)-p38 mitogen activated protein kinase (p38MAPK) pathway and thereby protecting against ISO-induced mitochondrialoxidative damage. Moreover, activation of SIRT3 by 2-APQC could facilitate AMP-activated protein kinase (AMPK)-Parkin axis to inhibit ISO-induced necrosis. Together, our results demonstrate that 2-APQC is a targeted SIRT3 activator that alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis, which may provide a new clue on exploiting a promising drug candidate for the future HF therapeutics.

Metformin induction of heat shock factor 1 activation and the mitochondrial unfolded protein response alleviate cardiac remodeling in spontaneously hypertensive rats.

Heart failure and cardiac remodeling are both characterized by mitochondrial dysfunction. Healthy mitochondria are required for adequate contractile activity and appropriate regulation of cell survival. In the mammalian heart, enhancement of the mitochondrial unfolded protein response (UPRmt) is cardioprotective under pressure overload conditions. We explored the UPRmt and the underlying regulatory mechanism in terms of hypertension-induced cardiac remodeling and the cardioprotective effect of metformin. Male spontaneously hypertensive rats and angiotensin II-treated neonatal rat cardiomyocytes were used to induce cardiac hypertrophy. The results showed that hypertension induced the formation of aberrant mitochondria, characterized by a reduced mtDNA/nDNA ratio and swelling, as well as lower levels of mitochondrial complexes I to V and inhibition of the expression of one protein subunit of each of complexes I to IV. Such changes eventually enlarged cardiomyocytes and increased cardiac fibrosis. Metformin treatment increased the mtDNA/nDNA ratio and regulated the UPRmt, as indicated by increased expression of activating transcription factor 5, Lon protease 1, and heat shock protein 60, and decreased expression of C/EBP homologous protein. Thus, metformin improved mitochondrial ultrastructure and function in spontaneously hypertensive rats. In vitro analyses revealed that metformin reduced the high levels of angiotensin II-induced mitochondrial reactive oxygen species in such animals and stimulated nuclear translocation of heat shock factor 1 (HSF1). Moreover, HSF1 small-interfering RNA reduced the metformin-mediated improvements in mitochondrial morphology and the UPRmt by suppressing hypertrophic signals and cardiomyocyte apoptosis. These results suggest that HSF1/UPRmt signaling contributes to the beneficial effects of metformin. Metformin-mediated targeting of mitochondrial protein homeostasis and modulation of HSF1 levels have potential therapeutic implications in terms of cardiac remodeling.

PM2.5 toxicity in blood impairs cardiac redox balance and promotes mitochondrial dysfunction in rat heart that further aggravates ischemia reperfusion injury by modulating PI3K/AKT/mTOR/NF-kB signaling axis.

According to the pathophysiological mechanisms linking particulate matter (PM2.5) exposure and cardiovascular diseases, PM2.5 may directly translocate into the blood stream and remote target organs and thereby induce cardiovascular effects. The toxicity of PM2.5 is known to induce oxidative stress in pulmonary tissue, but its impact on the redox state in heart (distant organ) is unknown and how it modulates the cardiac response to ischemia reperfusion (IR) remains unclear. In the present study, we evaluated the toxic effect of PM2.5 on cardiac physiology in the presence and absence of IR after introducing PM2.5 into the blood. Female Wistar rats were injected with diesel particulate matter (DPM) via i.p & i.v routes at a concentration of 10 µg/ml. The toxic impact of PM2.5 not only adversely affects the cardiac ultra-structure (leading to nuclear infiltration, edema, irregularities in heart muscle and nuclear infiltration), but also altered the cellular redox balance, elevated inflammation and promoted the upregulation of proapoptotic mediator genes at the basal level of myocardium. The results showed alterations in cardiac ultrastructure, elevated oxidative stress and significant redox imbalance, increased inflammation and proapoptotic mediators at the basal level of myocardium. Moreover, the cardioprotective pro survival signaling axis was declined along with an increased NF-kB activation at the basal level. IR inflicted further injury with deterioration of cardiac hemodynamic indices (Heart rate [HR], Left ventricular developed pressure [LVDP], Left ventricular end-diastolic pressure [LVEDP] and rate pressure product [RPP]) along with prominent inactivation of signaling pathways. Furthermore, the levels of GSH/GSSG, NADH/NAD, NADPH/NADP were significantly low along with increased lipid peroxidation in mitochondria of PM2.5 treated IR rat hearts. This observation was supported by downregulation of glutaredoxin and peroxiredoxin genes in the myocardium. Similarly the presence of oxidative stress inducing metals was found at a higher concentration in cardiac mitochondria. Thus, the toxic impact of PM2.5 in heart augment the IR associated pathological changes by altering the physiological response, initiating cellular metabolic alterations in mitochondria and modifying the signaling molecules.

Mitofilin in cardiovascular diseases: Insights into the pathogenesis and potential pharmacological interventions.

The impact of mitochondrial dysfunction on the pathogenesis of cardiovascular disease is increasing. However, the precise underlying mechanism remains unclear. Mitochondria produce cellular energy through oxidative phosphorylation while regulating calcium homeostasis, cellular respiration, and the production of biosynthetic chemicals. Nevertheless, problems related to cardiac energy metabolism, defective mitochondrial proteins, mitophagy, and structural changes in mitochondrial membranes can cause cardiovascular diseases via mitochondrial dysfunction. Mitofilin is a critical inner mitochondrial membrane protein that maintains cristae structure and facilitates protein transport while linking the inner mitochondrial membrane, outer mitochondrial membrane, and mitochondrial DNA transcription. Researchers believe that mitofilin may be a therapeutic target for treating cardiovascular diseases, particularly cardiac mitochondrial dysfunctions. In this review, we highlight current findings regarding the role of mitofilin in the pathogenesis of cardiovascular diseases and potential therapeutic compounds targeting mitofilin.

A potent and selective activator of large-conductance Ca2+-activated K+ channels induces preservation of mitochondrial function after hypoxia and reoxygenation by handling of calcium and transmembrane potential.

AIMS: Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. METHODS: Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. RESULTS: Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. CONCLUSION: The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.

Xinyang tablet alleviated cardiac dysfunction in a cardiac pressure overload model by regulating the receptor-interacting serum/three-protein kinase 3/FUN14 domain containing 1-mediated mitochondrial unfolded protein response and mitophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Xinyang tablet (XYT) has been used for heart failure (HF) for over twenty years in clinical practice, but the underlying molecular mechanism remains poorly understood. AIMS OF THE STUDY: In the present study, we aimed to explore the protective effects of XYT in HF in vivo and in vitro. MATERIALS AND METHODS: Transverse aortic constriction was performed in vivo to establish a mouse model of cardiac pressure overload. Echocardiography, tissue staining, and real-time quantitative PCR (qPCR) were examined to evaluate the protective effects of XYT on cardiac function and structure. Adenosine 5'-triphosphate production, reactive oxygen species staining, and measurement of malondialdehyde and superoxide dismutase was used to detect mitochondrial damage. Mitochondrial ultrastructure was observed by transmission electron microscope. Immunofluorescence staining, qPCR, and Western blotting were performed to evaluate the effect of XYT on the mitochondrial unfolded protein response and mitophagy, and to identify its potential pharmacological mechanism. In vitro, HL-1 cells and neonatal mouse cardiomyocytes were stimulated with Angiotensin II to establish the cell model. Western blotting, qPCR, immunofluorescence staining, and flow cytometry were utilized to determine the effects of XYT on cardiomyocytes. HL-1 cells overexpressing receptor-interacting serum/three-protein kinase 3 (RIPK3) were generated by transfection of RIPK3-overexpressing lentiviral vectors. Cells were then co-treated with XYT to determine the molecular mechanisms. RESULTS: In the present study, XYT was found to exerta protective effect on cardiac function and structure in the pressure overload mice. And it was also found XYT reduced mitochondrial damage by enhancing mitochondrial unfolded protein response and restoring mitophagy. Further studies showed that XYT achieved its cardioprotective role through regulating the RIPK3/FUN14 domain containing 1 (FUNDC1) signaling. Moreover, the overexpression of RIPK3 successfully reversed the XYT-induced protective effects and significantly attenuated the positive effects on the mitochondrial unfolded protein response and mitophagy. CONCLUSIONS: Our findings indicated that XYT prevented pressure overload-induced HF through regulating the RIPK3/FUNDC1-mediated mitochondrial unfolded protein response and mitophagy. The information gained from this study provides a potential strategy for attenuating mitochondrial damage in the context of pressure overload-induced heart failure using XYT.

Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

Resveratrol mediates mitochondrial function through the sirtuin 3 pathway to improve abnormal metabolic remodeling in atrial fibrillation.

This study investigated the impact of resveratrol on abnormal metabolic remodeling in atrial fibrillation (AF) and explored potential molecular mechanisms. An AF cell model was established by high-frequency electrical stimulation of HL-1 atrial muscle cells. Resveratrol concentrations were optimized using CCK-8 and flow cytometry. AF-induced increases in ROS and mitochondrial calcium, along with decreased adenosine triphosphate (ATP) and mitochondrial membrane potential, were observed. Resveratrol mitigated these changes and maintained normal mitochondrial morphology. Moreover, resveratrol acted through the SIRT3-dependent pathway, as evidenced by its ability to suppress AF-induced acetylation of key metabolic enzymes. SIRT3 overexpression controls acetylation modifications, suggesting its regulatory role. In conclusion, resveratrol's SIRT3-dependent pathway intervenes in AF-induced mitochondrial dysfunction, presenting a potential therapeutic avenue for AF-related metabolic disorders. This study sheds light on the role of resveratrol in mitigating AF-induced mitochondrial remodeling and highlights its potential as a novel treatment for AF.

Multiple-matrices metabolomics combined with serum pharmacochemistry for discovering the potential targets and active constituents of Qifu decoction against heart failure.

Qifu decoction (QFD) is an ancient traditional Chinese medicine (TCM) prescription for the treatment of heart failure. However, the mechanisms and active constituents of QFD are poorly understood. In this study, multi-matrices metabolomics (serum, urine, and myocardial mitochondria) based on ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOFMS), were employed for exploring the mechanisms of QFD against heart failure in rat model. Twenty-one, seventeen, and fifteen endogenous metabolite biomarkers associated with heart failure were identified from serum, urine, and myocardial mitochondria datasets, respectively. Fourteen, twelve, and ten of the identified serum, urine, and mitochondria biomarkers were significantly reversed by QFD, respectively. QFD-targeted pathways were involved in TCA cycle, branched chain amino acids metabolism, fatty acid ß-oxidation, sphingolipid metabolism, glycerophospholipid metabolism, arachidonic acid metabolism, tryptophan metabolism, purine metabolism. In addition, QFD-derived constituents in serum were fully analyzed by UHPLC-Q-TOFMS and SUS-plot, and 24 QFD-derived components were identified in serum. Then, the correlation analysis between the QFD-reversed serum biomarkers and QFD-derived constituents in serum was employed to dissect the active constituents of QFD. It was found that eight prototypical components and three metabolites were highly correlated with efficacy and could serve as the active constituents of QFD against heart failure. Finally, neoline and calycosin, which highly correlated with branched-chain amino acid metabolism and fatty acid ß-oxidation, were selected to validate in Na2S2O4-induced cell model. It was found that neoline and calycosin provided a significant protective effect against Na2S2O4-induced cell death in a low dose-dependent manner and increased the expressions of the pathway-related protein CPT1B and BCAT2 in the cell model. In conclusions, these findings provided light on the mechanisms and active constituents of QFD against heart failure. Neoline and calycosin could be selected as potential quality-markers of QFD against heart failure.

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles induce mitochondrial permeability and cardiac damage after oral exposure in rats.

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles (ZnO NPs) are found in diverse products for human use. E171 is used as whitening agent in food and cosmetics, and ZnO NPs in food packaging. Their potential multi-organ toxicity has raised concerns on their safety. Since mitochondrial dysfunction is a key aspect of cardio-pathologies, here, we evaluate the effect of chronic exposure to E171 and ZnO NPs in rats on cardiac mitochondria. Changes in cardiac electrophysiology and body weight were measured. E171 reduced body weight more than 10% after 5 weeks. Both E171 and ZnO NPs increased systolic blood pressure (SBP) from 110-120 to 120-140 mmHg after 45 days of treatment. Both NPs altered the mitochondrial permeability transition pore (mPTP), reducing calcium requirement for permeability by 60% and 93% in E171- and ZnO NPs-exposed rats, respectively. Treatments also affected conformational state of adenine nucleotide translocase (ANT). E171 reduced the binding of EMA to Cys 159 in 30% and ZnO NPs in 57%. Mitochondrial aconitase activity was reduced by roughly 50% with both NPs, indicating oxidative stress. Transmission electron microscopy (TEM) revealed changes in mitochondrial morphology including sarcomere discontinuity, edema, and hypertrophy in rats exposed to both NPs. In conclusion, chronic oral exposure to NPs induces functional and morphological damage in cardiac mitochondria, with ZnO NPs being more toxic than E171, possibly due to their dissociation in free Zn2+ ion form. Therefore, chronic intake of these food additives could increase risk of cardiovascular disease.

Dexmedetomidine alleviates Hypoxia/reoxygenation-induced mitochondrial dysfunction in cardiomyocytes via activation of Sirt3/Prdx3 pathway.

BACKGROUND: Myocardial ischemia/reperfusion injury (MIRI) seriously threatens the health of people. The mitochondrial dysfunction in cardiomyocytes can promote the progression of MIRI. Dexmedetomidine (Dex) could alleviate the myocardial injury, which was known to reverse mitochondrial dysfunction in lung injury. However, the function of Dex in mitochondrial dysfunction during MIRI remains unclear. OBJECTIVE: To assess the function of Dex in mitochondrial dysfunction during MIRI. METHODS: To investigate the function of Dex in MIRI, H9C2 cells were placed in condition of hypoxia/reoxygenation (H/R). CCK8 assay was performed to test the cell viability, and the mitochondrial membrane potential was evaluated by JC-1 staining. In addition, the binding relationship between Sirt3 and Prdx3 was explored by Co-IP assay. Furthermore, the protein expressions were examined using western blot. RESULTS: Dex could abolish H/R-induced mitochondrial dysfunction in H9C2 cells. In addition, H/R treatment significantly inhibited the expression of Sirt3, while Dex partially restored this phenomenon. Knockdown of Sirt3 or Prdx3 obviously reduced the protective effect of Dex on H/R-induced mitochondrial injury. Meanwhile, Sirt3 could enhance the function of Prdx3 via deacetylation of Prdx3. CONCLUSION: Dex was found to attenuate H/R-induced mitochondrial dysfunction in cardiomyocytes via activation of Sirt3/Prdx3 pathway. Thus, this study might shed new lights on exploring new strategies for the treatment of MIRI.

[Resveratrol pretreatment improves mitochondrial function and alleviates myocardial ischemia-reperfusion injury by up-regulating mi R-20b-5p to inhibit STIM2].

This study aimed to explore the mechanism of resveratrol(RES) pretreatment in improving mitochondrial function and alleviating myocardial ischemia-reperfusion(IR) injury by inhibiting stromal interaction molecule 2(STIM2) through microRNA-20 b-5 p(miR-20 b-5 p). Ninety rats were randomly assigned into sham group, IR group, IR+RES(50 mg·kg~(-1) RES) group, IR+RES+antagomir NC(50 mg·kg~(-1) RES+80 mg·kg~(-1) antagomir NC) group, and IR+RES+miR-20 b-5 p antagomir(50 mg·kg~(-1) RES+80 mg·kg~(-1) miR-20 b-5 p antagomir) group, with 18 rats/group. The IR rat model was established by ligation of the left anterior descending coronary artery. Two weeks before the operation, rats in the IR+RES group were intraperitoneally injected with 50 mg·kg~(-1) RES, and those in the sham and IR groups were injected with the same dose of normal saline, once a day. Ultrasonic instrument was used to detect the left ventricular internal diameter at end-diastole(LVIDd) and left ventricular internal diameter at end-systole(LVIDs) of rats in each group. The 2,3,5-triphenyte-trazoliumchloride(TTC) method and hematoxylin-eosin(HE) staining were employed to detect the myocardial infarction area and histopathology, respectively. Real-time quantitative PCR(qRT-PCR) was carried out to detect the expression of miR-20 b-5 p in myocardial tissue. Oxygen glucose deprivation/reoxygenation(OGD/R) was performed to establish an OGD/R model of H9 c2 cardiomyocytes. CCK-8 assay was employed to detect H9 c2 cell viability. H9 c2 cells were assigned into the control group, OGD/R group, OGD/R+RES group(25 µmol·L~(-1)), OGD/R+RES+inhibitor NC group, OGD/R+RES+miR-20 b-5 p inhibitor group, mimic NC group, miR-20 b-5 p mimic group, inhibitor NC group, and miR-20 b-5 p inhibitor group. Flow cytometry was employed to detect cell apoptosis. Western blot was employed to detect the expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-cysteine proteinase 3(cleaved-caspase-3), and STIM2 in cells. The mitochondrial membrane potential(MMP) assay kit, reactive oxygen species(ROS) assay kit, and adenosine triphosphate(ATP) assay kit were used to detect the MMP, ROS, and ATP levels, respectively. Dual luciferase reporter gene assay was adopted to verify the targeting relationship between miR-20 b-5 p and STIM2. Compared with the sham group, the modeling of IR increased the myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and down-regulated the expression of miR-20 b-5 p(P<0.05). These changes were alleviated in the IR+RES group(P<0.05). The IR+RES+miR-20 b-5 p antagomir group had higher myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and lower expression of miR-20 b-5 p than the IR+RES group(P<0.05). The OGD/R group had lower viability of H9 c2 cells than the control group(P<0.05) and the OGD/R+RES groups(25, 50, and 100 µmol·L~(-1))(P<0.05). Additionally, the OGD/R group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved caspase-3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the control group(P<0.05) and the OGD/R+RES group(P<0.05). The OGD/R+RES+miR-20 b-5 p inhibitor group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved-caspase 3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the OGD/R+RES group(P<0.05). miR-20 b-5 p had a targeting relationship with STIM2. The expression of STIM2 was lower in the miR-20 b-5 p mimic group than in the mimic NC group(P<0.05) and lower in the inhibitor NC group than in the miR-20 b-5 p inhibitor group(P<0.05). RES pretreatment can inhibit the expression of STIM2 by promoting the expression of miR-20 b-5 p, thereby improving the function of mitochondria and alleviating myocardial IR damage.

Mitochondrial remodeling and ischemic protection by G protein-coupled receptor 35 agonists.

Kynurenic acid (KynA) is tissue protective in cardiac, cerebral, renal, and retinal ischemia models, but the mechanism is unknown. KynA can bind to multiple receptors, including the aryl hydrocarbon receptor, the a7 nicotinic acetylcholine receptor (a7nAChR), multiple ionotropic glutamate receptors, and the orphan G protein-coupled receptor GPR35. Here, we show that GPR35 activation was necessary and sufficient for ischemic protection by KynA. When bound by KynA, GPR35 activated Gi- and G12/13-coupled signaling and trafficked to the outer mitochondria membrane, where it bound, apparantly indirectly, to ATP synthase inhibitory factor subunit 1 (ATPIF1). Activated GPR35, in an ATPIF1-dependent and pertussis toxin-sensitive manner, induced ATP synthase dimerization, which prevented ATP loss upon ischemia. These findings provide a rationale for the development of specific GPR35 agonists for the treatment of ischemic diseases.

Berberine exerts protective effects on cardiac senescence by regulating the Klotho/SIRT1 signaling pathway.

Berberine (BBR), an isoquinoline alkaloid, exerts protective effects on various cardiac injuries, and also extends the lifespan of individuals. However, the cardioprotective effect of BBR on cardiac senescence remains unknown. This study investigated the effects of BBR on cardiac senescence and its underlying mechanism. Senescent H9c2 cells induced by doxorubicin (DOX) and naturally aged rats were used to evaluate the protective effects of BBR on cardiac senescence. The results showed that BBR protected H9c2 cells against DOX-induced senescence. Exogenous Klotho (KL) exerts similar effects to those of BBR. BBR significantly increased in protein expression of KL, while transfection with KL-specific siRNA (siKL) inhibited the protective effect of BBR against senescence. Both BBR and exogenous KL decreased the levels of reactive oxygen species, inhibited apoptosis, and alleviated mitochondrial dysfunction in these cells; and transfection with siKL attenuated these effects of BBR. In naturally aged rats, BBR indeed protected the animals from cardiac aging, at least partially, through lowering the levels of cardiac hypertrophy markers, and increased the expression of KL in cardiac tissue. Additionally, BBR markedly reversed downregulation of sirtuin1 (SIRTI) in the aged heart. In vitro experiments revealed that BBR and exogenous KL also increased the expression of SIRT1, whereas siKL limited this effect of BBR in senescent H9c2 cell. In summary, BBR upregulated KL expression and prevented heart from cardiac senescence through anti-oxidative and anti-apoptotic effects, as well as alleviation of mitochondrial dysfunction. These effects may be mediated via regulation of the Klotho/SIRT1 signaling pathway.

Mitochondrial Sirtuin-3 (SIRT3) Prevents Doxorubicin-Induced Dilated Cardiomyopathy by Modulating Protein Acetylation and Oxidative Stress.

BACKGROUND: High doses of doxorubicin put cancer patients at risk for developing dilated cardiomyopathy. Previously, we showed that doxorubicin treatment decreases SIRT3 (sirtuin 3), the main mitochondrial deacetylase and increases protein acetylation in rat cardiomyocytes. Here, we hypothesize that SIRT3 expression can attenuate doxorubicin induced dilated cardiomyopathy in vivo by preventing the acetylation of mitochondrial proteins. METHODS: Nontransgenic, M3-SIRT3 (truncated SIRT3; short isoform), and M1-SIRT3 (full-length SIRT3; mitochondrial localized) transgenic mice were treated with doxorubicin for 4 weeks (8 mg/kg body weight per week). Echocardiography was performed to assess cardiac structure and function and validated by immunohistochemistry and immunofluorescence (n=4-10). Mass spectrometry was performed on cardiac mitochondrial peptides in saline (n=6) and doxorubicin (n=5) treated hearts. Validation was performed in doxorubicin treated primary rat and human induced stem cell derived cardiomyocytes transduced with adenoviruses for M3-SIRT3 and M1-SIRT3 and deacetylase deficient mutants (n=4-10). RESULTS: Echocardiography revealed that M3-SIRT3 transgenic mice were partially resistant to doxorubicin induced changes to cardiac structure and function whereas M1-SIRT3 expression prevented cardiac remodeling and dysfunction. In doxorubicin hearts, 37 unique acetylation sites on mitochondrial proteins were altered. Pathway analysis revealed these proteins are involved in energy production, fatty acid metabolism, and oxidative stress resistance. Increased M1-SIRT3 expression in primary rat and human cardiomyocytes attenuated doxorubicin-induced superoxide formation, whereas deacetylase deficient mutants were unable to prevent oxidative stress. CONCLUSIONS: Doxorubicin reduced SIRT3 expression and markedly affected the cardiac mitochondrial acetylome. Increased M1-SIRT3 expression in vivo prevented doxorubicin-induced cardiac dysfunction, suggesting that SIRT3 could be a potential therapeutic target for mitigating doxorubicin-induced dilated cardiomyopathy.

Mitochondrial ROS-Mediated Metabolic and Cytotoxic Effects of Isoproterenol on Cardiomyocytes Are p53-Dependent and Reversed by Curcumin.

Acute ß-adrenergic stimulation contributes to heart failure. Here, we investigated the role of p53 in isoproterenol (ISO)-mediated metabolic and oxidative stress effects on cardiomyocytes and explored the direct protective effects offered by the antioxidant nutraceutical curcumin. Differentiated H9C2 rat cardiomyocytes treated with ISO were assayed for glucose uptake, lactate release, and mitochondrial reactive oxygen species (ROS) generation. Survival was assessed by sulforhodamine B assays. Cardiomyocytes showed significantly decreased glucose uptake and lactate release, as well as increased cellular toxicity by ISO treatment. This was accompanied by marked dose-dependent increases of mitochondria-derived ROS. Scavenging with N-acetyl-L-cysteine (NAC) effectively lowered ROS levels, which completely recovered glycolytic metabolism and survival suppressed by ISO. Mechanistically, ISO reduced extracellular-signal-regulated kinase (ERK) activation, whereas it upregulated p53 expression in an ROS-dependent manner. Silencing of p53 with siRNA blocked the ability of ISO to stimulate mitochondrial ROS and suppress glucose uptake, and partially recovered cell survival. Finally, curcumin completely reversed the metabolic and ROS-stimulating effects of ISO. Furthermore, curcumin improved survival of cardiomyocytes exposed to ISO. Thus, ISO suppresses cardiomyocyte glycolytic metabolism and survival by stimulating mitochondrial ROS in a p53-dependent manner. Furthermore, curcumin can efficiently rescue cardiomyocytes from these adverse effects.

Mitochondrial-Targeted Therapy for Doxorubicin-Induced Cardiotoxicity.

Anthracyclines, such as doxorubicin, are effective chemotherapeutic agents for the treatment of cancer, but their clinical use is associated with severe and potentially life-threatening cardiotoxicity. Despite decades of research, treatment options remain limited. The mitochondria is commonly considered to be the main target of doxorubicin and mitochondrial dysfunction is the hallmark of doxorubicin-induced cardiotoxicity. Here, we review the pathogenic mechanisms of doxorubicin-induced cardiotoxicity and present an update on cardioprotective strategies for this disorder. Specifically, we focus on strategies that can protect the mitochondria and cover different therapeutic modalities encompassing small molecules, post-transcriptional regulators, and mitochondrial transfer. We also discuss the shortcomings of existing models of doxorubicin-induced cardiotoxicity and explore advances in the use of human pluripotent stem cell derived cardiomyocytes as a platform to facilitate the identification of novel treatments against this disorder.

Mitochondrial Toxicity Associated with Imatinib and Sorafenib in Isolated Rat Heart Fibers and the Cardiomyoblast H9c2 Cell Line.

Tyrosine kinase inhibitors (TKIs) are associated with cardiac toxicity, which may be caused by mitochondrial toxicity. The underlying mechanisms are currently unclear and require further investigation. In the present study, we aimed to investigate in more detail the role of the enzyme complexes of the electron transfer system (ETS), mitochondrial oxidative stress, and mechanisms of cell death in cardiac toxicity associated with imatinib and sorafenib. Cardiac myoblast H9c2 cells were exposed to imatinib and sorafenib (1 to 100 µM) for 24 h. Permeabilized rat cardiac fibers were treated with both drugs for 15 min. H9c2 cells exposed to sorafenib for 24 h showed a higher membrane toxicity and ATP depletion in the presence of galactose (favoring mitochondrial metabolism) compared to glucose (favoring glycolysis) but not when exposed to imatinib. Both TKIs resulted in a higher dissipation of the mitochondrial membrane potential in galactose compared to glucose media. Imatinib inhibited Complex I (CI)- and CIII- linked respiration under both conditions. Sorafenib impaired CI-, CII-, and CIII-linked respiration in H9c2 cells cultured with glucose, whereas it inhibited all ETS complexes with galactose. In permeabilized rat cardiac myofibers, acute exposure to imatinib and sorafenib decreased CI- and CIV-linked respiration in the presence of the drugs. Electron microscopy showed enlarged mitochondria with disorganized cristae. In addition, both TKIs caused mitochondrial superoxide accumulation and decreased the cellular GSH pool. Both TKIs induced caspase 3/7 activation, suggesting apoptosis as a mechanism of cell death. Imatinib and sorafenib impaired the function of cardiac mitochondria in isolated rat cardiac fibers and in H9c2 cells at plasma concentrations reached in humans. Both imatinib and sorafenib impaired the function of enzyme complexes of the ETS, which was associated with mitochondrial ROS accumulation and cell death by apoptosis.

Effect of l-carnitine and mildronate on the mitochondrial metabolism of heart and bacterial composition of the gut microbiome in ageing mice.

Ageing is the most significant risk factor for cardiovascular diseases. l-Carnitine has a potent cardioprotective effect and its synthesis decreases during ageing. At the same time, there are pharmaceuticals, such as mildronate which, on the contrary, are aimed at reducing the concentration of l-carnitine in the heart and lead to slows down the oxidation of fatty acids in mitochondria. Despite this, both l-carnitine and mildronate are positioned as cardio protectors. We showed that l-carnitine supplementation to the diet of 15-month-old mice increased expression of the PGC-1α gene, which is responsible for the regulation of fatty acid oxidation, and the Nrf2 gene, which is responsible for protecting mitochondria by regulating the expression of antioxidants and mitophagy, in the heart. Mildronate activated the expression of genes that regulate glucose metabolism. Probably, this metabolic shift may protect the mitochondria of the heart from the accumulation of acyl-carnitine, which occurs during the oxidation of fatty acids under oxygen deficiency. Both pharmaceuticals impacted the gut microbiome bacterial composition. l-Carnitine increased the level of Lachnoanaerobaculum and [Eubacterium] hallii group, mildronate increased the level of Bifidobacterium, Rikinella, Christensenellaceae. Considered, that these bacteria for protection the organism from various pathogens and chronic inflammation. Thus, we suggested that the positive effects of both drugs on the mitochondria metabolism and gut microbiome bacterial composition may contribute to the protection of the heart during ageing.

Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation.

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.