Local IGF-1 isoform protects cardiomyocytes from hypertrophic and oxidative stresses via SirT1 activity

Introduction

In response to age-associated stresses or dysfunction such as pressure/volume overload, myocardial infarction and cardiomyopathies, the heart undergoes adaptation processes that lead to pathological hypertrophy [1]. One of the main causes of cardiac dysfunction and cardiomyocytes loss is an imbalance between the generation of reactive oxygen species (ROS) and the antioxidant defenses in favor of the former [2]. Growing evidence demonstrate that oxidative stress and hypertrophy are mechanistically linked to each other in the heart [3,4]. Several therapeutic strategies are now employed to counteract the deleterious effects of cardiac hypertrophy and oxidative stress, making therefore the analysis of specific cell signaling imperative to generate novel drugs. In this scenario, the insulin like growth factor-1 (IGF-1) and Sirtuin -1 are novel important mediators of cell survival, oxidative stress, regeneration, and life-span regulation [5] in several tissues including the heart.

IGF-1 is a peptide hormone acting as a growth and differentiation factor [6]. The pleiotropic functions of IGF-1 are reflected in the intricate structure of the gene encoding it. The IGF-1 gene spans more than 70 kb, contains two promoters and has six exons, giving rise to multiple splicing variants. These splice variants all consist of the same unvarying core flanked by varying termini. IGF-1 isoforms are classified according to the N-terminal signal peptide (class 1 and 2) and to the C-terminal extension peptides, Ea and Eb [6]. It is established that IGF-1 is both a systemic growth factor produced primarily by liver and a local growth factor functioning in an autocrine/paracrine manner in tissues such as heart and skeletal muscle [6]. Post-transcriptionally, IGF-1 isoforms are cleaved to give a mature 70 amino acid core hormone (identical for all isoforms) devoid of both the signal peptide and the extension peptide. This mature hormone is released into the bloodstream and has been implicated in the restriction of life span [7]. Correspondingly, high levels of circulating IGF1 are associated with increased mortality and cardiovascular diseases in the elderly [8]. When expressed as transgenes in the cardiomyocytes, distinct IGF-1 isoforms result in diverse phenotypes, ranging from protection from hypertrophy to its exacerbation towards pathological states [6,9-11]. The role of IGF-1 in cardiac oxidative stress is also debated: cardiomyocyte-specific IGF-1 overexpression has been reported to protect from angiotensin II (Ang II)-mediated oxidative stress [12] but, on the contrary, severe circulating IGF-1 deficiency, as in hepatocyte-specific IGF-1 knock-out mice, antagonizes oxidative stress and cell death in cardiomyocytes triggered by the potent oxidant agent paraquat (PQ) [13].

The mIGF-1 isoform comprises a Class 1 signal peptide and a C-terminal Ea extension peptide [6]. It is highly expressed in neonatal tissues and in the adult liver, but decreases during aging in extra-hepatic tissues, where its expression is activated transiently in response to local damage [14]. Previous studies from our laboratory showed that continuous expression of mIGF-1 throughout postnatal life did not produce significant perturbations in normal heart physiology and, in contrast to previous studies with other IGF-1 transgenes. [11] did not progress to a pathological phenotype [15]. In response to injury however, molecular analysis revealed that mIGF-1 curtails the inflammatory response, enhances antioxidative cell-defense by upregulation of adiponectin, uncoupling protein 1 (UCP1) and methallothionein 2 (MT-2), and induces cardiac tissue restoration by increasing the number of proliferative cells at the border zone of the infarcted heart [15]. Given the benefits of cardiac restricted mIGF-1 expression, we sought to elucidate the molecular targets of this isoform.

Sirtuin 1 (SirT1) belongs to the sirtuin family of nicotinamide adenine dinucleotide NAD-dependent protein deacetylases, whose activation is considered beneficial for metabolic, neurodegenerative and inflammatory diseases and to augment longevity [5]. Moderate SirT1 activation in murine heart has been shown to protect from oxidative stress and angiotensin II (Ang II)-induced cell death [16,17]. Intriguingly, SirT1 expression is increased in the hypertrophic heart of rodents and monkeys [16,18], although its functional relevance is unclear. IGF-1 and SirT1 share downstream targets in cardiomyocytes, and this in turn may affect cardiovascular function [19]. It has been reported that SirT1 is also activated by the polyphenol resveratrol and by caloric restriction [20], whereas its induction is counteracted by circulating IGF-1 [20]. Moreover, the levels of circulating IGF-1 are lowered upon caloric restriction [21]. Hence, SirT1 and IGF-1 apparently play opposite biological roles, although there is no information on the impact of separate IGF-1 isoforms, acting locally or systemically, on cardiac SirT1. In particular, in this study we sought to test if the liver-produced and fully processed IGF-1 core protein isoform, circulating in the blood stream, and the locally acting mIGF-1 isoform [14], could display distinct effects in the protection from hypertrophic and oxidative stress. We tested this in mouse HL-1 and primary neonatal cardiomyocytes, using Ang II and PQ as hypertrophic and oxidative stressors.

We found that SirT1 and mIGF-1 co-regulate cardiomyocyte survival and protection from damage. mIGF-1 overexpression protects HL-1 cardiac cells and neonatal mouse cardiomyocytes from the deleterious effects induced by hypertrophic (Ang II) and oxidative (PQ) stressors in a SirT1-dependent fashion. The beneficial activity of SirT1 is mediated by the activation of protective molecules such as UCP1, adiponectin and MT2 and is dependent on mIGF-1 expression. Interestingly, the circulating IGF-1 isoform does not regulate SirT1 expression and activity and it is not beneficial during hypertrophy and oxidative stress conditions. The in vitro system herein described uncovers a novel signaling cross-talk that suggests potential pharmacological targets to modulate cardiac protection.

Results

Discussion

Hypertrophy and oxidative stress are intertwined processes in cardiomyocytes [3,4], contributing to heart disease progression [1,2]. In this study, we have identified a signaling pathway efficiently protecting mouse cardiomyocytes from oxidative and hypertrophic stresses (Ang II and PQ) that relies on the activation of NAD-dependent deacetylase SirT1 by the locally acting mGF-1 isoform. We show that mIGF-1-dependent SirT1 activation reduces ROS levels and cell death triggered by Ang II and PQ, and prevents Ang II-induced hypertrophic response (Figure 11). Our report is consistent with others showing that SirT1 display cardio-protective effects against oxidative stress-dependent cell death [16-18], and that SirT1 may elicit protection from cell hypertrophy by restoring MYH7 expression [38]. Interestingly, in smooth muscle cells SirT1 inhibits the expression of Ang II type 1 receptor [39], but if a similar mechanism occurs in cardiomyocytes remains to be established (Figure 11). We demonstrated that SirT1 counteracts both Ang II-induced cardiomyocyte hypertrophy and ROS-dependent cell death. The dual roles of Ang II as a prohypertrophic and pro-apoptotic agent may rely on the cross-talk between the EGF receptor and the different PI3K isoforms (a, b, gand δ) [40], leading in turn to hypertrophic growth or alternatively to cell death.Recently, it has been shown that other sirtuins family members (SirT3 and SirT7), play an important protective role against cardiac pathology [41,42], indicating that mIGF-1-mediated protective effects against oxidative and hypertrophic stresses could be in part due to other members of this family. However, our data showed that a specific SirT1 inhibitor (EX-527) or overexpression of dominant negative SirT1 protein (H363Y) can reverse mIGF-1 protective effects, supporting a SirT1-specific mechanism.

Importantly, our analysis showed that the locally produced mIGF-1 and the circulating IGF-1 have different roles in SirT1-mediated activity and cardiac protection. Although circulating IGF-1 and mIGF-1 trigger phosphorylation of the same receptor(s) (Figure 1C), differences in their respective signaling mechanisms leading to changes in SirT1 expression/activity must rely downstream of IGF-1 receptor(s). In this respect, it is important to stress that while circulating IGF-1 activates typically PI3K/AKT/mTOR and MAPK pathways [43], locally acting mIGF-1 does not activate these canonical pathways in cardiomyocytes, impinging instead on PDK1 and SGK1 signaling [15]. Thus, divergent signaling mechanisms could explain the apparently antagonistic roles of the two IGF-1 isoforms in cardiomyocytes, with mIGF-1 able to prevent circulating IGF-1-induced cell hypertrophy. We have previously reported that mIGF-1 induced accelerated cardiac growth, related to higher expression levels of ANP at 1 and 2 months, without any further significant change [15]. Interestingly, in the in vitro models herein described mIGF-1 did not elicit increased hypertrophic markers and cell size in both neonatal and adult (HL1) cardiomyocytes, indicating that the in vivo response is mainly due to specific physiological signaling occurring during cardiac development. Further work using in vivo and in vitro cardiac models is necessary to shed light on the intermediate players between mIGF-1-dependent signaling and SirT1 in cardiomyocytes (Figure 11). It would be important to understand whether some specific effects of mIGF-1 could be recapitulated by its N-terminal Class 1 signaling or C-terminal Ea extension peptides alone [6], which are absent in cleaved circulating IGF-1. It would be of interest also to ascertain if our data on mIGF-1/SirT1-dependent protection against oxidative and hypertrophic challenges can be confirmed in an in vivo setting, where circulating and autocrine/paracrine factors, absent in cultured cell systems, may have an impact.

mIGF-1 Tg mice display activation in the heart of genes involved in anti-apoptotic and anti-oxidant defenses [15]: among the most upregulated, we focused on adiponectin, UCP-1 and MT-2 [15]. Although these proteins are functionally unrelated (adiponectin is a hormone regulating metabolic processes, UCP-1 is a mitochondrial protein allowing protons to reenter the mitochondrial matrix short-circuiting the respiratory chain, and MT-2 is a zinc-binding protein), remarkably they have been reported independently to exert protection against hypertrophic and oxidative stresses in the heart [35-37]. Strikingly, we found that the activation of these genes by mIGF-1 relies on SirT1-dependent activation of their promoters, suggesting that at least some of the mIGF-1 dependent transcriptional program in cardiomyocytes is mediated by SirT1. Our data are in agreement with the finding that SirT1 upregulates adiponectin [44], whereas to our knowledge this is the first report about the SirT1-dependent regulation of UCP-1 and MT-2 transcripts.

In conclusion, there is increasing evidence that NAD-regulated enzymes such as SirT1 finely interplay in the regulation of cardiomyocyte function [17,45], and their role begins now to be appreciated. Consequently, research on the role of IGF-1 isoforms in this "NAD world" is also in its infancy. This domain is considered of clinical interest for the treatment of cardiovascular diseases [5,46], and the mIGF-1/SirT1 pathway presented in this study may represent a promising therapeutic target to fight cardiac hypertrophy and oxidative stress.

Materials and Methods

Animals. Transgenic FVB mice carrying a rat mIGF-1 cDNA driven by the mouse α-MyHC promoter were generated and maintained as previously described [11].

Western analyses. Protein extraction from whole cell or heart tissue preparation was performed in RIPA buffer (1% (w/w) Nonidet P40, 1% (w/w) Sodiumdeoxycate, 0.1% (w/v) SDS, 150mM NaCl, 50mM HEPES pH 7.0, 2mM EDTA pH 8.0, 100mM NaF, 10% glycerol, 1.5mM MgCl2, 100mM PMSF in ETOH, 200mM sodium orthovanadate, 1 μg/ml aprotinin). For analyses of nuclear proteins (SirT1, H1), nuclear fraction was isolated from cultured cells or heart tissues according to the following procedure: cells or liquid nitrogen-powderized heart tissues were dissolved in buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.05% NP-40, 100mM PMSF in ETOH, 200mM sodium orthovanadate, 1 μg/ml aprotinin, pH 7.9) and left on ice for 10 min. After centrifugation, cytoplasmic fraction (supernatant) was kept aside and frozen. Pellets were resuspended in buffer B (5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol (v/v), 100mM PMSF in ETOH, 200mM sodium orthovanadate, 1 mg/ml aprotinin, pH 7.9) plus NaCl to give a final concentration of 300mM NaCl. Lysates were then mechanically homogenized with a Dounce homogenizer on ice; samples were left on ice for 30 min. After a final centrifugation, supernatant (nuclear fraction) was collected for further analysis. Protein concentration was determined using Bradford method (Biorad) and 20 mg of protein lysates were separated in SDS polyacrylamide mini-gel (Biorad system) and transferred onto a hybond ECL nitrocellulose membrane (Amersham). Membranes were blocked with 5% milk, blotted with specific antibodies o/n at 4oC, washed 3 times with washing buffer (TBS and 0,1% Tween-20) for 30 min and blotted with specific secondary antibodies (horseradish peroxidase-conjugated, 1:5000) with 5% milk for 1h at RT. The membrane was incubated for 1 min using ECL reagent before exposure.

Cell cultures, transfections. Cardiac muscle cell line HL-1 was cultured as previously described, on gelatin/fibronectin coated flasks or multi-wells plates [22]. For transient plasmid transfection or co-transfection experiments, the lipid-based reagent LipofectamineTM 2000 (Invitrogen) was used, according to manufacturer instructions. For luciferase assays, 2 x 106 cells/well were transfected with 1 μg of luciferase reporter constructs and 1μg of pRL-TK (Renilla luciferase construct from Promega). Luciferase assays were performed 48 hours after transfection using a dual-luciferase reporter assay (Promega) and a luminescence counter VictorTM Light 1420 (Perkin Elmer). Firefly luciferase activity was normalized to renilla luciferase expression for each sample.

Preparation of primary neonatal cardiomyocytes culture. One-day-old C57/Bl6 or mIGF-1 transgenic mice were sacrificed and hearts were excised. After scalpel homogenization, ventricular cardiomyocytes were isolated following a series of collagenase/pancreatin digestions (Collagenase type II, CSL2, Worthington/Pancreatin 4x NF, GIBCO) and cells were collected by centrifugation (8.000rpm for 5min). Next, fibroblasts were removed from the culture after a 45 min pre-plating 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, 0.002M L-glutamine (Sigma) and 1x penicillin/streptomycin (Sigma)]. Alive cardiomyocytes were counted using Tryptan Blue solution (Sigma). Cells were transferred on 1% gelatin (Sigma) -coated 12- or 96-well plates.

Reactive oxygen species (ROS) measurements. The fluorescent probe dichlorofluorescein diacetate (CM-DCFDA, Sigma) was used to monitor the intracellular generation of reactive oxygen species (ROS). HL-1 or neonatal mouse cardiomyocytes, grown on coated 96-wells plates were transfected and/or treated for 60 minutes with Angiotensin II (1 mM, 60 min) or paraquat (100 mM) as described, with or without 10 mM superoxide scavenger Tiron. After washing with PBS, cells were incubated 20 min in the dark with 10 mM CM-DCFDA. Cells were washed again with PBS and fluorescence was detected at excitation/emission wavelength of 485-535nm in a fluorimeter Fluoroskan Ascent PL (Labsystems). Fluorescence values were normalized to protein content for each well.

[3H]-leucine incorporation. The cells (HL-1 or neonatal mouse cardiomyocytes) were plated on 12-well-coated dishes at a density of 100 cells/mm2. Protein synthesis was measured by [3H] Leucine (1 μCi/ml) incorporation as described elsewhere [23].

MF-20 immunostaining and confocal microscopy. Cells (HL-1 or neonatal mouse cardiomyocytes) were plated on coated coverslips. Upon the indicated treatment/transfection, cells were washed twice in PBS and fixed with 4% paraphormaldheyde for 10 min ice. Blocking was performed in PBS calcium free plus 10% goat serum, followed by 1 h incubation at RT with the MF-20 antibody diluted 1/250 in PBS calcium free plus 1.5% goat serum, and by 45 min incubation at RT with secondary Cy3 antibody (red) diluted 1/300 in PBS plus 1%BSA and 0.2% Triton-X. Coverslips were mounted on microscopy slides and confocal images were acquired on a Leica TCS SP5 microscope. Cell size (total area) and cell hypertrophy (total MF-20 staining intensity) were accurately quantified using the Metamorph® imaging software (Molecular Devices).

Cell viability assay. Cell viability/cell death was quantified by staining HL-1 cells or neonatal mouse cardiomyocytes with propidium iodide (Invitrogen) following cell transfections and/or treatments as indicated. Fluorescent intensity was analyzed using the BD FACSCanTM System. All FACS data was analyzed with FlowJo (Tree Star, USA).

Real-Time PCR. Total RNA was isolated from hearts using TRIzol (Invitrogen). Afterwards, the RNA was treated with DNaseI enzyme (Promega) for 1h at 37oC and then cleaned by column purification (Qiagen). The RNA concentration was determined with a spectrophotometer. After RNA quality verification, 1-2 mg was used to prepare cDNA (Ready-To-Go, T-Primed First-Strand Kit, Amersham Bioscience). Quantitative polymerase chain reaction (PCR) was performed using the SYBR Green (SIGMA) in a Light-Cycler (Roche). UbiC, Rn18S and GAPDH transcripts were used as internal controls, according to the GeNorm method [24]. Primer sequences were designed with the Primer 3 software (http://frodo.wi.mit.edu/) and are listed in Table 1.

Statistical analysis. Results are expressed as means ± S.E. Comparisons were made by using appropriated Student's t test. Differences were considered as significant when P<0.05 (*), P<0.01 (**) or P<0.001 (***).

Reagents, antibodies and plasmids. All reagents, antibodies and plasmids not described elsewhere in the text are listed below in Table 2.

Sources of funding

This work was supported by grants of the European Union (Heart Repair: LSHM-CT-2005-018630; EUMODIC: LSHG-CT-2006-037188) and of the Foundation Leducq (Transatlantic Networks of Excellence Program: 04 CVD 03)to NR. MV is the recipient of an EIPOD (EMBL Interdisciplinary POst-Doc) fellowship.