Nagalingam R. Sundaresan

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

OBJECTIVEnWe 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.nnnMETHODSnKeratin 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.nnnRESULTSnKeratin 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.nnnCONCLUSIONnWe demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.

Molecular profiling of sepsis in mice using Fourier Transform Infrared Microspectroscopy

Sepsis is a life threatening condition resulting from a high burden of infection. It is a major health care problem and associated with inflammation, organ dysfunction and significant mortality. However, proper understanding and delineating the changes that occur during this complex condition remains a challenge. A comparative study involving intra-peritoneal injection of BALB/c mice with Salmonella Typhimurium (infection), lipopolysaccharide (endotoxic shock) or thioglycollate (sterile peritonitis) was performed. The changes in organs and sera were profiled using immunological assays and Fourier Transform Infrared (FTIR) micro-spectroscopy. There is a rapid rise in inflammatory cytokines accompanied with lowering of temperature, respiratory rate and glucose amounts in mice injected with S. Typhimurium or lipopolysaccharide. FTIR identifies distinct changes in liver and sera: decrease in glycogen and protein/lipid ratio and increase in DNA and cholesteryl esters. These changes were distinct from the pattern observed in mice treated with thioglycollate and the differences in the data obtained between the three models are discussed. The combination of FTIR spectroscopy and other biomarkers will be valuable in monitoring molecular changes during sepsis.

SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart

Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/− mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1α. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/− hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1‐mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis.

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 β-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-β 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.

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.

Nitric oxide synthase 2 enhances the survival of mice during Salmonella Typhimurium infection-induced sepsis by increasing reactive oxygen species, inflammatory cytokines and recruitment of neutrophils to the peritoneal cavity

Sepsis, a leading cause of death in intensive care units, is primarily caused due to an exaggerated immune response. The hyperactive inflammatory response mediated by immune cells against infectious organisms and their toxins results in host cell death and tissue damage, the hallmarks of septic shock. Therefore, molecules that modulate inflammatory responses are attractive therapeutic targets for sepsis. Nitric oxide (NO) is a signaling molecule, which is implicated in regulating diverse immune functions. Although, the protective roles of NO in infectious diseases are well documented, its importance in sepsis is controversial. In the present study, the effects of intra-peritoneal injection of mice with Salmonella Typhimurium, a Gram-negative intracellular pathogen, were studied which leads to a rapid upregulation of serum cytokines and infiltration of neutrophils to the peritoneal cavity. Surprisingly, the induction of inflammatory cytokines and chemokines, e.g. IL6 and CCL2, and the infiltration of neutrophils into the peritoneal cavity are mitigated in mice lacking Nitric oxide synthase 2 (NOS2). The reduced inflammatory response in Nos2-/- mice is accompanied by greater bacterial burden in the peritoneal cavity, lower thymic atrophy, higher liver damage and cardiovascular dysfunction followed by decreased survival. However, no significant differences are observed in other responses between C57BL/6 wild type (WT) and Nos2-/- mice: induction of glucocorticoids, phagocytic ability and apoptosis of peritoneal cells. This study clearly highlights the NOS2-dependent and -independent responses in this mouse model of peritonitis induced sepsis. Importantly, pre-treatment of Nos2-/- mice with DETA-NO, a NO donor, upon infection, restores neutrophil recruitment, reduces bacterial numbers in the peritoneal cavity, improves liver and cardio-vascular function and enhances survival. Interestingly, DETA-NO treatment does not significantly increase the survival of infected WT mice. The implications of these results and the complex roles of NO as a target molecule during sepsis are discussed.

Subcutaneous Ehrlich Ascites Carcinoma mice model for studying cancer-induced cardiomyopathy

Cardiomyopathy is one of the characteristic features of cancer. In this study, we establish a suitable model to study breast cancer-induced cardiomyopathy in mice. We used Ehrlich Ascites Carcinoma cells to induce subcutaneous tumor in 129/SvJ mice and studied its effect on heart function. In Ehrlich Ascites Carcinoma bearing mice, we found significant reduction in left ventricle wall thickness, ejection fraction, and fractional shortening increase in left ventricle internal diameter. We found higher muscle atrophy, degeneration, fibrosis, expression of cell-adhesion molecules and cell death in tumor-bearing mice hearts. As observed in cancer patients, we found that mTOR, a key signalling molecule responsible for maintaining cell growth and autophagy was suppressed in this model. Tumor bearing mice hearts show increased expression and nuclear localization of TFEB and FoxO3a transcription factors, which are involved in the upregulation of muscle atrophy genes, lysosomal biogenesis genes and autophagy genes. We propose that Ehrlich Ascites Carcinoma induced tumor can be used as a model to identify potential therapeutic targets for the treatment of heart failure in patients suffering from cancer-induced cardiomyopathy. This model can also be used to test the adverse consequences of cancer chemotherapy in heart.

Elucidating molecular events underlying topography mediated cardiomyogenesis of stem cells on 3D nanofibrous scaffolds

Toward engineering a cardiac patch, the objective of this work was to assess stem cell response to a three-dimensional (3D) nanofibrous scaffold and probe the underlying molecular mechanisms including both cell signaling and epigenetic changes. Cardiomyogenesis of human mesenchymal stem cells (hMSCs) in 3D poly(ε-caprolactone) (PCL) nanofibers and macroporous scaffolds was compared with two-dimensional (2D) PCL films. In addition, nanofiber mats of PCL and its blend with gelatin (PCL-Gel) were prepared with fibers of random or unidirectional alignment to assess the roles of topography (fibrous architecture and its alignment) and biochemical cue (cell-adhesive sites) in directing cell functions. Cells on 3D random nanofibers, exhibited elevated expression of known cardiac markers such as cardiac actinin, cardiac troponin and β-myocardial heavy chain compared to cells on 2D films suggesting enhanced differentiation that was further accentuated on the aligned fibers. 3D macroporous scaffolds did not enhance the cardiomyogenic differentiation. However, minimal differences were noted between cells on PCL and PCL-Gel fibers, irrespective of alignment. Co-culture with neonatal rat cardiomyocytes induced beating in the differentiated cells. The use of small molecule inhibitors revealed that cytoskeletal elements F-actin, microtubules and downstream ROCK protein are essential for the cardiomyogenesis of hMSCs on the nanofibers. The activation of ERK, AKT and mTOR was observed during cardiomyogenesis. Interestingly, enhanced differentiation on the aligned nanofibers was associated with increased level of the histone deacytelase SIRT6 and decreased level of the acetylated histone H3K9 suggesting a role for epigenetic regulation. This study demonstrates that aligned nanofibrous scaffolds augment cardiomyogenic differentiation wherein topography plays a critical role in driving stem cell function. In addition, this study offers insight into molecular pathways driving the cellular response.

SIRT2 regulates oxidative stress-induced cell death through deacetylation of c-Jun NH2-terminal kinase

Abstractc-Jun NH2-terminal kinases (JNKs) are responsive to stress stimuli and their activation regulate key cellular functions, including cell survival, growth, differentiation and aging. Previous studies demonstrate that activation of JNK requires dual phosphorylation by the mitogen-activated protein kinase kinases. However, other post-translational mechanisms involved in regulating the activity of JNK have been poorly understood. In this work, we studied the functional significance of reversible lysine acetylation in regulating the kinase activity of JNK. We found that the acetyl transferase p300 binds to, acetylates and inhibits kinase activity of JNK. Using tandem mass spectrometry, molecular modelling and molecular dynamics simulations, we found that acetylation of JNKxa0at Lys153 would hinder the stable interactions of the negatively charged phosphates and prevent the adenosine binding to JNK. Our screening for the deacetylases found SIRT2 as a deacetylase for JNK. Mechanistically, SIRT2-dependent deacetylation enhances ATP binding and enzymatic activity of JNK towards c-Jun. Furthermore, SIRT2-mediated deacetylation favours the phosphorylation of JNK by MKK4, an upstream kinase. Our results indicate that deacetylation of JNK by SIRT2 promotes oxidative stress-induced cell death. Conversely, SIRT2 inhibition attenuates H2O2-mediated cell death in HeLa cells. SIRT2-deficient (SIRT2-KO) mice exhibit increased acetylationxa0of JNK, which is associated with markedly reduced catalytic activity of JNK in the liver. Interestingly, SIRT2-KO mice were resistant to acetaminophen-induced liver toxicity. SIRT2-KO mice show lower cell death, minimal degenerative changes, improved liver function and survival following acetaminophen treatment. Overall, our work identifies SIRT2-mediated deacetylation of JNK as a critical regulator of cell survival during oxidative stress.

SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

Glycogen synthase kinase 3 (GSK3) is a critical regulator of diverse cellular functions involved in the maintenance of structure and function. Enzymatic activity of GSK3 is inhibited by N-terminal serine phosphorylation. However, alternate post-translational mechanism(s) responsible for GSK3 inactivation are not characterized. Here, we report that GSK3α and GSK3β are acetylated at Lys246 and Lys183, respectively. Molecular modeling and/or molecular dynamics simulations indicate that acetylation of GSK3 isoforms would hinder both the adenosine binding and prevent stable interactions of the negatively charged phosphates. We found that SIRT2 deacetylates GSK3β, and thus enhances its binding to ATP. Interestingly, the reduced activity of GSK3β is associated with lysine acetylation, but not with phosphorylation at Ser9 in hearts of SIRT2-deficient mice. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2 in cardiomyocytes. Overall, our study identified lysine acetylation as a novel post-translational modification regulating GSK3 activity.