PARP1 inhibition protects mice against Japanese encephalitis virus infection.

Japanese encephalitis (JE) is a vector-borne viral disease that causes acute encephalitis in children. Although vaccines have been developed against the JE virus (JEV), no effective antiviral therapy exists. Our study shows that inhibition of poly(ADP-ribose) polymerase 1 (PARP1), an NAD+-dependent (poly-ADP) ribosyl transferase, protects against JEV infection. Interestingly, PARP1 is critical for JEV pathogenesis in Neuro-2a cells and mice. Small molecular inhibitors of PARP1, olaparib, and 3-aminobenzamide (3-AB) significantly reduce clinical signs and viral load in the serum and brains of mice and improve survival. PARP1 inhibition confers protection against JEV infection by inhibiting autophagy. Mechanistically, upon JEV infection, PARP1 PARylates AKT and negatively affects its phosphorylation. In addition, PARP1 transcriptionally upregulates PTEN, the PIP3 phosphatase, negatively regulating AKT. PARP1-mediated AKT inactivation promotes autophagy and JEV pathogenesis by increasing the FoxO activity. Thus, our findings demonstrate PARP1 as a potential mediator of JEV pathogenesis that can be effectively targeted for treating JE.

Sirtuin 6 inhibition protects against glucocorticoid-induced skeletal muscle atrophy by regulating IGF/PI3K/AKT signaling.

Chronic activation of stress hormones such as glucocorticoids leads to skeletal muscle wasting in mammals. However, the molecular events that mediate glucocorticoid-induced muscle wasting are not well understood. Here, we show that SIRT6, a chromatin-associated deacetylase indirectly regulates glucocorticoid-induced muscle wasting by modulating IGF/PI3K/AKT signaling. Our results show that SIRT6 levels are increased during glucocorticoid-induced reduction of myotube size and during skeletal muscle atrophy in mice. Notably, overexpression of SIRT6 spontaneously decreases the size of primary myotubes in a cell-autonomous manner. On the other hand, SIRT6 depletion increases the diameter of myotubes and protects them against glucocorticoid-induced reduction in myotube size, which is associated with enhanced protein synthesis and repression of atrogenes. In line with this, we find that muscle-specific SIRT6 deficient mice are resistant to glucocorticoid-induced muscle wasting. Mechanistically, we find that SIRT6 deficiency hyperactivates IGF/PI3K/AKT signaling through c-Jun transcription factor-mediated increase in IGF2 expression. The increased activation, in turn, leads to nuclear exclusion and transcriptional repression of the FoxO transcription factor, a key activator of muscle atrophy. Further, we find that pharmacological inhibition of SIRT6 protects against glucocorticoid-induced muscle wasting in mice by regulating IGF/PI3K/AKT signaling implicating the role of SIRT6 in glucocorticoid-induced muscle atrophy.

Emerging roles of Sirtuin 2 in cardiovascular diseases.

Sirtuins are a family of NAD+ -dependent deacetylases implicated in a wide variety of age-associated pathologies, including cardiovascular disorders. Among the seven mammalian sirtuins, SIRT2 modulates various cellular processes through the deacetylation or deacylation of their target proteins. Notably, the levels of SIRT2 in the heart decline with age and other pathological conditions, leading to cardiovascular dysfunction. In the present review, we discuss the emerging roles of SIRT2 in cardiovascular dysfunction and heart failure associated with factors like age, hypertension, oxidative stress, and diabetes. We also discuss the potential of using inhibitors to study the unexplored role of SIRT2 in the heart. While SIRT2 undoubtedly plays a crucial role in the cardiovascular system, its functions are only beginning to be understood, making it an attractive candidate for further research in the field.

Isolation and Culture of Neonatal Murine Primary Cardiomyocytes.

The cardiomyocyte is the main cell type in the heart responsible for its contractile function. Culturing primary cardiomyocytes from mammalian sources to study their function remains challenging as they are terminally differentiated and cease to multiply soon after birth. The major technical hurdles associated with primary cardiomyocyte culture include attaining high yields, obtaining healthy/viable cells that show spontaneous contractions upon culture, and avoiding contamination by non-myocyte cardiac cell types such as fibroblasts and endothelial cells. The yield and the quality of the cardiomyocytes obtained are impacted by a variety of factors, such as the purity of the reagents, composition of the digestion mixture, the digestion conditions, and the temperature of the tissue during different steps of isolation. Here, we provide a simplified workflow to isolate, culture, and maintain neonatal primary cardiomyocytes from rats/mice in culture dishes, which can then be used to study, for instance, cardiac hypertrophy and drug-induced cardiotoxicity. © 2021 Wiley Periodicals LLC. Basic Protocol: Isolation and culture of primary cardiomyocytes from rat/mouse pups Support Protocol: Coating of tissue culture plates with extracellular matrix substrates for efficient cardiomyocyte attachment.

SIRT6 transcriptionally regulates fatty acid transport by suppressing PPARγ.

Pathological lipid accumulation is often associated with enhanced uptake of free fatty acids via specific transporters in cardiomyocytes. Here, we identify SIRT6 as a critical transcriptional regulator of fatty acid transporters in cardiomyocytes. We find that SIRT6 deficiency enhances the expression of fatty acid transporters, leading to enhanced fatty acid uptake and lipid accumulation. Interestingly, the haploinsufficiency of SIRT6 is sufficient to induce the expression of fatty acid transporters and cause lipid accumulation in murine hearts. Mechanistically, SIRT6 depletion enhances the occupancy of the transcription factor PPARγ on the promoters of critical fatty acid transporters without modulating the acetylation of histone 3 at Lys 9 and Lys 56. Notably, the binding of SIRT6 to the DNA-binding domain of PPARγ is critical for regulating the expression of fatty acid transporters in cardiomyocytes. Our data suggest exploiting SIRT6 as a potential therapeutic target for protecting the heart from metabolic diseases.

Role of FoxO transcription factors in aging-associated cardiovascular diseases.

Aging constitutes a major risk factor toward the development of cardiovascular diseases (CVDs). The aging heart undergoes several changes at the molecular, cellular and physiological levels, which diminishes its contractile function and weakens stress tolerance. Further, old age increases the exposure to risk factors such as hypertension, diabetes and hypercholesterolemia. Notably, research in the past decades have identified FoxO subfamily of the forkhead transcription factors as key players in regulating diverse cellular processes linked to cardiac aging and diseases. In the present chapter, we discuss the important role of FoxO in the development of various aging-associated cardiovascular complications such as cardiac hypertrophy, cardiac fibrosis, heart failure, vascular dysfunction, atherosclerosis, hypertension and myocardial ischemia. Besides, we will also discuss the role of FoxO in cardiometabolic alterations, autophagy and proteasomal degradation, which are implicated in aging-associated cardiac dysfunction.

Recapitulating pathophysiology of skeletal muscle diseases in vitro using primary mouse myoblasts on a nanofibrous platform.

Tissue engineering approaches are used to mimic the microenvironment of the skeletal muscle in vitro. However, the validation of a bioengineered muscle as a model to study diseases is inadequate. Here, we present polycaprolactone nanofibers as a robust platform that mimics cellular organization and recapitulates critical functions of the myotubes observed in vivo. We isolated myoblasts from mice following a simplified protocol and cultured them on aligned nanofibers. Myotubes grown on aligned nanofibers maintained alignment for 14 days and exhibited a time-dependent increase in levels of p-AKT upon insulin stimulation. Treatment with matrix-assisted integrin inhibitor led to reduction in p-AKT levels, underscoring the critical role of environment on the biological processes. We demonstrate the suitability of myotubes grown on nanofibrous platform to study corticosteroid-induced muscle degeneration. This study, thus, demonstrates that aligned nanofibers retain myotubes in culture for longer duration and recapitulate the functions of skeletal muscle under pathophysiological conditions.

Measuring Protein Synthesis in Cultured Cells and Mouse Tissues Using the Non-radioactive SUnSET Assay.

Changes in protein synthesis occur under diverse physiological and pathological conditions. For example, translation can increase in response to growth signals or decrease in response to pathological states. Such changes have traditionally been measured by tracking the incorporation of radiolabeled amino acids. However, use of radioactivity is increasingly disfavored, and a simple and efficient puromycin-based, non-radioactive method called the SUnSET assay has gained popularity for measuring protein synthesis in diverse cell types and tissues. Here, we describe the principles, procedures, and troubleshooting steps for measuring protein synthesis using the SUnSET assay in cultured cells and mouse tissues. © 2020 Wiley Periodicals LLC Basic Protocol 1: Measuring protein synthesis in cultured cells by western blotting Support Protocol 1: Ponceau staining Support Protocol 2: Testing the specificity of the anti-puromycin antibody Basic Protocol 2: Measuring protein synthesis in cultured cells by immunofluorescence Basic Protocol 3: Measuring protein synthesis in mouse tissues by western blotting.

Preparation and characterization of bioactive silk fibroin/paramylon blend films for chronic wound healing.

A major challenge in the chronic wound healing is a self-care wound dressing. The problem in chronic wounds is the lack of functional extracellular matrix to stimulate healing. The extracellular matrix is a network of proteins and polysaccharides that serves as physical framework and provides regulatory signals. The primary goal is to mimic the property of the extracellular matrix with Silk Fibroin and Paramylon to produce a novel bioactive film. Silk fibroin is a fibrous protein having unique mechanical properties and tunable biodegradability that makes it favourable for tailoring to specific applications. Paramylon is a ß-glucan that possess a broad spectrum of biological activity that enhances the immune response. Silk fibroin/paramylon blend films have been prepared by solution casting and were examined for structural, thermal, mechanical and other physical properties for knowing its transformation for chronic wound healing. It was observed that the fibroin films shows high thermal stability, high hydrophobicity, high stiffness value whereas the paramylon film show good water absorption property and both fibroin and paramylon shows good blood compatibility and was non-toxic as well. It also confirms that the blended films showed a combination of fibroin and paramylon property at different concentrations without any chemical interaction.

Sirtuin 6 deficiency transcriptionally up-regulates TGF-ß signaling and induces fibrosis in mice.

Caloric restriction has been associated with increased life span and reduced aging-related disorders and reduces fibrosis in several diseases. Fibrosis is characterized by deposition of excess fibrous material in tissues and organs and is caused by aging, chronic stress, injury, or disease. Myofibroblasts are fibroblast-like cells that secrete high levels of extracellular matrix proteins, resulting in fibrosis. Histological studies have identified many-fold increases of myofibroblasts in aged organs where myofibroblasts are constantly generated from resident tissue fibroblasts and other cell types. However, it remains unclear how aging increases the generation of myofibroblasts. Here, using mouse models and biochemical assays, we show that sirtuin 6 (SIRT6) deficiency plays a major role in aging-associated transformation of fibroblasts to myofibroblasts, resulting in tissue fibrosis. Our findings suggest that SIRT6-deficient fibroblasts transform spontaneously to myofibroblasts through hyperactivation of transforming growth factor ß (TGF-ß) signaling in a cell-autonomous manner. Importantly, we noted that SIRT6 haploinsufficiency is sufficient for enhancing myofibroblast generation, leading to multiorgan fibrosis and cardiac dysfunction in mice during aging. Mechanistically, SIRT6 bound to and repressed the expression of key TGF-ß signaling genes by deacetylating SMAD family member 3 (SMAD3) and Lys-9 and Lys-56 in histone 3. SIRT6 binding to the promoters of genes in the TGF-ß signaling pathway decreased significantly with age and was accompanied by increased binding of SMAD3 to these promoters. Our findings reveal that SIRT6 may be a potential candidate for modulating TGF-ß signaling to reduce multiorgan fibrosis during aging and fibrosis-associated diseases.

SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Sirtuin 6 mediated stem cell cardiomyogenesis on protein coated nanofibrous scaffolds.

The cellular niche provides combination of biomolecular and biophysical cues to control stem cell fate. Three-dimensional (3D) aligned nanofibrous scaffolds can effectively augment stem cell cardiomyogenesis. This work aims to understand the role of biomolecular signals from extracellular matrix (ECM) proteins and leverage them to further promote cardiomyogenesis on nanofibrous scaffolds. Human mesenchymal stem cells (hMSCs) were cultured on 3D aligned polycaprolactone scaffolds coated with different ECM proteins. Among multiple coatings tested, collagen coated fibers were most effective in promoting cardiomyogenesis as determined from increased expression of cardiac biomarkers and intracellular calcium flux. At molecular level, enhanced differentiation on collagen coated fibers was associated with an increased level of sirtuin 6 (SIRT6). Depletion of SIRT6 using siRNA attenuated the differentiation process through activation of Wnt signaling pathway. This study, thus, demonstrates that protein coated scaffolds can augment cardiomyogenic differentiation of stem cells through a combination of topographical and biomolecular signals.

Systematic evaluation of the adaptability of the non-radioactive SUnSET assay to measure cardiac protein synthesis.

Heart is a dynamic organ that undergoes remodeling in response to both physiological and pathological stimuli. One of the fundamental cellular processes that facilitates changes in the size and shape of this muscular organ is the protein synthesis. Traditionally changes in cardiac protein synthesis levels were measured by radiolabeled tracers. However, these methods are often cumbersome and suffer from radioactive risk. Recently a nonradioactive method for detecting protein synthesis under in vitro conditions called the Surface Sensing of Translation (SUnSET) was described in cell lines of mouse dendrites and T cells. In this work, we provide multiple lines of evidence that the SUnSET assay can be applied to reliably detect changes in protein synthesis both in isolated neonatal primary cardiomyocytes and heart. We successfully tracked the changes in protein synthesis by western blotting as well as immunohistochemical variants of the SUnSET assay. Applying the SUnSET assay, we measured the cardiac protein synthesis during the different ages of mice. Further, we successfully tracked the increase in cardiac protein synthesis during different stages of a well-established model for pathological hypertrophy. Overall, we propose SUnSET assay as a simple, reliable and robust method to measure protein synthesis in the cardiac milieu.

SIRT2 deacetylase represses NFAT transcription factor to maintain cardiac homeostasis.

Heart failure is an aging-associated disease that is the leading cause of death worldwide. Sirtuin family members have been largely studied in the context of aging and aging-associated diseases. Sirtuin 2 (SIRT2) is a cytoplasmic protein in the family of sirtuins that are NAD+-dependent class III histone deacetylases. In this work, we studied the role of SIRT2 in regulating nuclear factor of activated T-cells (NFAT) transcription factor and the development of cardiac hypertrophy. Confocal microscopy analysis indicated that SIRT2 is localized in the cytoplasm of cardiomyocytes and SIRT2 levels are reduced during pathological hypertrophy of the heart. SIRT2-deficient mice develop spontaneous pathological cardiac hypertrophy, remodeling, fibrosis, and dysfunction in an age-dependent manner. Moreover, young SIRT2-deficient mice develop exacerbated agonist-induced hypertrophy. In contrast, SIRT2 overexpression attenuated agonist-induced cardiac hypertrophy in cardiomyocytes in a cell-autonomous manner. Mechanistically, SIRT2 binds to and deacetylates NFATc2 transcription factor. SIRT2 deficiency stabilizes NFATc2 and enhances nuclear localization of NFATc2, resulting in increased transcription activity. Our results suggest that inhibition of NFAT rescues the cardiac dysfunction in SIRT2-deficient mice. Thus, our study establishes SIRT2 as a novel endogenous negative regulator of NFAT transcription factor.

A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces.

OBJECTIVE: We aim to develop a simple, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which can be used for studying cardiac hypertrophy in vitro. METHODS: Keratin was extracted from human hair and applied as nanoscale coating onto the culture dishes. Physical parameters such as surface morphology and roughness of the coating were studied by SEM and AFM. Cardiomyocyte specific markers were assessed by immunofluorescence. Signaling pathways activated in hypertrophy were analyzed by western blotting and changes in the expression of fetal genes were analyzed by qPCR. The changes in the calcium fluxes were observed microscopically using Fluo-4. RESULTS: Keratin coated surfaces displayed a uniform coating and comparable roughness across dishes. Our optimized protocol for isolating cardiomyocytes yielded up to ~106 cells per heart. Characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contractions on keratin-coated substrates similar to fibronectin-coated surfaces. Phenylephrine (PE) treated cardiomyocytes grown on keratin-coated substrates exhibited increased cell size, sarcomere organization and perinuclear ANP expression indicating the development of cardiac hypertrophy. In addition, we observed increased activation of Akt and ERK pathways, induction of the fetal genes and increased protein synthesis upon PE treatment, which are characteristics of cardiomyocyte hypertrophy. The protocol was extended to mouse cardiomyocytes and found to show similar results upon examination. CONCLUSION: We demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.

Keratin mediated attachment of stem cells to augment cardiomyogenic lineage commitment.

The objective of this work was to develop a simple surface modification technique using keratin derived from human hair for efficient cardiomyogenic lineage commitment of human mesenchymal stem cells (hMSCs). Keratin was extracted from discarded human hair containing both the acidic and basic components along with the heterodimers. The extracted keratin was adsorbed to conventional tissue culture polystyrene surfaces at different concentration. Keratin solution of 500µg/ml yielded a well coated layer of 12±1nm thickness with minimal agglomeration. The keratin coated surfaces promoted cell attachment and proliferation. Large increases in the mRNA expression of known cardiomyocyte genes such as cardiac actinin, cardiac troponin and ß-myosin heavy chain were observed. Immunostaining revealed increased expression of sarcomeric α-actinin and tropomyosin whereas Western blots confirmed higher expression of tropomyosin and myocyte enhancer factor 2C in cells on the keratin coated surface than on the non-coated surface. Keratin promoted DNA demethylation of the Atp2a2 and Nkx2.5 genes thereby elucidating the importance of epigenetic changes as a possible molecular mechanism underlying the increased differentiation. A global gene expression analysis revealed a significant alteration in the expression of genes involved in pathways associated in cardiomyogenic commitment including cytokine and chemokine signaling, cell-cell and cell-matrix interactions, Wnt signaling, MAPK signaling, TGF-ß signaling and FGF signaling pathways among others. Thus, adsorption of keratin offers a facile and affordable yet potent route for inducing cardiomyogenic lineage commitment of stem cells with important implications in developing xeno-free strategies in cardiovascular regenerative medicine.

Synergistic antimicrobial profiling of violacein with commercial antibiotics against pathogenic micro-organisms.

CONTEXT: Chromobacterium violaceum Bergonzini (Neisseriaceae), a Gram-negative bacterium, secretes a spectacular pigment called violacein. Violacein is a quorum-sensing metabolite and is also an active antimicrobial, anticancer agent. However, its efficiency as a potential drug, alone or in synergy with other active principles, has not being completely exploited. With the advent of different multi-drug resistant strains, it becomes essential to find a new natural product(s) that could be effectively used as a therapeutic agent. OBJECTIVE: This work focused on the extraction of violacein from an isolated strain of C. violaceum and determined the combinatory effect of violacein with commercial antibiotics against various pathogens. MATERIALS AND METHODS: Violacein production was optimized and was later extracted using ethanol and characterized by liquid chromatography-mass spectrometry and infrared spectroscopy. Then, individual minimum inhibitory concentration (MIC) values for each of the antibiotics were determined followed by violacein-commercial antibiotics (1:1) combinations, tested at different concentrations starting from 500 to 1 µg/ml against major pathogens. RESULTS AND DISCUSSION: The individual MIC data for violacein was found to be 5.7 µg/ml (Staphylococcus aureus), 15.6 µg/ml (Klebsiella pneumoniae), 18.5 µg/ml (Pseudomonas aeruginosa), 20.0 µg/ml (Vibrio cholerae) and 5.7 µg/ml (Salmonella typhi). Violacein-gentamicin and violacein-cefadroxil combinations had MIC of 1.0 µg/ml against S. aureus. Most violacein-macrolide and violacein--aminoglycoside class combinations revealed fractional inhibitory concentration indices (FICI) of <0.5, thus exhibiting synergism. Furthermore, violacein-azithromycin and violacein-kanamycin combination, exhibited significant synergy (FICI-0.3) against S. typhi. CONCLUSION: Violacein works synergistically with most commercial antibiotics and could be used as drug in combination with other antimicrobial agents.