Deoxyelephantopin-a novel PPARγ agonist regresses pressure overload-induced cardiac fibrosis via IL-6/STAT-3 pathway in crosstalk with PKCδ.

Pathological cardiac hypertrophy is associated with ventricular fibrosis leading to heart failure. The use of thiazolidinediones as Peroxisome Proliferator-Activated Receptor-gamma (PPARγ)-modulating anti-hypertrophic therapeutics has been restricted due to major side-effects. The present study aims to evaluate the anti-fibrotic potential of a novel PPARγ agonist, deoxyelephantopin (DEP) in cardiac hypertrophy. AngiotensinII treatment in vitro and renal artery ligation in vivo were performed to mimic pressure overload-induced cardiac hypertrophy. Myocardial fibrosis was evaluated by Masson's trichrome staining and hydroxyproline assay. Our results showed that DEP treatment significantly improves the echocardiographic parameters by ameliorating ventricular fibrosis without any bystander damage to other major organs. Following molecular docking, all-atomistic molecular dynamics simulation, reverse transcription-polymerase chain reaction and immunoblot analyses, we established DEP as a PPARγ agonist stably interacting with the ligand-binding domain of PPARγ. DEP specifically downregulated the Signal Transducer and Activator of Transcription (STAT)-3-mediated collagen gene expression in a PPARγ-dependent manner, as confirmed by PPARγ silencing and site-directed mutagenesis of DEP-interacting PPARγ residues. Although DEP impaired STAT-3 activation, it did not have any effect on the upstream Interleukin (IL)-6 level implying possible crosstalk of the IL-6/STAT-3 axis with other signaling mediators. Mechanistically, DEP increased the binding of PPARγ with Protein Kinase C-delta (PKCδ) which impeded the membrane translocation and activation of PKCδ, downregulating STAT-3 phosphorylation and resultant fibrosis. This study, therefore, for the first time demonstrates DEP as a novel cardioprotective PPARγ agonist. The therapeutic potential of DEP as an anti-fibrotic remedy can be exploited against hypertrophic heart failure in the future.

Cardiomyocyte-specific regression of nitrosative stress-mediated S-Nitrosylation of IKKγ alleviates pathological cardiac hypertrophy.

IKKγ prototypically promotes NFκBp65 activity by regulating the assembly of the IKK holocomplex. In hypertrophied cardiomyocytes, the p65-p300 complex-induced regenerative efforts are neutralized by the p53-p300 complex-mediated apoptotic load resulting in compromised cardiac function. The present study reports that nitrosative stress leads to S-Nitrosylation of IKKγ in hypertrophied cardiomyocytes in a pre-clinical model. Using a cardiomyocyte-targeted nanoconjugate, IKKγ S-Nitrosylation-resistant mutant plasmids were delivered to the pathologically hypertrophied heart that resulted in improved cardiac function by amelioration of cardiomyocyte apoptosis and simultaneous induction of their cell cycle re-entry machinery. Mechanistically, in IKKγ S-Nitrosyl mutant-transfected hypertrophied cells, increased IKKγ-p300 binding downregulated the binding of p53 and p65 with p300. This shifted the binding preference of p65 from p300 to HDAC1 resulting in upregulated expression of cyclin D1 and CDK2 via the p27/pRb pathway. This approach has therapeutic advantage over mainstream anti-hypertrophic remedies which concomitantly reduce the regenerative prowess of resident cardiomyocytes during hypertrophy upon downregulation of myocyte apoptosis. Therefore, cardiomyocyte-targeted delivery of IKKγ S-Nitrosyl mutants during hypertrophy can be exploited as a novel strategy to re-muscularize the diseased heart.

Cardiac-specific overexpression of HIF-1α during acute myocardial infarction ameliorates cardiomyocyte apoptosis via differential regulation of hypoxia-inducible pro-apoptotic and anti-oxidative genes.

HIF-1α acts as the cellular rheostat for oxygen sensing in cardiomyocytes. Overexpression of HIF-1α in the heart during acute myocardial infarction (MI) is known to attenuate cardiac dysfunction by upregulating pro-angiogenic HIF-1α target genes. However, the effect of HIF-1α overexpression on hypoxic cardiomyocyte apoptosis still remains obscure. In this study, we report for the first time that myocardium-targeted nanotized HIF-1α overexpression during MI downregulates cardiomyocyte apoptosis. HIF-1α overexpression attenuates bnip3-mediated apoptosis indirectly by promoting HO-1-induced anti-oxidant response. Chromatin immunoprecipitation experiment revealed that HIF-1α overexpression in hypoxic cardiomyocytes increases binding of HIF-1α to the hypoxia-responsive element in the promoter of its target anti-oxidant gene ho-1 which is known to attenuate ROS accumulation. ROS accumulation in hypoxic cardiomyocytes causes cysteine oxidation of the DNA-binding p50 subunit of NFκB, which hampers NFκB binding to κB-responsive genes like bnip3. Downregulated oxidative stress due to HIF-1α overexpression leads to decline in cysteine oxidation of NFκBp50, causing NFκB to bind to the promoter of bnip3 as a transcriptional repressor. Therefore HIF-1α overexpression-mediated attenuation of cardiomyocyte apoptosis occurs by transcriptional repression of bnip3 by NFκB. Our study thus reveals that downregulation of bnip3-mediated cardiomyocyte apoptosis occurs via ho-1 upregulation upon HIF-1α overexpression during MI, despite both being HIF-1α target genes. The cross-regulation of HIF-1α and NFκB-mediated pathways effectively downregulates apoptosis due to HIF-1α overexpression during MI, which can be exploited for possible therapeutic intervention.

Severity and duration of hypoxic stress differentially regulates HIF-1α-mediated cardiomyocyte apoptotic signaling milieu during myocardial infarction.

BACKGROUND: The severity and duration of hypoxia is known to determine apoptotic fate in heart; however, its implication during myocardial infarction (MI) remains unaddressed. Therefore the aim of the study was to determine apoptotic regulation in cardiomyocytes under varied hypoxic intensity and duration and to unravel the role of HIF-1α in such modulation. METHODS: Treatment of cardiomyocytes to varied hypoxic intensity and duration was carried out in vitro, which was mimicked in vivo by dose-dependent Isoproterenol hydrochloride treatment for varied time-points. Myocardium-targeted HIF-1α knockdown in vivo was performed to decipher its role in cardiomyocyte apoptosis under varied stress. Signaling intermediates were analyzed by RT-PCR, immunoblotting and co-immunoprecipitation. DCFDA-based ROS assay, Griess assay for NO release and biochemical assays for estimating caspase activity were performed. RESULTS: Severe stress resulted in cardiomyocyte apoptosis in both shorter and longer time-points. Moderate stress, on the other hand, induced apoptosis only in the shorter time-point which was downregulated in the longer time-point. ROS activity was upregulated under severe hypoxic stress for both time-points and only in the early time-point under moderate hypoxia. Increased ROS accumulation activated ERK-1/2 which stabilized nuclear HIF-1α, promoting bnip3-mediated apoptosis. Stable HSP90-IRE-1 association in such cells caused elevated endoplasmic reticulum stress-related caspase-12 activity. Sustained moderate hypoxia caused decline in ROS activity, but upregulated NFκB-dependent NO generation. NO-stabilized HIF-1α was predominantly cytosolic, since low ROS levels downregulated ERK-1/2 activity, thereby suppressing bnip3 expression. Cytosolic HIF-1α in such cells sequestered HSP90 from IRE-1, downregulating caspase-12 activity due to proteasomal degradation of IRE-1. Accordingly, myocardium-specific in vivo silencing of HIF-1α was beneficial at both time-points under severe stress as also for lesser duration of moderate stress. However, silencing of HIF-1α aggravated apoptotic injury during sustained moderate stress. CONCLUSION: ROS-mediated HIF-1α stabilization promotes cardiomyocyte apoptosis on one hand while NO-mediated stabilization of HIF-1α disrupts apoptosis depending upon the severity and duration of hypoxia. Therefore the outcome of modulation of cardiac HIF-1α activity is regulated by both the severity and duration of ischemic stress.

Metabolic impairment in response to early induction of C/EBPß leads to compromised cardiac function during pathological hypertrophy.

Chronic pressure overload-induced left ventricular hypertrophy in heart is preceded by a metabolic perturbation that prefers glucose over lipid as substrate for energy requirement. Here, we establish C/EBPß (CCAAT/enhancer-binding protein ß) as an early marker of the metabolic derangement that triggers the imbalance in fatty acid (FA) oxidation and glucose uptake with increased lipid accumulation in cardiomyocytes during pathological hypertrophy, leading to contractile dysfunction and endoplasmic reticulum (ER) stress. This is the first study that shows that myocardium-targeted C/EBPß knockdown prevents the impaired cardiac function during cardiac hypertrophy led by maladaptive metabolic response with persistent hypertrophic stimuli, whereas its targeted overexpression in control increases lipid accumulation significantly compared to control hearts. A new observation from this study was the dual and opposite transcriptional regulation of the alpha and gamma isoforms of Peroxisomal proliferator activated receptors (PPARα and PPARγ) by C/EBPß in hypertrophied cardiomyocytes. Before the functional and structural remodeling sets in the diseased myocardium, C/EBPß aggravates lipid accumulation with the aid of the increased FA uptake involving induced PPARγ expression and decreased fatty acid oxidation (FAO) by suppressing PPARα expression. Glucose uptake into cardiomyocytes was greatly increased by C/EBPß via PPARα suppression. The activation of mammalian target of rapamycin complex-1 (mTORC1) during increased workload in presence of glucose as the only substrate was prevented by C/EBPß knockdown, thereby abating contractile dysfunction in cardiomyocytes. Our study thus suggests that C/EBPß may be considered as a novel cellular marker for deranged metabolic milieu before the heart pathologically remodels itself during hypertrophy.

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy.

Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats (Rattus norvegicus) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy.