Oxidative stress contributes to the pathogenesis of aging-associated heart failure. Among various signaling pathways mediating oxidative stress, the NAD+-dependent protein deacetylase SirT1 has been implicated in the protection of heart muscle. Expression of a locally acting insulin-like growth factor-1 (IGF-1) propeptide (mIGF-1) helps the heart to recover from infarct and enhances SirT1 expression in cardiomyocytes (CM) in vitro, exerting protection from hypertrophic and oxidative stresses. To study the role of mIGF-1/SirT1 signaling in vivo, we generated cardiac-specific mIGF-1 transgenic mice in which SirT1 was depleted from adult CM in a tamoxifen-inducible and conditional fashion. Analysis of these mice confirmed that mIGF-1-induced SirT1 activity is necessary to protect the heart from paraquat (PQ)-induced oxidative stress and lethality. In cultured CM, mIGF-1 increases SirT1 expression through a c-Jun NH(2)-terminal protein kinase 1 (JNK1)-dependent signaling mechanism. Thus, mIGF-1 protects the heart from oxidative stress via SirT1/JNK1 activity, suggesting new avenues for cardiac therapy during aging and heart failure.
Chronic congestive heart failure carries a poor prognosis and is a leading cause of cardiovascular death (McMurray & Stewart, 2000). Despite advances in treatment, the underlying age-dependent process that leads to cardiac dysfunction remains not fully understood. The early pioneering free radical theory of aging implicates multi-organ accumulation of free radical damage (Harman, 1956), which has been more recently expanded to encompass oxidative damage from mitochondrial reactive oxygen species (ROS). Accumulating evidence suggests that ROS signaling plays an important role in the development and progression of age-associated heart failure, regardless of the etiology (Anilkumar et al., 2009).
Studies in rodent models implicate ROS in the development of cardiac hypertrophy, cardiomyocyte apoptosis, and the remodeling of the failing heart following myocardial infarction, pressure overload, or aging (Giordano, 2005; Takimoto & Kass, 2007) and have suggested a possible causal role for increased ROS in the development of age-mediated cardiac vulnerability and contractile dysfunction (Jahangir et al., 2007).
From a therapeutic perspective, the precise antioxidant potential of cardioprotective pathways is not completely understood. Elucidation of specific cell signaling pathways that counteract the deleterious effects of oxidative stress is therefore important for developing strategies to limit myocardial dysfunction in the elderly.
IGF-1 is a peptide hormone acting as a systemic growth factor produced mainly by the adult liver and as a local growth and differentiation factor functioning in an autocrine/paracrine manner in tissues such as heart muscle (Winn et al., 2002; Barton, 2006; Santini et al., 2007). Multiple IGF-1 propeptides produced by alternate exon splicing are cleaved to yield a common 70-amino acid core hormone that is released into the bloodstream and has been implicated in the restriction of lifespan and in cardiovascular diseases (Andreassen et al., 2009; Kenyon, 2010). By contrast, the locally acting mIGF-1 propeptide (Musaro et al., 2001) includes the C-terminal Ea extension peptide and is highly expressed in neonatal tissues and adult liver but decreases during aging. In skeletal muscle, adult mIGF-1 expression levels increase transiently in response to local damage (Winn et al., 2002; Matheny et al., 2010).
The fact that IGF-1 can act either as a circulating hormone or as a local growth factor has led to conflicting results from analyses of its role in cardiovascular function (during development or in response to oxidative challenge) and from studies in which different transgenic IGF-1 isoforms were synthesized in extrahepatic mouse tissues (Delaughter et al., 1999; Kajstura et al., 2001; Li et al., 2007), underscoring the physiological differences between IGF-1 propeptides versus mature IGF-1 peptide.
We have previously shown that continuous cardiomyocyte (CM)-restricted expression of the mIGF-1 propeptide throughout postnatal life did not perturb cardiac physiology or induce a pathological phenotype (Santini et al., 2007). Molecular analysis revealed further that mIGF-1 enhanced antioxidative cell defenses by upregulating a subset of protecting genes such as adiponectin, uncoupling protein 1 (UCP1), and metallothionein 2 (MT-2) (Santini et al., 2007).
We recently uncovered SirT1 as another downstream mediator of mIGF-1 action in the heart (Vinciguerra et al., 2009). SirT1 belongs to the sirtuin family of nicotinamide adenine dinucleotide NAD-dependent protein deacetylases, whose activation appears beneficial for aging-associated metabolic, inflammatory, and cardiac diseases, and to increase lifespan in model organisms (Donmez & Guarente, 2010). Moderate transgenic SirT1 protein overexpression (from 2.5- to 7.5-fold) in mouse heart protected from paraquat (PQ)-induced oxidative stress (Alcendor et al., 2007). Conversely, cardiac depletion of SirT1 aggravates ischemia/reperfusion (I/R)-induced oxidative injury (Hsu et al., 2010). Other sirtuins (SirT3 and SirT7) also play an important protective role against oxidative stress–induced cardiac pathology (Schug & Li, 2010), suggesting a conserved role of the sirtuin family against cardiac oxidative stress.
IGF-1 and SIRT1 share molecular downstream targets in cardiomyocytes (CM), such as the Forkhead box protein O1 (FOXO1), a key player during cellular response to stress, and this cross talk in turn may affect cardiovascular function (Ni et al., 2007). SirT1 is classically activated by the polyphenol resveratrol and by caloric restriction (Cohen et al., 2004), whereas the levels of circulating IGF-1 are lowered upon caloric restriction (Huffman et al., 2008). Moreover, SirT1 induction by caloric restriction is counteracted by circulating IGF-1 (Cohen et al., 2004). Hence, SirT1 and IGF-1 may play opposite biological roles.
Indeed, mouse CM are protected from oxidative stress through the activation of SirT1 by the mIGF-1 isoform, which reduces ROS levels and cell death triggered by Ang II and Paraquat (PQ), whereas the circulating IGF-1 core protein does not regulate SirT1 expression and activity and is not protective in oxidative stress conditions (Vinciguerra et al., 2009). These results suggest that SirT1 is a specific downstream effector of mIGF-1 propeptide action in protection against oxidative stress.
To test this hypothesis in vivo, we generated cardiac-specific mIGF-1 transgenic mice where SirT1 was depleted specifically from adult CM using Cre/loxP technology. Cardiac basal function in these mice was not affected, whereas in the absence of SirT1 expression in CM, mIGF-1 activity could not protect the heart from PQ-induced oxidative stress and lethality. We further defined c-Jun N-Terminal Protein Kinase 1 (JNK1) as the signaling intermediate whereby the mIGF-1 propeptide upregulates SirT1 expression in CM. These findings underscore the relevance of a mIGF-1/JNK1/SirT1 signaling pathway as a potential therapeutic target to affect cardioprotection against oxidative stress.
Materials and methods
Transgenic FVB mice carrying a rat mIGF-1 cDNA driven by the mouse αMyHC promoter (αMyHC/mIGF-1) were generated and maintained as previously described (Santini et al., 2007). SirT1 floxed (Fl/Fl) mice were previously described (Cheng et al., 2003) and acquired from The Jackson Laboratory (Bar Harbor, ME, USA): A loxP-flanked neomycin cassette immediately upstream of exon 4 (encoding an evolutionarily conserved Sir2 motif) and a third loxP site downstream of exon 4 were inserted to create the targeted mutant SirT1 allele. Homozygous SirT1 floxed (Fl/Fl) mice were viable and fertile. Τamoxifen-inducible αMyHC/mER-CRE-mER transgenic mice were crossed to SirT1 floxed (Fl/Fl) mice to deplete SirT1 expression in adult CM (Sohal et al., 2001) upon tamoxifen administration. Mice were placed on tamoxifen-containing chow (Harlan Special Diet TD.55125) at 3–4 months of age. Two weeks of tamoxifen-enriched diet led to efficient and reproducible gene recombination (Kratsios et al., 2010a). The mIGF-1 transgene was introduced by three-way crosses to generate αMyHC/mIGF-1 Tg × αMyHC/mER-CRE-mER SirT1Fl/Fl mice (hereafter referred to as mIGF-1 Tg × SirT1 CKO). Genotyping was performed by PCR using genomic DNA from tail biopsies.
Oxidative stress in vivo
For oxidative stress and survival experiments, PQ was administered intraperitoneally at 30 mg kg−1 concentration. All mouse procedures were approved by the European Molecular Biology Laboratory Monterotondo Ethical Committee (Monterotondo, Italy) and were in accordance with national and European regulations.
Oxidative stress in vitro
The fluorescent probe dichlorofluorescein diacetate (CM-DCFDA; Sigma, Milano, Italy) was used to monitor the intracellular generation of ROS in neonatal mouse CM, grown on coated 96-well plates and treated for 60 min with PQ (100 μm). After washing with PBS, cells were incubated 20 min in the dark with 10 μm CM-DCFDA. Cells were washed again with PBS and fluorescence was detected at excitation/emission wavelength of 485–535 nm in a fluorimeter Fluoroskan Ascent PL (Labsystems, Ramsey, MN, USA). Fluorescence values were normalized to protein content for each well. Aconitase activity was measured in isolated heart mitochondria as previously described (Bugger et al., 2008). Malondialdehyde (MDA) content was assessed in heart protein samples with the OxiSelect™ MDA Adduct ELISA Kit (Cell Biolabs, San Diego, CA, USA), according to manufacturer’s instructions.
Animals were anesthetized with 2% isofluorane, and the left hemithorax was shaved. The mice were placed on a temperature-controlled pad, and heart rate was continuously monitored (400–550 bpm). Ultrasound transmission gel (Parker Laboratories Inc., Fairfield, NJ, USA) was used, and the heart was imaged in the parasternal short-axis view. Two-dimensional B-mode images were obtained at the papillary muscle level using the Vevo 770 Ultrasound system (VisualSonics, Amsterdam, the Netherlands). Fractional shortening (FS) and left ventricular transversal area (LVAT), relative wall thickness (RWT) in diastole and systole, and fractional shortening (FS) were calculated using the Vevo 770 V2.2.3 software (VisualSonics).
Western blot analyses
Cytoplasmic and nuclear protein extraction from whole cell or heart tissue preparations and immunoblotting analyses were performed as previously described (Vinciguerra et al., 2009).
Preparation of primary neonatal cardiomyocyte cultures
One-day-old wild-type, mIGF-1 Tg, and mIGF-1 Tg × SirT1 CKO mice were sacrificed and hearts were excised. After scalpel homogenization, ventricular CM were isolated following a series of collagenase/pancreatin digestions (Collagenase type II, CSL2, Worthington/Pancreatin 4 × NF; GIBCO, Monza, Italy), and cells were collected by centrifugation (12 800 g for 5 min). Fibroblasts were removed from the culture after a 45-min preplating step at 37 °C in complete medium [DMEM/199 medium (5/1 ratio) supplemented with 10% heat-inactivated horse serum (Sigma), 5% heat-inactivated fetal calf serum (Sigma), 0.025 m HEPES, 0.002 m l-glutamine (Sigma), and 1 × penicillin/streptomycin (Sigma)]. Live CM were counted using Trypan Blue solution (Sigma). Cells were transferred on 1% gelatin (Sigma)-coated 12- or 96-well plates.
Primary cardiomyocyte isolation procedure
Excised mouse hearts were perfused using a Langendorff perfusion apparatus with calcium-free KRB solution containing collagenase (1 mg mL−1) until they became flaccid. The hearts were then chopped finely, and the mince was agitated gently in the same medium to dissociate individual cells. The resulting cell suspension was filtered to remove undigested material, and the CM were separated from noncardiomyocytes (NCM) by sedimentation of CM at 500 g for 2 min. Supernatant containing NCM was separated and further centrifuged at 1500 g for 5 min to pellet cells. Calcium tolerance of sediment containing CM was restored gently by resuspending it in KRB containing a progressively higher concentration of calcium ion to a final concentration of 1 mm. Both CM and NCM sediments were stored in TRIzol (Invitrogen, Monza, Italy) for RNA analyses.
Cardiac muscle cell line HL-1 was cultured as previously described, on gelatin/fibronectin-coated flasks or multiwell plates (Claycomb et al., 1998). For transient plasmid and siRNA transfection experiments, the lipid-based reagent Lipofectamine™ 2000 (Invitrogen) was used, according to manufacturer’s instructions. Chemical inhibitors were added at the indicated concentration; An equal volume of vehicle (DMSO or ethanol) was added to the controls.
Total RNA was isolated from hearts using TRIzol (Invitrogen). Afterward, the RNA was treated with DNaseI enzyme (Promega, Milano, Italy) for 1 h at 37 °C and then cleaned by column purification (Qiagen, Milano, Italy). The RNA concentration was determined with a spectrophotometer. After RNA quality verification, 1–2 μg was used to prepare cDNA (Ready-To-Go, T-Primed First-Strand Kit; Amersham Bioscience, Milano, Italy). Quantitative polymerase chain reaction (PCR) for SirT1 was performed using the SYBR Green (Sigma) in a Light-Cycler (Roche, Monza, Italy). UbiC, Rn18S, and GAPDH transcripts were used as internal controls, according to the GeNorm method; primer sequences were previously described (Vinciguerra et al., 2009).
Results are expressed as means ± SE. Comparisons were made by using Student’s t-test. Differences were considered as significant when P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***). For the estimation of the effect of the treatment on mice survival rate upon PQ-induced stress, Kaplan–Meier statistics was used to build survival curves, and the statistical relative risk (RR), standard error (SE), and P values were assessed.
Reagents, antibodies, and plasmids
All reagents not described elsewhere in the text are listed in Table S1.
Cardiac conditional/inducible SirT1 depletion in the adult mouse does not generate functional imbalances
To extend the analysis of mIGF-1-mediated cardioprotection from oxidative insults through SirT1 action in vivo, we exploited conditional knockout mice and depleted SirT1 in CM of the adult heart to limit the impact of confounding phenotypes owing to developmental processes. When generated in an inbred background, whole-body SirT1 knockout (KO) mice carrying two null alleles of SirT1 mostly die prenatally or during the early postnatal period (Cheng et al., 2003), owing to cardiac defects at the level of the ventricular septum and of the atrioventricular valve (Cheng et al., 2003).
However, on outbred backgrounds, whole-body SirT1 KO produces viable mice with diverse phenotypes, such as imperfect gametogenesis and sterility, an autoimmune-like condition and an impairment in benefiting from the positive CR-induced metabolic effects [reviewed in (Vinciguerra et al., 2010)]. Conversely, mice moderately overexpressing SirT1 in the myocardium are protected from oxidative stress (Alcendor et al., 2007).
To ablate SirT1 activity exclusively in the CM compartment, we crossed CM-specific, tamoxifen-inducible αMyHC/mER-CRE-mER transgenic mice with conditional SirT1Fl/Fl KO mice (Sohal et al., 2001; Cheng et al., 2003), to produce SirT1 CKO mice, which were born at expected Mendelian ratio and were phenotypically undistinguishable from their SirT1Fl/Fl or wild-type littermates. Cardiac function and SirT1 expression in these mice were unaltered during growth, as assessed by Western blot and echocardiography (data not shown).
After 2 weeks on a tamoxifen-enriched diet, 4-month-old SirT1 CKO mice displayed efficient and reproducible deletion of the catalytic domain (exon 4) of SirT1 in the CM, giving rise to faster migrating bands on the immunoblots corresponding to an inactive truncated form of the protein (Cheng et al., 2003) (Fig. 1A). This excision did not occur in tamoxifen-fed wild-type, αMyHC/mER-CRE-mER, and SirT1Fl/Fl mice and was not observed in unrelated organs (liver and skeletal muscle) (Fig. 1A,B). Echocardiographic analyses of cardiac functional parameters did not detect any statistical difference between tamoxifen-fed mice among experimental groups (Fig. 1C,D).
Functional parameters and SirT1 expression were unaltered up to 8 months after tamoxifen administration (1 year of age – data not shown). Thus, tamoxifen-dependent inducible KO of cardiac SirT1 in the adult is efficient and does not generate any evident functional perturbations. Notably, SirT1 levels were detectable at low levels by immunoblotting in whole-heart lysates upon tamoxifen-induced CM-specific gene excision (Fig. 1A), despite the fact that SirT1 is known to be expressed in all noncardiomyocyte (NCM) cell types (endothelial cells, fibroblasts, macrophages, smooth muscle cells), which comprises ∼50% of total cardiac cell number (Banerjee et al., 2007). In cells from wild-type hearts fractionated to separate the CM from NCM fractionation, SirT1 mRNA expression was found ∼12 times higher in the CM fraction compared to the NCM fraction, as shown by qRT–PCR (Fig. S1), which may contribute to explain the low levels of SirT1 upon Cre-mediated CM deletion by immunoblotting analysis on whole-heart lysates.
Cardiac-specific SirT1 deletion in mIGF-1 Tg mice abrogates mIGF-1-dependent cardioprotection from oxidative stress
We next assessed the role of SirT1 in mIGF-1-dependent signaling by three-way crossing of mIGF-1 Tg mice (Santini et al., 2007) with SirT1 CKO to generate mIGF-1 Tg × SirT1 CKO mice. All progeny were born at expected ratio and did not display any abnormalities. We further analyzed four groups of mice: wild-type, mIGF-1 Tg × SirT1Fl/Fl, SirT1 CKO, and mIGF-1 Tg × SirT1 CKO mice. SirT1 overexpression protects the murine heart from oxidative stress induced by the herbicide PQ (Alcendor et al., 2007), one of the most potent known oxidative stressors that compromise cardiac functions in humans and rodents (Bismuth et al., 1988). Using chemical inhibitors (EX-527, sirtinol), we previously demonstrated that mIGF-1 protects against PQ-induced oxidative stress in CM cultures via SirT1 activity (Vinciguerra et al., 2009). To test the effects of SirT1 deletion on mIGF-1-mediated protection from PQ-induced oxidative stress, wild-type, mIGF-1 Tg × SirT1Fl/Fl, SirT1 CKO, and mIGF-1 Tg × SirT1 CKO mice were fed a tamoxifen-enriched diet for 2 weeks, which led to SirT1 deletion specifically in the heart of mIGF-1 Tg × SirT1 CKO (data not shown). We then intraperitoneally injected wild-type, mIGF-1 Tg × SirT1Fl/Fl, SirT1 CKO, and mIGF-1 Tg × SirT1 CKO mice with 30 mg kg−1 of PQ. After 24 h, the mice were sacrificed, the hearts explanted, and lipid/protein peroxidation adduct levels [increasing upon ROS generation (Vinciguerra et al., 2009)] were assessed. Immunoblot analyses showed that lipid peroxidation of 4-hydroxy-2-nonenal (4-HNE) and MDA protein adducts was significantly increased in the heart of PQ-injected wild-type and SirT1 CKO mice, whereas hearts of mIGF-1 Tg × SirT1Fl/Fl mice were significantly protected from forming these compounds upon PQ injection (Fig. 2A,B). However, PQ induced the formation of 4-HNE and MDA in the hearts of mIGF-1 Tg × SirT1 CKO mice, indicating that the presence of SirT1 is important for mIGF-1-mediated protection against PQ insult.
As illustrated in Fig. 3A,B, wild-type and SirT1 CKO mice responded to PQ with a decrease in cardiac aconitase activity and an increase in cardiac MDA levels, while mIGF-1 Tg × SirT1Fl/Fl were completely protected from these changes. Conversely, PQ decreased aconitase activity and increased MDA in the hearts of mIGF-1 Tg × SirT1 CKO mice.
We next measured ROS levels in neonatal primary CM isolated from wild-type, SirT1 CKO, mIGF-1 Tg × SirT1Fl/Fl, and mIGF-1 Tg × SirT1 CKO mice and treated with tamoxifen (10 μm) for 24 h. Tamoxifen treatment was not toxic to the cells and led to Cre-mediated SirT1 excision (data not shown). Subsequently, cells were exposed to PQ, and ROS production was monitored. Wild-type and SirT1 CKO CM displayed a > 2 fold increase in ROS levels upon treatment with PQ, which was blocked in neonatal CM from mIGF-1 Tg × SirT1Fl/Fl mice but not in CM from mIGF-1 Tg × SirT1 CKO mice (Fig. 3C). Taken together, these findings indicate that mIGF-1 requires SirT1 activity to protect the murine heart from oxidative stress in vivo.
SirT1 cardiac expression is required for mIGF-1-dependent increase in mice survival upon PQ oxidative insult
As shown above, PQ oxidative insult triggers stress in the heart on the short term (24 h after injection); however, it becomes lethal at longer times (Li et al., 2007). To determine the extent to which mIGF-1/SirT1 signaling protects against long-term PQ-induced lethality, we injected 2-week tamoxifen-fed wild-type, mIGF-1 Tg × SirT1Fl/Fl, SirT1 CKO, and mIGF-1 Tg × SirT1 CKO mice with 30 mg kg−1 PQ and monitored the survival every 12 h for several days until the animals deceased. We found that within 7 days after PQ injection, 100% of the mice of the four experimental groups invariably died (Fig. 4A); however, as shown by the Kaplan–Meier cumulative survival curve (PQ tolerance test), while wild-type, SirT1 CKO, and mIGF-1 Tg × SirT1 CKO died on average 3–4 days after PQ administration, mIGF-1 Tg × SirT1Fl/Fl mice lived 1–2 days longer, surviving 5–6 days in total to PQ lethal insult (Fig. 4, P value <0.001, for mIGF-1 Tg × SirT1Fl/Fl mice versus wild-type mice).
To assess whether an increased resistance to heart failure might be involved in the greater survival of mIGF-1 Tg mice, we examined by qRT–PCR the expression of genes of the ‘fetal’ program [adult α-myosin heavy chain 6 and 7 (MYH6 and MYH7), atrial and brain natriuretic peptides (ANP and BNP), sarco/endoplasmic reticulum calcium ATPase-2 (SERCA2), and α-skeletal actin (ACTA-1)], which typically display the changes in an adult failing myocardium (Vinciguerra et al., 2010), in the hearts of tamoxifen-fed wild-type, mIGF-1 Tg × SirT1Fl/Fl, SirT1 CKO, and mIGF-1 Tg × SirT1 CKO mice upon PQ-induced death (Fig. 4B). Whereas the induction of a typical fetal gene expression pattern (increases in MYH7, ANP, BNP and ACTA-1 levels; decreases in MYH6 and SERCA2 levels) was observed in WT, SirT1 CKO, and mIGF-1xSirT1 CKO mice, mIGF-1 Tg mice displayed a marked attenuated activation of the fetal gene program upon PQ injection (Fig. 4B). These findings demonstrate that SirT1 induction is necessary for the increased survival, and it might be involved in the cardiac resistance to heart failure of mIGF-1 cardiac Tg mice upon lethal PQ challenge.
mIGF-1 and IGF-1 activate both common and divergent intracellular signaling cascades in HL-1 cardiomyocytes
Determining the intermediates responsible for the SirT1-mediated beneficial effects of mIGF-1 could provide novel therapeutic targets for intervention in the adverse consequences of cardiac oxidative stress. Our previous analysis showed that the locally produced mIGF-1 and circulating IGF-1 have different roles in SirT1-mediated activity and CM protection from oxidative stress (Vinciguerra et al., 2009).
Both circulating IGF-1 and mIGF-1 trigger phosphorylation of the same receptor (Vinciguerra et al., 2009), implicating differences in the respective signaling mechanisms that lead to the changes in SirT1 expression/activity downstream of IGF-1 receptor. While circulating IGF-1 activates PI3K/AKT/mTOR and MAPK pathways (Glass, 2010), locally acting mIGF-1 does not activate these canonical pathways in CM, impinging instead on PDK1 and SGK1 signaling (Santini et al., 2007). These differences may also depend on the time frame of cell exposure to mIGF-1: PI3K/AKT/mTOR activation is triggered as an acute response, whereas PDK-1 and SGK1 remain activated upon chronic exposure (Schulze et al., 2005; Santini et al., 2007).
We extended our previous analysis of cellular signaling transduction pathways in HL-1 CM (Vinciguerra et al., 2009) by transfecting with expression vectors encoding SirT1, a catalytic inactive SirT1 protein (H363Y) (Cohen et al., 2004), or mouse mIGF-1, or by treating them with 20 ng mL−1 70-aa recombinant IGF-1 core peptide (Fig. 5A). As previously shown (Vinciguerra et al., 2009), transfected mIGF-1 increased SirT1 protein levels, while recombinant IGF-1 administration did not.
Interestingly, mIGF-1-dependent increase in SirT1 protein was not paralleled by an increase in SirT1 mRNA (data not shown), indicating a mechanism independent from transcription.
Of note, transfection of HL-1 CM with an expression vector encoding the 70-aa IGF-1 core peptide was equally ineffective in modulating SirT1 expression (Fig. S2). Enzymatic activities of Akt, mTOR, PDK1, SGK1, S6K, JNK1, JNK2, ERK1/2 expression levels and phosphorylation on key residues (Mourkioti & Rosenthal, 2005; Santini et al., 2007) were assessed in HL-1 lysates (Fig. 5A). Whereas SirT1 or SirT1 H363Y overexpression did not affect the total or phosphorylation levels of any of the signaling effectors analyzed, both mIGF-1 and IGF-1 triggered the phosphorylation of S6K and ERK1/2 kinases (Fig. 5A,B).
However, mIGF-1 specifically induced the phosphorylation of PDK1, SGK1, and JNK1, whereas IGF-1 triggered phosphorylation events on AKT, mTOR, and JNK2 (Fig. 5A), as assessed by densitometric quantification of their respective phosphoprotein/protein levels ratio (Fig. 5B). Focusing on the divergent signaling intermediates induced by the mIGF-1 propeptide vs. fully processed IGF-1 may help to understand their distinct roles in cardioprotection from oxidative stress.
JNK-1 activity is required for mIGF-1-dependent increase in SirT1 expression and phosphorylation levels in HL-1 cardiomyocytes
To determine whether mIGF-1-mediated phosphorylation of PDK1, SGK1, or JNK1 might account for SirT1 induction, we treated mIGF-1-transfected HL-1 CM with OSU03012 (PDK1 inhibitor), GSK650394 (SGK1 inhibitor), or SP600125 (JNK inhibitor).
At the concentrations used, these compounds did not exhibit toxic effects but efficiently blocked mIGF-1-induced phosphorylation events on the respective kinases (Fig. 6A). SP600125, but not OSU03012 or GSK650394, blocked mIGF-1-mediated SirT1 induction, implicating JNK1 in the mIGF-1 signaling response (Fig. 6B). Deacetylation levels of two SirT1 targets, p53 and histone H1 (Luo 2001; Vaquero et al., 2004), correlated with this result, while total protein levels remained unchanged (Fig. 6B). SirT1 predominantly localized to the nucleus in cultured neonatal mouse and HL-1 CM (Fig. S3).
As human SirT1 has been recently reported to be directly phosphorylated by nuclear JNK1 on two serine residues (Ser27 and Ser47) (Nasrin et al., 2009), one of which is conserved in mouse (Ser46, corresponding to human Ser47) and is detectable with an antibody raised against human phospho-Ser47 (Gao et al., 2011), we examined SirT1 phosphorylation in HL-1 cardiomyocytes transfected with mIGF-1. As seen in Fig. S4A, mIGF-1 triggered the phosphorylation of SirT1 on Ser46 (Ser47 in humans), which was fully prevented by incubation of the cells with the JNK inhibitor SP600125.
As SP600125 inhibits indiscriminately JNK isoforms, we performed JNK1 or JNK2 knockdown in HL-1 CM using specific siRNA to clarify their respective involvement in the mIGF-1-induced phosphorylation of SirT1 on Ser46. JNK1 or JNK2 were silenced of ∼80% compared to untransfected cells (Fig. S4B). We detected a decrease in SirT1 phosphorylation on Ser46 to baseline levels uniquely in JNK-1-deficient cells, but not in JNK2-deficient cells, in mIGF-1 expressing HL-1 CM (Fig. S4B).
In summary, these data show that JNK1 activity is required for the increase in SirT1 expression and phosphorylation induced by mIGF-1, and also these data support the notion that JNK1 activity mediates the response of SirT1 to mIGF-1 signaling, affording cardioprotection from oxidative stress.
In this study, we analyzed the role of a locally acting insulin growth factor propeptide (mIGF-1) and of the NAD+-dependent protein deacetylase SirT1 in cardioprotection from oxidative stress, a major risk factor of age-associated heart failure. We have previously reported that mIGF-1 protects cultured mouse CM from oxidative stress through an increase in SirT1 expression/activity (Vinciguerra et al., 2009).
Here we show are that depleting SirT1 from adult CM in a tamoxifen-inducible and conditional fashion (SirT1 CKO) did not alter cardiac basal functions. We report that SirT1 is required for the mIGF-1-dependent protection against PQ-induced challenge. Further, we established a functional link between JNK1 activity and the mIGF-1-dependent increase in SirT1 expression/activity.
The role of mIGF-1/SirT1 pathway in oxidative cardioprotection
In the challenged heart, SirT1 activity is required for mIGF-1-dependent protection from changes in oxidative stress markers, such as MDA and 4-HNE protein adduct products, MDA and ROS levels, and aconitase enzymatic activity upon PQ challenge (Figs 2 and 3).
These data exclude a redundant role of other sirtuins and are consistent with our previous in vitro data showing that a specific SirT1 inhibitor (EX-527) can reverse mIGF-1 protective effects (Vinciguerra et al., 2009). The mIGF-1/SirT1 protection from oxidative stress is transient as mIGF-1 Tg × SirT1Fl/Fl mice die within 1 week upon PQ injection.
However, mIGF-1 Tg × SirT1Fl/Fl show a small but statistically significant increase in survival compared to tamoxifen-fed mIGF-1 Tg × SirT1 CKO and SirT1 CKO mice upon PQ insult (5–6 days against 3–4 days on average, Fig. 4A). PQ-induced cardiac fetal gene expression program, a hallmark that accompanies the dysfunctional failing heart, was attenuated in mIGF-1 Tg × SirT1Fl/Fl, compared to the other conditions, which may correlate functionally with the delay in succumbing.
Notably, animals succumb to PQ toxic effects not only from cardiac failure, but also from other irreversible organ failure (i.e., lungs). Ongoing efforts are aimed at understanding the role of this newly characterized mIGF-1/SirT1 pathway in the protection against ‘milder’ physiological hypertrophic and oxidative challenges to the heart (e.g., pressure overload model, Ang II infusion, transient ischemia, ischemia/reperfusion injury), relevant to clinical settings.
mIGF-1 enhances SirT1 expression in cardiomyocytes through JNK1 activity
By identifying the signaling cascades involved in the mIGF-1-induced increase in SirT1 expression, we have uncovered that divergent signaling mechanisms could explain the apparently antagonistic roles of the two IGF-1 isoforms on SirT1 expression in the heart. Both IGF-1 and mIGF-1 triggered the phosphorylation of S6K and ERK1/2 kinases.
However, while circulating IGF-1 specifically induced the phosphorylation and activation of AKT, mTOR, and JNK2, locally produced mIGF-1 overexpression induced the phosphorylation of PDK1, SGK1, and JNK1 (Fig. 5). We cannot exclude the possibility that IGF-1 and/or mIGF-1 could cross talk with G-protein-coupled receptor–dependent signaling (Rozengurt et al., 2010).
However it is likely that mIGF-1-triggered cytoplasmic phosphorylation events, with their consequent downstream signal transduction, mediate the increase in SirT1 expression, and not vice versa, because we observed mostly nuclear SirT1 localization upon biochemical nuclear fractionation in neonatal mouse and HL-1 CM (Fig. S3). Tanno et al. (2010) have recently shown that SirT1 is localized predominantly in the cytoplasm of adult mouse CM and shuttles to the nucleus upon stress: Perhaps this discrepancy may rely on the different experimental settings, as SirT1 localization may vary even in the same cell type (C2C12) upon differentiation [(Tanno et al., 2010) and references therein].
Activation of JNK1 is evidently required for mIGF-1-mediated induction of SirT1, because only the JNK inhibitor SP600125 (but not PDK1 and SGK1 inhibitors) was effective in abrogating this induction (Fig. 6). SP600125 inhibits the activity of all JNK isoforms; however, as mIGF-1 did not affect JNK2 phosphorylation levels (Fig. 5) and siRNA-mediated silencing of JNK2 did not affect mIGF-1-dependent increase in SirT1 (Fig. S4), the observed effects on SirT1 rely specifically on the JNK1 isoform. A third JNK isoform exists in mammals, JNK3, whose function seems to be relevant to the nervous system rather than to the cardiovascular system (Bogoyevitch, 2006). SirT1 can be phosphorylated by JNK1 on serine residues, promoting its catalytic activity (Nasrin et al., 2009; Gao et al., 2011). Consistently, we observed that mIGF-1 triggered the phosphorylation of mouse SirT1 on Ser46 in a JNK1-dependent fashion in CM.
Given that SirT1 transcript levels are unaffected by mIGF-1 (data not shown), it will be necessary to establish whether mIGF-1-driven JNK1 activity affects SirT1 protein expression specifically in CM by impinging on its stability/turnover or whether additional post-transcriptional and/or post-translational regulatory mechanisms are involved.
In any case, our data, together with others (Nasrin et al., 2009), support the notion that JNK1-mediated phosphorylation of SirT1 is implicated in the cellular protection from oxidative stress. IGF-1 is known to activate JNK signaling pathways (Mourkioti & Rosenthal, 2005), but we show here that different forms of IGF-1 trigger the phosphorylation of distinct JNK isoforms (1 and 2). JNK isoforms differ slightly in their molecular weights and display both specific and redundant functions, and in line with our study, cardiac-specific genetic activation or inactivation of JNK1 (but not other JNK isoforms) in mice demonstrated a protective role for this kinase against pressure-overload-induced deterioration of cardiac function (Bogoyevitch, 2006), which involves oxidative-stress-mediated remodeling of the tissue (Takimoto & Kass, 2007).
However, other studies using small-molecule JNK inhibitor showed that JNK inhibition, irrespective of the isoform targeted, may be beneficial in ischemic heart disease (Bogoyevitch, 2006). Nevertheless, in the context of mIGF-1 signaling, the JNK1/SirT1 pathway presented here represents a promising therapeutic target to fight cardiac oxidative stress in clinical settings.
Interactions of mIGF-1/SirT1 with other signaling pathways during cardiac stress
The networks of transcriptional factors and coactivators involved in regulating SirT1 activity and in cardiac plasticity upon stress are quite intricate. SirT1 was shown to block cardiac stress by inhibiting NF-κBp65 activity (Planavila et al., 2010).
Our laboratory has shown that conditional deletion in the heart of the NF-κB essential modulator (NEMO) leads to oxidative stress and to adult-onset dilated cardiomyopathy (Kratsios et al., 2010b), a condition that was attenuated by feeding the animals an antioxidant diet (Kratsios et al., 2010b).
A recent breakthrough study (Guarani et al., 2011) demonstrated that, in several cell types, SirT1 provide an important adaptation mechanism linking tissue oxygenation and nutritional supply through Notch-dependent signaling, a key cell–cell communication mechanism essential for cell specification and tissue patterning, including the heart.
We have recently found that inducible transgenic overexpression of Notch1 or intramyocardial delivery of a Notch1 pseudoligand improves cardiac performance, fibrosis and apoptosis upon myocardial infarction, a deleterious ischemic and oxidative stress (Kratsios et al., 2010a). Furthermore, our laboratory demonstrated for the first time that genetic disruption of SGK1, which is activated in the myocardium by mIGF-1 overexpression (in parallel with PDK1 and JNK1), reduces the expression of Notch signaling genes in mouse embryos, impairing heart structure and function (Catela et al., 2010).
Thus, SirT1-dependent signaling is at the crossroads of a complex signaling network in the CM, to be further untangled and to be exploited to combat age-associated cardiac dysfunctions.
We are indebted to Esfir Slonimsky and Melanie Leuener for invaluable technical help.
Sources of funding
This work was supported by grants from the European Union (Heart Repair: LSHM-CT-2005-018630; EUMODIC: LSHG-CT-2006-037188), of the Foundation Leducq (Transatlantic Networks of Excellence Program: 04 CVD 03), and of the British Heart Foundation (Project Grant numbers PG/08/111/26226 and PG/10/019) to NR. MV is the recipient of an EIPOD (EMBL Interdisciplinary POst-Doc) fellowship. NR is an NHMRC Australia Fellow.