Nanotherapeutics for the Myocardium: A Potential Alternative for Treating Cardiac Diseases.

ABSTRACT: Cardiovascular diseases (CVDs) are the foremost cause of morbidity and mortality worldwide. Current clinical interventions include invasive approaches for progressed conditions and pharmacological assistance for initial stages, which has systemic side effects. Preventive, curative, diagnostic, and theranostic (therapeutic + diagnostic) approaches till date are not very useful in combating the ongoing CVD epidemic, which demands a promising efficient alternative approach. To combat the growing CVD outbreak globally, the ideal strategy is to make the therapeutic intervention least invasive and direct to the heart to reduce the bystander effects on other organs and increase the bioavailability of the therapeutics to the myocardium. The application of nanoscience and nanoparticle-mediated approaches have gained a lot of momentum because of their efficient passive and active myocardium targeting capability owing to their improved specificity and controlled release. This review provides extensive insight into the various types of nanoparticles available for CVDs, their mechanisms of targeting (eg, direct or indirect), and the utmost need for further development of bench-to-bedside cardiac tissue-based nanomedicines. Furthermore, the review aims to summarize the different ideas and methods of nanoparticle-mediated therapeutic approaches to the myocardium till date with present clinical trials and future perspectives. This review also reflects the potential of such nanoparticle-mediated tissue-targeted therapies to contribute to the sustainable development goals of good health and well-being.

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.

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.

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.

Arjunolic acid, a peroxisome proliferator-activated receptor α agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-ß signaling.

Cardiac hypertrophy and associated heart fibrosis remain a major cause of death worldwide. Phytochemicals have gained attention as alternative therapeutics for managing cardiovascular diseases. These include the extract from the plant Terminalia arjuna, which is a popular cardioprotectant and may prevent or slow progression of pathological hypertrophy to heart failure. Here, we investigated the mode of action of a principal bioactive T. arjuna compound, arjunolic acid (AA), in ameliorating hemodynamic load-induced cardiac fibrosis and identified its intracellular target. Our data revealed that AA significantly represses collagen expression and improves cardiac function during hypertrophy. We found that AA binds to and stabilizes the ligand-binding domain of peroxisome proliferator-activated receptor α (PPARα) and increases its expression during cardiac hypertrophy. PPARα knockdown during AA treatment in hypertrophy samples, including angiotensin II-treated adult cardiac fibroblasts and renal artery-ligated rat heart, suggests that AA-driven cardioprotection primarily arises from PPARα agonism. Moreover, AA-induced PPARα up-regulation leads to repression of TGF-ß signaling, specifically by inhibiting TGF-ß-activated kinase1 (TAK1) phosphorylation. We observed that PPARα directly interacts with TAK1, predominantly via PPARα N-terminal transactivation domain (AF-1) thereby masking the TAK1 kinase domain. The AA-induced PPARα-bound TAK1 level thereby shows inverse correlation with the phosphorylation level of TAK1 and subsequent reduction in p38 MAPK and NF-κBp65 activation, ultimately culminating in amelioration of excess collagen synthesis in cardiac hypertrophy. In conclusion, our findings unravel the mechanism of AA action in regressing hypertrophy-associated cardiac fibrosis by assigning a role of AA as a PPARα agonist that inactivates non-canonical TGF-ß signaling.

Modulation of NFKB1/p50 by ROS leads to impaired ATP production during MI compared to cardiac hypertrophy.

Pathological hypertrophy and myocardial infarction (MI) are two etiologically different cardiac disorders having differential molecular mechanisms of disease manifestation. However, no study has been conducted so far to analyze and compare the differential status of energy metabolism in these two disease forms. It was shown recently by our group that production of ATP is significantly impaired during MI along with inhibition of pyruvate dehydrogenase E1-ß (PDHE1 B) by pyruvate dehydrogenase kinase 4 (PDK4). However, the ATP levels showed no significant change during pathological hypertrophy compared to control group. To seek a plausible explanation of this phenomenon, the peroxisome proliferator-activated receptor alpha (PPAR) pathway was studied in all the experimental groups which revealed that PGC1α- ERRα axis remains active in MI while the same remained inactive during pathological hypertrophy possibly by NF-κB that plays a significant role in deactivating this pathway during hypertrophy. At the same time, it was observed that reactive oxygen species (ROS) negatively regulates NF-κB activity during MI by oxidation of cysteine residues of p50- the DNA binding subunit of NF-κB. Thus, this study reports for the first time, a possible mechanism for the differential status of energy metabolism during two etiologically different cardiac pathophysiological conditions involving PGC1α-ERRα axis along with p50 subunit of NF-κB.

Field Populations of Wild Apis cerana Honey Bees Exhibit Increased Genetic Diversity Under Pesticide Stress Along an Agricultural Intensification Gradient in Eastern India.

Pesticides have been reported to be one of the major drivers in the global pollinator losses. The large-scale decline in honey bees, an important pollinator group, has resulted in comprehensive studies on honey bee colonies. Lack of information on native wild pollinators has paved the way for this study, which highlights the underlying evolutionary changes occurring in the wild honey bee populations exposed to pesticides along an agricultural intensification landscape. The study reports an increased genetic diversity in native Apis cerana Fabricius (Hymenoptera: Apidae) populations continually exposed to pesticide stress. An increased heterozygosity, evidenced by a higher electrophoretic banding pattern, was observed in the pesticide-exposed populations for two isozymes involved with xenobiotic metabolism-esterase and glucose-6-phosphate dehydrogenase. Differential banding patterns also revealed a higher percentage of polymorphic loci, number of polymorphic bands, Nei's genetic distance, etc. observed in these populations in the Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) experiments using three random decamer primers. Higher heterozygosity, being indicative of a more resistant population, implies population survival within the threshold pesticide stress. This study reports such changes for the first time in native wild Indian honey bee populations exposed to pesticides and has far-reaching implications on the population adaptability under pesticide stress.

Improved bioavailability of targeted Curcumin delivery efficiently regressed cardiac hypertrophy by modulating apoptotic load within cardiac microenvironment.

Cardiomyocyte apoptosis acts as a prime modulator of cardiac hypertrophy leading to heart failure, a major cause of human mortality worldwide. Recent therapeutic interventions have focussed on translational applications of diverse pharmaceutical regimes among which, Curcumin (from Curcuma longa) is known to have an anti-hypertrophic potential but with limited pharmacological efficacies due to low aqueous solubility and poor bioavailability. In this study, Curcumin encapsulated by carboxymethyl chitosan (CMC) nanoparticle conjugated to a myocyte specific homing peptide was successfully delivered in bioactive form to pathological myocardium for effective regression of cardiac hypertrophy in a rat (Rattus norvegicus) model. Targeted nanotization showed higher cardiac bioavailability of Curcumin at a low dose of 5 mg/kg body weight compared to free Curcumin at 35 mg/kg body weight. Moreover, Curcumin/CMC-peptide treatment during hypertrophy significantly improved cardiac function by downregulating expression of hypertrophy marker genes (ANF, ß-MHC), apoptotic mediators (Bax, Cytochrome-c) and activity of apoptotic markers (Caspase 3 and PARP); whereas free Curcumin in much higher dose showed minimal improvement during compromised cardiac function. Targeted Curcumin treatment significantly lowered p53 expression and activation in diseased myocardium via inhibited interaction of p53 with p300-HAT. Thus attenuated acetylation of p53 facilitated p53 ubiquitination and reduced the apoptotic load in hypertrophied cardiomyocytes; thereby limiting cardiomyocytes' need to enter the regeneration cycle during hypertrophy. This study elucidates for the first time an efficient targeted delivery regimen for Curcumin and also attributes towards probable mechanistic insight into its therapeutic potential as a cardio-protective agent for regression of cardiac hypertrophy.

Cardioprotective role of P38 MAPK during myocardial infarction via parallel activation of α-crystallin B and Nrf2.

Myocardial infarction (MI) is defined as cardiac cell death due to prolonged ischemia. Although necrotic cell death was considered to be solely responsible for myocyte death during MI, it was recently revealed that apoptosis also plays its part in this death process. Our laboratory has recently shown that endoplasmic reticulum (ER) stress-induced apoptosis is the predominant route for apoptosis during MI and the conventional mitochondrial pathway is bypassed by activation of a small heat shock protein α-crystallin B (CRYAB). Since CRYAB is a direct target of P38 mitogen-activated protein kinase (MAPK) cascade, we were prompted to check the role of P38 MAPK in 20-week-old male Wister rats immediately after infarct formation. Interestingly, parallel activation of mitochondrial apoptotic pathway with an increase in ER stress-induced apoptotic load was observed along with decreased activation of CRYAB and Nrf2 (a pro-survival protein activated in response to ER stress) in MI rats treated with SB203580, a specific inhibitor of P38α and P38ß compared to the MI alone. As a cumulative effect, this inhibitor treatment also resulted in significant increase in the levels of caspase3 activity and TUNEL positivity, the end point apoptotic markers. Furthermore, SB203580-treated hypoxic adult cardiomyocytes showed formation of desmin aggregates which were previously associated with impaired cardiac function. Thus, this study shows for the first time the precise mechanism by which P38 MAPK plays a pro-survival role and confers protection of cardiomyocytes, during infarct formation.

Antiviral activity of baicalin against influenza virus H1N1-pdm09 is due to modulation of NS1-mediated cellular innate immune responses.

OBJECTIVES: Baicalin, a flavonoid, has been shown to have antiviral and anti-inflammatory activities, although the mechanism of action has been unknown. Therefore, attempts were made to analyse the mechanism behind the antiviral effects of baicalin using an influenza A virus (IAV) model in vitro and in vivo. METHODS: Baicalin's anti-influenza activity was elucidated (in vitro and in vivo) utilizing pandemic influenza strain A/H1N1/Eastern India/66/pdm09 (H1N1-pdm09). Anti-influenza activity was measured by plaque inhibition, fluorescent focus-forming units (ffu) and quantifying viral transcripts using quantitative real-time PCR following treatment with baicalin in a dose- and time-dependent manner. The role of the IAV non-structural protein 1 (NS1) gene in modulating host responses was measured by immunoblotting, co-immunoprecipitation and molecular docking. RESULTS: Baicalin treatment following IAV infection revealed up-regulation of interferon (IFN)-induced antiviral signalling and decreased phosphoinositide 3-kinase/Akt (PI3K/Akt) activation compared with infected, untreated controls. Baicalin exerts its antiviral effects by modulating the function of the IAV-encoded NS1 protein. NS1 has been shown to counteract cellular antiviral responses by down-regulating IFN induction and up-regulating PI3K/Akt signalling. Baicalin disrupted NS1-p85ß binding. Molecular docking predicted the binding site of baicalin in the RNA binding domain (RBD) of NS1. Site-directed mutagenesis within the RBD region of NS1 and the difference in the fluorescence quenching pattern of full-length NS1 and mutant NS1 proteins in the presence of baicalin confirmed the interaction of baicalin with the NS1 RBD. Amino acid residues 39-43 of the NS1 RBD were found to be crucial for the baicalin-NS1 interaction. CONCLUSIONS: Overall, this study highlights that baicalin exerts its anti-influenza virus activity by modulating viral protein NS1, resulting in up-regulation of IFN-induced antiviral signalling and a decrease in PI3K/Akt signalling in cells.

Bioinformatics analysis of hypoxia associated genes and inflammatory cytokine profiling in COPD-PH.

Pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) is associated with worse clinical outcomes and decreased survival rates. In absence of disease specific diagnostic/therapeutic targets and unclear pathophysiology, there is an urgent need for the identification of potential genetic/molecular markers and disease associated pathways. The present study aims to use a bioinformatics approach to identify and validate hypoxia-associated gene signatures in COPD-PH patients. Additionally, hypoxia-related inflammatory profile is also explored in these patients. Microarray dataset obtained from the Gene Expression Omnibus repository was used to identify differentially expressed genes (DEGs) in a hypoxic PH mice model. The top three hub genes identified were further validated in COPD-PH patients, with chemokine (C-X-C motif) ligand 9 (CXCL9) and CXCL12 showing significant changes in comparison to healthy controls. Furthermore, multiplexed analysis of 10 inflammatory cytokines, tumor necrosis factor alpha (TNF-α), transforming growth factor ß (TGF-ß), interleukin 1-beta (IL-1ß), IL-4, IL-5, IL-6, IL-13, IL-17, IL-18 and IL-21 was also performed. These markers showed significant changes in COPD-PH patients as compared to controls. They also exhibited the ability to differentially diagnose COPD-PH patients in comparison to COPD. Additionally, IL-6 and IL-17 showed significant positive correlation with systolic pulmonary artery pressure (sPAP). This study is the first report to assess the levels of CXCL9 and CXCL12 in COPD-PH patients and also explores their link with the inflammatory profile of these patients. Our findings could be extended to better understand the underlying disease mechanism and possibly used for tailoring therapies exclusive for the disease.

Inhibition of signal transducer and activator of transcription 3 (STAT3) attenuates interleukin-6 (IL-6)-induced collagen synthesis and resultant hypertrophy in rat heart.

IL-6 has been shown to play a major role in collagen up-regulation process during cardiac hypertrophy, although the precise mechanism is still not known. In this study we have analyzed the mechanism by which IL-6 modulates cardiac hypertrophy. For the in vitro model, IL-6-treated cultured cardiac fibroblasts were used, whereas the in vivo cardiac hypertrophy model was generated by renal artery ligation in adult male Wistar rats (Rattus norvegicus). During induction of hypertrophy, increased phosphorylation of STAT1, STAT3, MAPK, and ERK proteins was observed both in vitro and in vivo. Treatment of fibroblasts with specific inhibitors for STAT1 (fludarabine, 50 µM), STAT3 (S31-201, 10 µM), p38 MAPK (SB203580, 10 µM), and ERK1/2 (U0126, 10 µM) resulted in down-regulation of IL-6-induced phosphorylation of specific proteins; however, only S31-201 and SB203580 inhibited collagen biosynthesis. In ligated rats in vivo, only STAT3 inhibitors resulted in significant decrease in collagen synthesis and hypertrophy markers such as atrial natriuretic factor and ß-myosin heavy chain. In addition, decreased heart weight to body weight ratio and improved cardiac function as measured by echocardiography was evident in animals treated with STAT3 inhibitor or siRNA. Compared with IL-6 neutralization, more pronounced down-regulation of collagen synthesis and regression of hypertrophy was observed with STAT3 inhibition, suggesting that STAT3 is the major downstream signaling molecule and a potential therapeutic target for cardiac hypertrophy.

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.

Analysis of p53 and NF-κB signaling in modulating the cardiomyocyte fate during hypertrophy.

Cardiac hypertrophy leading to eventual heart failure is the most common cause of mortality throughout the world. The triggering mechanisms for cardiac hypertrophy are not clear but both apoptosis and cell proliferation have been reported in sections of failing hearts. In this study, we utilized both angiotensin II (AngII) treatment of cardiomyocytes and aortic ligation in rats (Rattus norvegicus, Wistar strain) for induction of hypertrophy to understand the cellular factors responsible for activation of apoptotic or anti-apoptotic pathway. Hypertrophy markers (ANF, ß-MHC), apoptotic proteins (Bax, Bad, Fas, p53, caspase-3, PARP), and anti-apoptotic or cell proliferation marker proteins (Bcl2, NF-κB, Ki-67) were induced significantly during hypertrophy, both in vitro as well as in vivo. Co-localization of both active caspase-3 and Ki-67 was observed in hypertrophied myocytes. p53 and NF-κBp65 binding to co-activator p300 was also increased in AngII treated myocytes. Inhibition of p53 resulted in downregulation of apoptosis, NF-κB activation, and NF-κB-p300 binding; however, NF-κB inhibition did not inhibit apoptosis or p53-p300 binding. Blocking of either p53 or NF-κB by specific inhibitors resulted in decrease in cell proliferation and hypertrophy markers, suggesting that p53 initially binds to p300 and then this complex recruits NF-κB. Thus, these results indicate the crucial role of p53 in regulating both apoptotic and cell proliferation during hypertrophy.

Rotavirus nonstructural protein 1 suppresses virus-induced cellular apoptosis to facilitate viral growth by activating the cell survival pathways during early stages of infection.

Following virus infection, one of the cellular responses to limit the virus spread is induction of apoptosis. In the present study, we report role of rotavirus nonstructural protein 1 (NSP1) in regulating apoptosis by activating prosurvival pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB (nuclear factor kappaB) during early hours of infections (2 to 8 hpi). The NSP1 mutant strain A5-16 induces weak and transient activation of Akt (protein kinase B) and p65 NF-kappaB compared to the isogenic wild-type strain A5-13 in MA104 or HT29 cells. The weak NF-kappaB promoter activity or Akt phosphorylation after A5-16 infection could be complemented in cells transfected with plasmid expressing NSP1 after infection with the rotavirus A5-16 strain. In cells either infected with A5-13 or transfected with pcD-NSP1, coimmunoprecipitation of NSP1 with phosphoinositide 3-kinase (PI3K) was observed, indicating that strong activation of PI3K/Akt could be due to its interaction with NSP1. In addition, after infection with same multiplicity of infection, A5-16 showed reduced number of viral particles compared to the A5-13 strain at the end of the replication cycle. A lower growth rate could be due to weak induction of PI3K/Akt and NF-kappaB, since the A5-13 strain also showed reduced growth in the presence of PI3K or NF-kappaB inhibitors. This effect was interferon independent; however, it was partly due to significantly higher caspase-3 activity, poly-ADP ribose polymerase (PARP) cleavage, and apoptosis during earlier stages of infection with the NSP1 mutant. Thus, our data suggest that NSP1 positively supports rotavirus growth by suppression of premature apoptosis for improved virus growth after infection.