Skeletal muscle-specific over-expression of the nuclear sirtuin SIRT6 blocks cancer-associated cachexia by regulating multiple targets.

BACKGROUND: During cancer cachexia, cytokines released from tumour cells can alter body's metabolism, which can lead to onset of this disease process. Biological basis of cachexia is multifactorial; hence, it is important to identify and modulate multiple targets to curtail the process of cachexia. Previously, we reported that the nuclear sirtuin, SIRT6, blocks expression of myostatin, a negative regulator of muscle growth, through modulation of the NF-κB signalling. This study was undertaken to test whether muscle-specific over-expression of SIRT6 can block the cancer-associated muscle wasting in vivo and to identify additional relevant targets of SIRT6, which can explain its ability to maintain muscle health. METHODS: We generated a skeletal muscle-specific SIRT6 over-expressing transgenic mouse line (Sk.T6Tg) expressing SIRT6 at a moderate (two-fold to four-fold) level, compared with its control littermates. To generate a cancer-cachexia model, B16F10 mouse melanoma cells were injected subcutaneously in the flanks of mice. Gastrocnemius muscle tissues from non-tumour and tumour controls and Sk.T6Tg mice (n = 5-20) were analysed by histology, immunoblotting, and RT-qPCR. Plasma samples of mice were evaluated using cytokine arrays and ELISA in both non-tumour and tumour conditions. RESULTS: Our results demonstrate dual benefits of muscle-specific moderate over-expression of SIRT6 in a mouse model of cancer-cachexia. In tumour-bearing mice, SIRT6 over-expression preserved muscle weight (P < 0.001) and fibre size (P < 0.005) as well as suppressed tumour growth (P < 0.05). SIRT6 over-expression significantly reduced myostatin expression and plasma free fatty acids levels but maintained plasma insulin levels in tumour-bearing mice. These positive effects of SIRT6 were associated with downregulation of the circulatory chemokine, CXCL10, and the myokine, WNT4. SIRT6 also upregulated expression of GLUT4, the major glucose transporter in the skeletal muscle. These results for the first time demonstrate that SIRT6 regulates multiple targets to limit tumour growth and cancer-associated muscle atrophy. CONCLUSION: Given the multifactorial nature of cachexia, SIRT6, which concurrently controls multiple pathways, can be a valuable therapeutic target to overcome this debilitating syndrome.

Cellular mechanisms promoting cachexia and how they are opposed by sirtuins 1.

Many chronic diseases are associated with unintentional loss of body weight, which is termed "cachexia". Cachexia is a complex multifactorial syndrome associated with the underlying primary disease, and characterized by loss of skeletal muscle with or without loss of fat tissue. Patients with cachexia face dire symptoms like dyspnea, fatigue, edema, exercise intolerance, and low responsiveness to medical therapy, which worsen quality of life. Because cachexia is not a stand-alone disorder, treating primary disease - such as cancer - takes precedence for the physician, and it remains mostly a neglected illness. Existing clinical trials have demonstrated limited success mostly because of their monotherapeutic approach and late detection of the syndrome. To conquer cachexia, it is essential to identify as many molecular targets as possible using the latest technologies we have at our disposal. In this review, we have discussed different aspects of cachexia, which include various disease settings, active molecular pathways, and recent novel advances made in this field to understand consequences of this illness. We also discuss roles of the sirtuins, the NAD+-dependent lysine deacetylases, microRNAs, certain dietary options, and epigenetic drugs as potential approaches, which can be used to tackle cachexia as early as possible in its course.

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 GSK3ß 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 GSK3ß, and thus enhances its binding to ATP. Interestingly, the reduced activity of GSK3ß 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.

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

c-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 JNK at 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 acetylation of 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.

The histone deacetylase SIRT6 blocks myostatin expression and development of muscle atrophy.

Muscle wasting, also known as cachexia, is associated with many chronic diseases, which worsens prognosis of primary illness leading to enhanced mortality. Molecular basis of this metabolic syndrome is not yet completely understood. SIRT6 is a chromatin-bound member of the sirtuin family, implicated in regulating many cellular processes, ranging from metabolism, DNA repair to aging. SIRT6 knockout (SIRT6-KO) mice display loss of muscle, fat and bone density, typical characteristics of cachexia. Here we report that SIRT6 depletion in cardiac as well as skeletal muscle cells promotes myostatin (Mstn) expression. We also observed upregulation of other factors implicated in muscle atrophy, such as angiotensin-II, activin and Acvr2b, in SIRT6 depleted cells. SIRT6-KO mice showed degenerated skeletal muscle phenotype with significant fibrosis, an effect consistent with increased levels of Mstn. Additionally, we observed that in an in vivo model of cancer cachexia, Mstn expression coupled with downregulation of SIRT6. Furthermore, SIRT6 overexpression downregulated the cytokine (TNFα-IFNγ)-induced Mstn expression in C2C12 cells, and promoted myogenesis. From the ChIP assay, we found that SIRT6 controls Mstn expression by attenuating NF-κB binding to the Mstn promoter. Together, these data suggest a novel role for SIRT6 in maintaining muscle mass by controlling expression of atrophic factors like Mstn and activin.

Honokiol, an activator of Sirtuin-3 (SIRT3) preserves mitochondria and protects the heart from doxorubicin-induced cardiomyopathy in mice.

Doxorubicin is the chemotherapeutic drug of choice for a wide variety of cancers, and cardiotoxicity is one of the major side effects of doxorubicin treatment. One of the main cellular targets of doxorubicin in the heart is mitochondria. Mitochondrial sirtuin, SIRT3 has been shown to protect against doxorubicin-induced cardiotoxicity. We have recently identified honokiol (HKL) as an activator of SIRT3, which protects the heart from developing pressure overload hypertrophy. Here, we show that HKL-mediated activation of SIRT3 also protects the heart from doxorubicin-induced cardiac damage without compromising the tumor killing potential of doxorubicin. Doxorubicin-induced cardiotoxicity is associated with increased ROS production and consequent fragmentation of mitochondria and cell death. HKL-mediated activation of SIRT3 prevented Doxorubicin induced ROS production, mitochondrial damage and cell death in rat neonatal cardiomyocytes. HKL also promoted mitochondrial fusion. We also show that treatment with HKL blocked doxorubicin-induced cardiac toxicity in mice. This was associated with reduced mitochondrial DNA damage and improved mitochondrial function. Furthermore, treatments of mice, bearing prostrate tumor-xenografts, with HKL and doxorubicin showed inhibition of tumor growth with significantly reduced cardiac toxicity. Our results suggest that HKL-mediated activation of SIRT3 protects the heart from doxorubicin-induced cardiotoxicity and represents a potentially novel adjunct for chemotherapy treatments.

SIRT3 blocks myofibroblast differentiation and pulmonary fibrosis by preventing mitochondrial DNA damage.

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic diseases. Transforming growth factor-ß1 (TGF-ß1) is a powerful inducer of myofibroblast differentiation and is implicated in pathogenesis of tissue fibrosis. This study was undertaken to determine the role of mitochondrial deacetylase SIRT3 in TGF-ß1-induced myofibroblast differentiation in vitro and lung fibrosis in vivo. Treatment of human lung fibroblasts with TGF-ß1 resulted in increased expression of fibrosis markers, smooth muscle α-actin (α-SMA), collagen-1, and fibronectin. TGF-ß1 treatment also caused depletion of endogenous SIRT3, which paralleled with increased production of reactive oxygen species (ROS), DNA damage, and subsequent reduction in levels of 8-oxoguanine DNA glycosylase (OGG1), an enzyme that hydrolyzes oxidized guanine (8-oxo-dG) and thus protects DNA from oxidative damage. Overexpression of SIRT3 by adenovirus-mediated transduction reversed the effects of TGF-ß1 on ROS production and mitochondrial DNA damage and inhibited TGF-ß1-induced myofibroblast differentiation. To determine the antifibrotic role of SIRT3 in vivo, we used the bleomycin-induced mouse model of pulmonary fibrosis. Compared with wild-type controls, Sirt3-knockout mice showed exacerbated fibrosis after intratracheal instillation of bleomycin. Increased lung fibrosis was associated with decreased levels of OGG1 and concomitant accumulation of 8-oxo-dG and increased mitochondrial DNA damage. In contrast, the transgenic mice with whole body Sirt3 overexpression were protected from bleomycin-induced mtDNA damage and development of lung fibrosis. These data demonstrate a critical role of SIRT3 in the control of myofibroblast differentiation and lung fibrosis.

Sirt3 protects mitochondrial DNA damage and blocks the development of doxorubicin-induced cardiomyopathy in mice.

Doxorubicin (Doxo) is a chemotherapeutic drug widely used to treat variety of cancers. One of the most serious side effects of Doxo is its dose-dependent and delayed toxicity to the heart. Doxo is known to induce cardiac mitochondrial damage. Recently, the mitochondrial sirtuin SIRT3 has been shown to protect mitochondria from oxidative stress. Here we show that overexpression of SIRT3 protects the heart from toxicity of Doxo by preventing the drug-induced mitochondrial DNA (mtDNA) damage. Doxo treatment caused depletion of Sirt3 levels both in primary cultures of cardiomyocytes and in mouse hearts, which led to massive acetylation of mitochondrial proteins. Doxo-induced toxicity to cardiomyocytes was associated with increased reactive oxygen species (ROS) production, mitochondrial fragmentation, and cell death. Overexpression of SIRT3 helped to attenuate Doxo-induced ROS levels and cardiomyocyte death. Sirt3 knockout (Sirt3.KO) mice could not endure the full dose of Doxo treatment, developed exacerbated cardiac hypertrophy, and died during the course of treatment, whereas Sirt3 transgenic (Sirt3.tg) mice were protected against Doxo-induced cardiotoxicity. Along with Sirt3, we also observed a concomitant decrease in levels of oxoguanine-DNA glycosylase-1 (OGG1), a major DNA glycosylase that hydrolyzes oxidized-guanine (8-oxo-dG) to guanine. Depletion of OGG1 levels was associated with increased mtDNA damage. Sirt3.KO mice and Doxo-treated mice showed increased 8-oxo-dG adducts in DNA and corresponding increase in mtDNA damage, whereas, 8-oxo-dG adducts and mtDNA damage were markedly reduced in Sirt3 overexpressing transgenic mice hearts. These results thus demonstrated that Sirt3 activation protects the heart from Doxo-induced cardiotoxicity by maintaining OGG1 levels and protecting mitochondria from DNA damage.

In situ constructive myocardial remodeling of extracellular matrix patch enhanced with controlled growth factor release.

OBJECTIVE: In an effort to expand treatment for advanced heart failure, we sought to develop a tissue-engineered cardiac patch for constructive and functional in situ myocardial regeneration. METHODS: An extracellular matrix patch derived from porcine small intestine submucosa was incorporated with a controlled release of basic fibroblast growth factor. The patch was surgically implanted into the porcine right ventricle (group B, n = 5). Untreated extracellular matrix (group U) and Dacron (group D) patches served as control (n = 5/group). Cardiovascular magnetic resonance was performed in all 3 groups 60 days postsurgery to evaluate regional contractility with peak longitudinal strain, perfusion with relative maximum upslope, and extent of fibrosis/edema with extracellular volume fraction. Electrophysiologic-anatomic mapping was performed in group B. Histology and quantitative reverse transcription-polymerase chain reaction were performed for further tissue characterization. RESULTS: Cardiovascular magnetic resonance-derived parameters were significantly better in group B compared with groups U and D (strain: group B = -16.6% ± 1.8%, group U = -14.7% ± 1.2%, group D = -9.0% ± 1.5%, P < .001; upslope: group B = 13.7% ± 1.1%, group U = 10.8% ± 1.3%, group D = 6.4% ± 1.8%, P < .001; extracellular volume: group B = 45% ± 7%, group U = 54% ± 10%, group D = 70% ± 10%, P = .003). Histology in group B showed a homogenous distribution of host cells, including tropomyosin and α-sarcomeric actinin-positive maturing cardiomyocytes. Group B demonstrated the greatest degree of vasculogenesis as determined by capillary density analysis (group B = 19.5 ± 6.2/mm(3), group U = 12.7 ± 2.5/mm(3), group D = 6.9 ± 3.7/mm(3), P < .001). Quantitative reverse transcription-polymerase chain reaction supported the histologic findings. Electrophysiologic-anatomic mapping in group B indicated positive electrical conductivity in the patch area. CONCLUSIONS: The extracellular matrix patch enhanced with controlled release of fibroblast growth factor facilitated in situ constructive repopulation of the host cells, including cardiomyocyte and functional regeneration, increased regional contractility and tissue perfusion, and positive electrical activity in a porcine preparation.

Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3.

Honokiol (HKL) is a natural biphenolic compound derived from the bark of magnolia trees with anti-inflammatory, anti-oxidative, anti-tumour and neuroprotective properties. Here we show that HKL blocks agonist-induced and pressure overload-mediated, cardiac hypertrophic responses, and ameliorates pre-existing cardiac hypertrophy, in mice. Our data suggest that the anti-hypertrophic effects of HKL depend on activation of the deacetylase Sirt3. We demonstrate that HKL is present in mitochondria, enhances Sirt3 expression nearly twofold and suggest that HKL may bind to Sirt3 to further increase its activity. Increased Sirt3 activity is associated with reduced acetylation of mitochondrial Sirt3 substrates, MnSOD and oligomycin-sensitivity conferring protein (OSCP). HKL-treatment increases mitochondrial rate of oxygen consumption and reduces ROS synthesis in wild type, but not in Sirt3-KO cells. Moreover, HKL-treatment blocks cardiac fibroblast proliferation and differentiation to myofibroblasts in a Sirt3-dependent manner. These results suggest that HKL is a pharmacological activator of Sirt3 capable of blocking, and even reversing, the cardiac hypertrophic response.

Posttranslational modification of Sirt6 activity by peroxynitrite.

The mammalian sirtuin 6 (Sirt6) is a site-specific histone deacetylase that regulates chromatin structure and many fundamental biological processes. It inhibits endothelial cell senescence and inflammation, prevents development of cardiac hypertrophy and heart failure, modulates glucose metabolism, and represses tumor growth. The basic molecular mechanisms underlying regulation of Sirt6 enzymatic function are largely unknown. Here we hypothesized that Sirt6 function can be regulated via posttranslational modification, focusing on the role of peroxynitrite, one of the major reactive nitrogen species formed by excessive nitric oxide and superoxide generated during disease processes. We found that incubation of purified recombinant Sirt6 protein with 3-morpholinosydnonimine (SIN-1; a peroxynitrite donor that generates nitric oxide and superoxide simultaneously) increased Sirt6 tyrosine nitration and decreased its intrinsic catalytic activity. Similar results were observed in SIN-1-treated Sirt6, which was overexpressed in HEK293 cells, and in endogenous Sirt6 when human retinal microvascular endothelial cells were treated with SIN-1. To further investigate whether Sirt6 nitration occurs under pathological conditions, we determined Sirt6 nitration and activity in retina using a model of endotoxin-induced retinal inflammation. Our data showed that Sirt6 nitration was increased, whereas its activity was decreased, in this model. With mass spectrometry, we identified that tyrosine 257 in Sirt6 was nitrated after SIN-1 treatment. Mutation of tyrosine 257 to phenylalanine caused loss of Sirt6 activity and abolished SIN-1-induced nitration and decrease in its activity. Mass spectrometry analysis also revealed oxidation of methionine and tryptophan in Sirt6 after SIN-1 treatment. Our results demonstrate a novel regulatory mechanism controlling Sirt6 activity through reactive nitrogen species-mediated posttranslational modification under oxidative and nitrosative stress.

SIRT6 promotes COX-2 expression and acts as an oncogene in skin cancer.

SIRT6 is a SIR2 family member that regulates multiple molecular pathways involved in metabolism, genomic stability, and aging. It has been proposed previously that SIRT6 is a tumor suppressor in cancer. Here, we challenge this concept by presenting evidence that skin-specific deletion of SIRT6 in the mouse inhibits skin tumorigenesis. SIRT6 promoted expression of COX-2 by repressing AMPK signaling, thereby increasing cell proliferation and survival in the skin epidermis. SIRT6 expression in skin keratinocytes was increased by exposure to UVB light through activation of the AKT pathway. Clinically, we found that SIRT6 was upregulated in human skin squamous cell carcinoma. Taken together, our results provide evidence that SIRT6 functions as an oncogene in the epidermis and suggest greater complexity to its role in epithelial carcinogenesis.

Deficiency of cardiomyocyte-specific microRNA-378 contributes to the development of cardiac fibrosis involving a transforming growth factor ß (TGFß1)-dependent paracrine mechanism.

Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during heart failure. Previously we identified miR-378 as a cardiomyocyte-abundant miRNA down-regulated in several experimental models of cardiac hypertrophy and in patients with heart failure. To understand the consequence of miR-378 down-regulation during cardiac remodeling, our current study employed a locked nucleic acid-modified antimiR to target miR-378 in vivo. Results showed development of cardiomyocyte hypertrophy and fibrosis in mouse hearts. Mechanistically, miR-378 depletion was found to induce TGFß1 expression in mouse hearts and in cultured cardiomyocytes. Among various secreted cytokines in the conditioned-media of miR-378-depleted cardiomyocytes, only TGFß1 levels were found to be increased. The increase was prevented by miR-378 expression. Treatment of cardiac fibroblasts with the conditioned media of miR-378-depleted myocytes activated pSMAD2/3 and induced fibrotic gene expression. This effect was counteracted by including a TGFß1-neutralizing antibody in the conditioned-medium. In cardiomyocytes, adenoviruses expressing dominant negative N-Ras or c-Jun prevented antimiR-mediated induction of TGFß1 mRNA, documenting the importance of Ras and AP-1 signaling in this response. Our study demonstrates that reduction of miR-378 during pathological conditions contributes to cardiac remodeling by promoting paracrine release of profibrotic cytokine, TGFß1 from cardiomyocytes. Our data imply that the presence in cardiomyocyte of miR-378 plays a critical role in the protection of neighboring fibroblasts from activation by pro-fibrotic stimuli.

Regulation of Akt signaling by sirtuins: its implication in cardiac hypertrophy and aging.

Cardiac hypertrophy is a multifactorial disease characterized by multiple molecular alterations. One of these alterations is change in the activity of Akt, which plays a central role in regulating a variety of cellular processes ranging from cell survival to aging. Akt activation is mainly achieved by its binding to phosphatidylinositol (3,4,5)-triphosphate. This results in a conformational change that exposes the kinase domain of Akt for phosphorylation and activation by its upstream kinase, 3-phosphoinositide-dependent protein kinase 1, in the cell membrane. Recent studies have shown that sirtuin isoforms, silent information regulator (SIRT) 1, SIRT3, and SIRT6, play an essential role in the regulation of Akt activation. Although SIRT1 deacetylates Akt to promote phosphatidylinositol (3,4,5)-triphosphate binding and activation, SIRT3 controls reactive oxygen species-mediated Akt activation, and SIRT6 transcriptionally represses Akt at the level of chromatin. In the first part of this review, we discuss the mechanisms by which sirtuins regulate Akt activation and how they influence other post-translational modifications of Akt. In the latter part of the review, we summarize the implications of sirtuin-dependent regulation of Akt signaling in the control of major cellular processes such as cellular growth, angiogenesis, apoptosis, autophagy, and aging, which are involved in the initiation and progression of several diseases.

SIRT3 deacetylates and activates OPA1 to regulate mitochondrial dynamics during stress.

Mitochondrial morphology is regulated by the balance between two counteracting mitochondrial processes of fusion and fission. There is significant evidence suggesting a stringent association between morphology and bioenergetics of mitochondria. Morphological alterations in mitochondria are linked to several pathological disorders, including cardiovascular diseases. The consequences of stress-induced acetylation of mitochondrial proteins on the organelle morphology remain largely unexplored. Here we report that OPA1, a mitochondrial fusion protein, was hyperacetylated in hearts under pathological stress and this posttranslational modification reduced the GTPase activity of the protein. The mitochondrial deacetylase SIRT3 was capable of deacetylating OPA1 and elevating its GTPase activity. Mass spectrometry and mutagenesis analyses indicated that in SIRT3-deficient cells OPA1 was acetylated at lysine 926 and 931 residues. Overexpression of a deacetylation-mimetic version of OPA1 recovered the mitochondrial functions of OPA1-null cells, thus demonstrating the functional significance of K926/931 acetylation in regulating OPA1 activity. Moreover, SIRT3-dependent activation of OPA1 contributed to the preservation of mitochondrial networking and protection of cardiomyocytes from doxorubicin-mediated cell death. In summary, these data indicated that SIRT3 promotes mitochondrial function not only by regulating activity of metabolic enzymes, as previously reported, but also by regulating mitochondrial dynamics by targeting OPA1.

Defective Nrf2-dependent redox signalling contributes to microvascular dysfunction in type 2 diabetes.

AIMS: In type 2 diabetes, antioxidant depletion contributes to increased oxidative stress in the microvasculature. The current study was designed to assess how oxidative stress contributes to functional changes in the microvasculature, and determine the importance, and the effects of pharmacologically targeting, the transcription factor Nrf2. METHODS AND RESULTS: Pressure myography was used to measure myogenic constriction in mesenteric arterioles from diabetic (db/db) and non-diabetic (db/m) mice. Compared with db/m, myogenic constriction was larger in db/db, independent of the endothelial cell layer, and directly correlated with elevated basal and pressure-induced reactive oxygen species (ROS) production. Nrf2 was depleted in db/db vessels and associated with down-regulation of Nrf2-regulated genes. Notably, expression of GCLC and GCLM, enzymes important for glutathione (GSH) synthesis, was dramatically reduced, as was total cellular GSH. Normal myogenic function was restored to db/db arterioles by incubation with cell-permeant GSH. Similarly, the db/db myogenic phenotype was recapitulated in the db/m vessels by pharmacological GSH depletion. Treatment with the Nrf2-activator sulforaphane increased Nrf2 and promoted its nuclear localization and increased GCLC and GCLM expression in both db/m and db/db. Sulforaphane dramatically lowered ROS signalling in db/db and reduced myogenic tone to levels similar to that seen in db/m vessels. CONCLUSION: Depleted Nrf2 and expression of its dependent genes compromises antioxidant capacity resulting in dysfunctional myogenic tone in diabetes that is reversed by the Nrf2-activator sulforaphane.

A cardiac-enriched microRNA, miR-378, blocks cardiac hypertrophy by targeting Ras signaling.

Understanding the regulation of cardiomyocyte growth is crucial for the management of adverse ventricular remodeling and heart failure. MicroRNA-378 (miR-378) is a newly described member of the cardiac-enriched miRNAs, which is expressed only in cardiac myocytes and not in cardiac fibroblasts. We have previously shown that miR-378 regulates cardiac growth during the postnatal period by direct targeting of IGF1R (Knezevic, I., Patel, A., Sundaresan, N. R., Gupta, M. P., Solaro, R. J., Nagalingam, R. S., and Gupta, M. (2012) J. Biol. Chem. 287, 12913-12926). Here, we report that miR-378 is an endogenous negative regulator of cardiac hypertrophy, and its levels are down-regulated during hypertrophic growth of the heart and during heart failure. In primary cultures of cardiomyocytes, overexpression of miR-378 blocked phenylephrine (PE)-stimulated Ras activity and also prevented activation of two major growth-promoting signaling pathways, PI3K-AKT and Raf1-MEK1-ERK1/2, acting downstream of Ras signaling. Overexpression of miR-378 suppressed PE-induced phosphorylation of S6 ribosomal kinase, pERK1/2, pAKT, pGSK-3ß, and nuclear accumulation of NFAT. There was also suppression of the fetal gene program that was induced by PE. Experiments carried out to delineate the mechanism behind the suppression of Ras, led us to identify Grb2, an upstream component of Ras signaling, as a bona fide direct target of miR-378-mediated regulation. Deficiency of miR-378 alone was sufficient to induce fetal gene expression, which was prevented by knocking down Grb2 expression and blocking Ras activation, thus suggesting that miR-378 interferes with Ras activation by targeting Grb2. Our study demonstrates that miR-378 is an endogenous negative regulator of Ras signaling and cardiac hypertrophy and its deficiency contributes to the development of cardiac hypertrophy.

Nampt secreted from cardiomyocytes promotes development of cardiac hypertrophy and adverse ventricular remodeling.

Nicotinamide phosphoribosyltransferase (Nampt) is an important coenzyme involved in cellular redox reactions. Inside the cell, Nampt (iNampt) functions as a rate-limiting enzyme in the NAD salvage pathway, and outside the cell (eNampt), it acts as a proinflammatory cytokine. High-circulating levels of Nampt are reported in different pathological conditions. This study was designed to examine the role of Nampt in the development of cardiac hypertrophy and ventricular remodeling. We studied the hypertrophic response in Nampt heterozygous (+/-) knockout and cardiac-specific overexpressing Nampt transgenic mice. Whereas Nampt(+/-) mice were protected against agonist (isoproterenol and angiotensin II)-induced hypertrophy, Nampt transgenic mice spontaneously developed cardiac hypertrophy at 6 mo of age. Experiments conducted to gain insight into the mechanism revealed that treatment of cardiomyocytes with recombinant (eNampt) or overexpression with Nampt-synthesizing adenovirus vector (Ad.Nampt) induced cardiomyocyte hypertrophy. The prohypertrophic effects of eNampt and Ad.Nampt were blocked by the addition of a Nampt-blocking antibody into cultures, thus suggesting that Nampt was in fact invoking hypertrophic response of cardiomyocytes by acting on the cell surface receptors. We also found increased Nampt levels in the supernatant of cardiomyocyte cultures subjected to stress by either serum starvation or H(2)O(2) treatment. Exploration of signaling pathways in Nampt-induced cardiac hypertrophy and fibrosis revealed increased activation of mitogen-activated protein kinases, namely, JNK1, p38, and ERK. This was also associated with increased calcineurin levels and nuclear factor of activated T-cell localization into the nucleus. From these studies we conclude that cardiomyocytes are capable of secreting Nampt during stress, and exogenous Nampt is a positive regulator of cardiac hypertrophy and adverse ventricular remodeling.

The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun.

Abnormal activation of insulin-like growth factor (IGF)-Akt signaling is implicated in the development of various diseases, including heart failure. However, the molecular mechanisms that regulate activation of this signaling pathway are not completely understood. Here we show that sirtuin 6 (SIRT6), a nuclear histone deacetylase, functions at the level of chromatin to directly attenuate IGF-Akt signaling. SIRT6-deficient mice developed cardiac hypertrophy and heart failure, whereas SIRT6 transgenic mice were protected from hypertrophic stimuli, indicating that SIRT6 acts as a negative regulator of cardiac hypertrophy. SIRT6-deficient mouse hearts showed hyperactivation of IGF signaling-related genes and their downstream targets. Mechanistically, SIRT6 binds to and suppresses the promoter of IGF signaling-related genes by interacting with c-Jun and deacetylating histone 3 at Lys9 (H3K9). We also found reduced SIRT6 expression in human failing hearts. These findings disclose a new link between SIRT6 and IGF-Akt signaling and implicate SIRT6 in the development of cardiac hypertrophy and failure.

Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro.

Cardiac myosin binding protein-C (cMyBP-C) plays a role in sarcomeric structure and stability, as well as modulating heart muscle contraction. The 150 kDa full-length (FL) cMyBP-C has been shown to undergo proteolytic cleavage during ischemia-reperfusion injury, producing an N-terminal 40 kDa fragment (mass 29 kDa) that is predominantly associated with post-ischemic contractile dysfunction. Thus far, the pathogenic properties of such truncated cMyBP-C proteins have not been elucidated. In the present study, we hypothesized that the presence of these 40 kDa fragments is toxic to cardiomyocytes, compared to the 110 kDa C-terminal fragment and FL cMyBP-C. To test this hypothesis, we infected neonatal rat ventricular cardiomyocytes and adult rabbit ventricular cardiomyocytes with adenoviruses expressing the FL, 110 and 40 kDa fragments of cMyBP-C, and measured cytotoxicity, Ca(2+) transients, contractility, and protein-protein interactions. Here we show that expression of 40 kDa fragments in neonatal rat ventricular cardiomyocytes significantly increases LDH release and caspase 3 activity, significantly reduces cell viability, and impairs Ca(2+) handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca(2+) handling along with a significant reduction of sarcomere length shortening, relaxation velocity, and contraction velocity. Pull-down assays using recombinant proteins showed that the 40 kDa fragment binds significantly to sarcomeric actin, comparable to C0-C2 domains. In addition, we discovered several acetylation sites within the 40 kDa fragment that could potentially affect actomyosin function. Altogether, our data demonstrate that the 40 kDa cleavage fragments of cMyBP-C are toxic to cardiomyocytes and significantly impair contractility and Ca(2+) handling via inhibition of actomyosin function. By elucidating the deleterious effects of endogenously expressed cMyBP-C N-terminal fragments on sarcomere function, these data contribute to the understanding of contractile dysfunction following myocardial injury.