Berberine Promotes Cardiac Function by Upregulating PINK1/Parkin-Mediated Mitophagy in Heart Failure.
ABSTRACT: Berberine has been verified to protect cardiac function in patients with heart failure (HF). However, the mechanism(s) involved in berberine-mediated cardioprotective effects has not been clearly elucidated. The aim of this study was to further investigate the mechanism(s) involved in the beneficial effects of berberine on transverse aortic contraction (TAC)-induced chronic HF. Mice were randomly divided into four groups. Berberine was administered at a dose of 50 mg/kg/day for 4 weeks via oral gavage. Our findings showed that TAC-induced pressure overload (PO) prompted cardiac dysfunction, cardiac hypertrophy, interstitial fibrosis, cardiomyocyte apoptosis and mitochondrial injury, accompanied with suppressed mitophagy, the effects of which were attenuated by berberine. Furthermore, mitophagy regulators PINK1 and mito-Parkin were downregulated in TAC-induced HF, while berberine upregulated PINK1/Parkin-mediated mitophagy. Notably, knockdown of PINK1 by small interfering RNA significantly suppressed Parkin-mediated mitochondrial ubiquitination and nullified the beneficial actions on HF exerted by berberine. Taken together, our results indicated that berberine plays a critical role in attenuating cardiac hypertrophy and preserving cardiac function from PO induced HF. The potential underlying mechanism is the activation of mitochondrial autophagy via PINK1/Parkin/Ubiquitination pathway.
Project description:<h4>Background</h4>Inflammation and oxidative stress are involved in the initiation and progress of heart failure (HF). However, the role of the IL6/STAT3 pathway in the pressure overload-induced HF remains controversial.<h4>Methods and results</h4>Transverse aortic constriction (TAC) was used to induce pressure overload-HF in C57BL/6J mice. 18 mice were randomized into three groups (Sham, TAC, and TAC+raloxifene, <i>n</i> = 6, respectively). Echocardiographic and histological results showed that cardiac hypertrophy, fibrosis, and left ventricular dysfunction were manifested in mice after TAC treatment of eight weeks, with aggravation of macrophage infiltration and interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-<i>α</i>) expression in the myocardium. TAC (four and eight weeks) elevated the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) and prohibitin2 (PHB2) protein expression. Importantly, IL-6/gp130/STAT3 inhibition by raloxifene alleviated TAC-induced myocardial inflammation, cardiac remodeling, and dysfunction. <i>In vitro</i>, we demonstrated cellular hypertrophy with STAT3 activation and oxidative stress exacerbation could be elicited by IL-6 (25 ng/mL, 48 h) in H9c2 myoblasts. Sustained IL-6 stimulation increased intracellular reactive oxygen species, repressed mitochondrial membrane potential (MMP), decreased intracellular content of ATP, and led to decreased SOD activity, an increase in iNOS protein expression, and increased protein expression of Pink1, Parkin, and Bnip3 involving in mitophagy, all of which were reversed by raloxifene.<h4>Conclusion</h4>Inflammation and IL-6/STAT3 signaling were activated in TAC-induced HF in mice, while sustained IL-6 incubation elicited oxidative stress and mitophagy-related protein increase in H9c2 myoblasts, all of which were inhibited by raloxifene. These indicated IL-6/STAT3 signaling might be involved in the pathogenesis of myocardial hypertrophy and HF.
Project description:RATIONALE:Mitochondrial dysfunction plays an important role in heart failure (HF). However, the molecular mechanisms regulating mitochondrial functions via selective mitochondrial autophagy (mitophagy) are poorly understood. OBJECTIVE:We sought to determine the role of AMPK (AMP-activated protein kinase) in selective mitophagy during HF. METHODS AND RESULTS:An isoform shift from AMPK?2 to AMPK?1 was observed in failing heart samples from HF patients and transverse aortic constriction-induced mice, accompanied by decreased mitophagy and mitochondrial function. The recombinant adeno-associated virus Serotype 9-mediated overexpression of AMPK?2 in mouse hearts prevented the development of transverse aortic constriction-induced chronic HF by increasing mitophagy and improving mitochondrial function. In contrast, AMPK?2-/- mutant mice exhibited an exacerbation of the early progression of transverse aortic constriction-induced HF via decreases in cardiac mitophagy. In isolated adult mouse cardiomyocytes, AMPK?2 overexpression mechanistically rescued the impairment of mitophagy after phenylephrine stimulation for 24 hours. Genetic knockdown of AMPK?2, but not AMPK?1, by short interfering RNA suppressed the early phase (6 hours) of phenylephrine-induced compensatory increases in mitophagy. Furthermore, AMPK?2 specifically interacted with phosphorylated PINK1 (PTEN-induced putative kinase 1) at Ser495 after phenylephrine stimulation. Subsequently, phosphorylated PINK1 recruited the E3 ubiquitin ligase, Parkin, to depolarized mitochondria, and then enhanced the role of the PINK1-Parkin-SQSTM1 (sequestosome-1) pathway involved in cardiac mitophagy. This increase in cardiac mitophagy was accompanied by the elimination of damaged mitochondria, improvement in mitochondrial function, decrease in reactive oxygen species production, and apoptosis of cardiomyocytes. Finally, Ala mutation of PINK1 at Ser495 partially suppressed AMPK?2 overexpression-induced mitophagy and improvement of mitochondrial function in phenylephrine-stimulated cardiomyocytes, whereas Asp (phosphorylation mimic) mutation promoted mitophagy after phenylephrine stimulation. CONCLUSIONS:In failing hearts, the dominant AMPK? isoform switched from AMPK?2 to AMPK?1, which accelerated HF. The results show that phosphorylation of Ser495 in PINK1 by AMPK?2 was essential for efficient mitophagy to prevent the progression of HF.
Project description:BRD4 is a member of the BET family of epigenetic regulators. Inhibition of BRD4 by the selective bromodomain inhibitor JQ1, alleviates thoracic aortic constriction-induced cardiac hypertrophy and heart failure. However, whether BRD4 inhibition by JQ1 has therapeutic effect on diabetic cardiomyopathy, a major cause of heart failure in patients with Type 2 diabetes, remains unknown. Here, we discover a novel link between BRD4 and PINK1/Parkin-mediated mitophagy during diabetic cardiomyopathy. Upregulation of BRD4 in diabetic mouse hearts inhibits PINK1/Parkin-mediated mitophagy, resulting in accumulation of damaged mitochondria and subsequent impairment of cardiac structure and function. BRD4 inhibition by JQ1 improves mitochondrial function, and repairs the cardiac structure and function of the diabetic heart. These effects depended on rewiring of the BRD4-driven transcription and repression of PINK1. Deletion of Pink1 suppresses mitophagy, exacerbates cardiomyopathy, and abrogates the therapeutic effect of JQ1 on diabetic cardiomyopathy. Our results illustrate a valid therapeutic strategy for treating diabetic cardiomyopathy by inhibition of BRD4.
Project description:Pathological cardiac hypertrophy, the adaptive response of the myocardium to various pathological stimuli, is one of the primary predictors and predisposing factors of heart failure. However, its molecular mechanisms underlying pathogenesis remain poorly understood. Here, we studied the function of Samm50 in mitophagy during Ang II-induced cardiomyocyte hypertrophy via lentiviruses mediated knockdown and overexpression of Samm50 protein. We first found that Samm50 is a key positive regulator of cardiac hypertrophy, for western blot and real-time quantitative PCR detection revealed Samm50 was downregulated both in pressure-overload-induced hypertrophic hearts and Ang II-induced cardiomyocyte hypertrophy. Then, Samm50 overexpression exhibits enhanced induction of cardiac hypertrophy marker genes and cell enlargement in primary mouse cardiomyocytes by qPCR and immunofluorescence analysis, respectively. Meanwhile, Samm50 remarkably reduced Ang II-induced autophagy as indicated by decreased mitophagy protein levels and autophagic flux, whereas the opposite phenotype was observed in Samm50 knockdown cardiomyocytes. However, the protective role of Samm50 deficiency against cardiac hypertrophy was abolished by inhibiting mitophagy through Vps34 inhibitor or Pink1 knockdown. Moreover, we further demonstrated that Samm50 interacted with Pink1 and stimulated the accumulation of Parkin on mitochondria to initiate mitophagy by co-immunoprecipitation analysis and immunofluorescence. Thus, these results suggest that Samm50 regulates Pink1-Parkin-mediated mitophagy to promote cardiac hypertrophy, and targeting mitophagy may provide new insights into the treatment of cardiac hypertrophy.
Project description:In developing hearts, changes in the cardiac metabolic milieu during the perinatal period redirect mitochondrial substrate preference from carbohydrates to fatty acids. Mechanisms responsible for this mitochondrial plasticity are unknown. Here, we found that PINK1-Mfn2-Parkin-mediated mitophagy directs this metabolic transformation in mouse hearts. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary for Parkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressing mitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expression of Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrial maturation essential to survival. Five-week-old Mfn2 AA hearts retained a fetal mitochondrial transcriptional signature without normal increases in fatty acid metabolism and mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels and cardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed. Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondria undergo perinatal Parkin-mediated mitophagy and replacement by mature adult mitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocyte mitochondrial plasticity and metabolic transitioning of perinatal hearts.
Project description:Maintaining proper mitochondrial respiratory function is crucial for alleviating cardiac metabolic disorders during obesity, and mitophagy is critically involved in this process. Long non-coding RNA H19 (H19) is crucial for metabolic regulation, but its roles in cardiac disorders, mitochondrial respiratory function, and mitophagy during obesity are largely unknown. In this study, palmitic acid (PA)-treated H9c2 cell and Lep<sup>-/-</sup> mice were used to investigate cardiac metabolic disorders in vitro and in vivo, respectively. The effects of H19 on metabolic disorders, mitochondrial respiratory function, and mitophagy were investigated. Moreover, the regulatory mechanisms of PA, H19, mitophagy, and respiratory function were examined. The models tested displayed a reduction in H19 expression, respiratory function and mitochondrial number and volume, while the expression of mitophagy- and Pink1/Parkin signaling-related proteins was upregulated, as indicated using quantitative real-time PCR, Seahorse mitochondrial stress test analyzer, transmission electron microscopy, fluorescence indicators and western blotting. Forced expression of H19 helped to the recoveries of respiratory capacity and mitochondrial number while inhibited the levels of mitophagy- and Pink1/Parkin signaling-related proteins. Pink1 knockdown also attenuated PA-induced mitophagy and increased respiratory capacity. Mechanistically, RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation assays showed that H19 could hinder the binding of eukaryotic translation initiation factor 4A, isoform 2 (eIF4A2) with Pink1 mRNA, thus inhibiting the translation of Pink1 and attenuation of mitophagy. PA significantly increased the methylation levels of the H19 promoter region by upregulation Dnmt3b methylase levels, thereby inhibiting H19 transcription. Collectively, these findings suggest that DNA methylation-mediated the downregulation of H19 expression plays a crucial role in cardiomyocyte or H9c2 cells metabolic disorders and induces cardiac respiratory dysfunction by promoting mitophagy. H19 inhibits excessive mitophagy by limiting Pink1 mRNA translation, thus alleviating this cardiac defect that occurs during obesity.
Project description:Pressure overload (PO) first causes cardiac hypertrophy and then heart failure (HF), which are associated with sex differences in cardiac morphology and function. We aimed to identify genes that may cause HF-related sex differences. We used a transverse aortic constriction (TAC) mouse model leading to hypertrophy without sex differences in cardiac function after 2 weeks, but with sex differences in hypertrophy 6 and 9 weeks after TAC. Cardiac gene expression was analyzed 2 weeks after surgery. Deregulated genes were classified into functional gene ontology (GO) categories and used for pathway analysis. Classical marker genes of hypertrophy were similarly upregulated in both sexes (alpha-actin, ANP, BNP, CTGF). Thirty-five genes controlling mitochondrial function (PGC-1, cytochrome oxidase, carnitine palmitoyl transferase, acyl-CoA dehydrogenase, pyruvate dehydrogenase kinase) had lower expression in males compared to females after TAC. Genes encoding ribosomal proteins and genes associated with extracellular matrix remodeling exhibited relative higher expression in males (collagen 3, matrix metalloproteinase 2, TIMP2, and TGFbeta2, all about twofold) after TAC. We confirmed 87% of the gene expression by real-time polymerase chain reaction. By GO classification, female-specific genes were related to mitochondria and metabolism and males to matrix and biosynthesis. Promoter studies confirmed the upregulation of PGC-1 by E2. Less downregulation of metabolic genes in female hearts and increased protein synthesis capacity and deregulation of matrix remodeling in male hearts characterize the sex-specific early response to PO. These differences could contribute to subsequent sex differences in cardiac function and HF.
Project description:Mitophagy alleviates neuronal damage after cerebral ischemia by selectively removing dysfunctional mitochondria. Phosphatase and tensin homolog (PTEN) induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy is the most well-known type of mitophagy. However, little is known about the role of PINK1/Parkin-mediated mitophagy in ischemic tolerance induced by hypoxic postconditioning (HPC) with 8% O<sub>2</sub> against transient global cerebral ischemia (tGCI). Hence, we aimed to test the hypothesis that HPC-mediated PINK1/Parkin-induced mitochondrial ubiquitination and promotes mitophagy, thus exerting neuroprotection in the hippocampal CA1 subregion against tGCI. We found that mitochondrial clearance was disturbed at the late phase of reperfusion after tGCI, which was reversed by HPC, as evidenced by the reduction of the translocase of outer mitochondrial membrane 20 homologs (TOMM20), translocase of inner mitochondrial membrane 23 (TIMM23) and heat shock protein 60 (HSP60) in CA1 after HPC. In addition, HPC further increased the ratio of LC3II/I in mitochondrial fraction and promoted the formation of mitophagosomes in CA1 neurons after tGCI. The administration of lysosome inhibitor chloroquine (CQ) intraperitoneally or mitophagy inhibitor (Mdivi-1) intracerebroventricularly abrogated HPC-induced mitochondrial turnover and neuroprotection in CA1 after tGCI. We also found that HPC activated PINK1/Parkin pathway after tGCI, as shown by the augment of mitochondrial PINK1 and Parkin and the promotion of mitochondrial ubiquitination in CA1. In addition, PINK1 or Parkin knockdown with small-interfering RNA (siRNA) suppressed the activation of PINK1/Parkin pathway and hampered mitochondrial clearance and attenuated neuroprotection induced by HPC, whereas PINK1 overexpression promoted PINK1/Parkin-mediated mitophagy and ameliorated neuronal damage in CA1 after tGCI. Taken together, the new finding in this study is that HPC-induced neuroprotection against tGCI through promoting mitophagy mediated by PINK1/Parkin-dependent pathway.
Project description:Chronic pressure overload (PO) induces pathological left ventricular hypertrophy (LVH) leading to congestive heart failure (HF). Overexpression of FKBP12.6 (FK506-binding protein [K]) in mice should prevent Ca2+-leak during diastole and may improve overall cardiac function. In order to decipher molecular mechanisms involved in thoracic aortic constriction (TAC)-induced cardiac remodeling and the influence of gender and genotype, we performed a proteomic analysis using two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and bioinformatics techniques to identify alterations in characteristic biological networks. Wild-type (W) and K mice of both genders underwent TAC. Thirty days post-TAC, the altered cardiac remodeling was accompanied with systolic and diastolic dysfunction in all experimental groups. A gender difference in inflammatory protein expression (fibrinogen, ?-1-antitrypsin isoforms) and in calreticulin occurred (males > females). Detoxification enzymes and cytoskeletal proteins were noticeably increased in K mice. Both non- and congestive failing mouse heart exhibited down- and upregulation of proteins related to mitochondrial function and purine metabolism, respectively. HF was characterized by a decrease in enzymes related to iron homeostasis, and altered mitochondrial protein expression related to fatty acid metabolism, glycolysis, and redox balance. Moreover, two distinct differential protein profiles characterized TAC-induced pathological LVH and congestive HF in all TAC mice. FKBP12.6 overexpression did not influence TAC-induced deleterious effects. Huntingtin was revealed as a potential mediator for HF. A broad dysregulation of signaling proteins associated with congestive HF suggested that different sets of proteins could be selected as useful biomarkers for HF progression and might predict outcome in PO-induced pathological LVH.
Project description:The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy.