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• W2804205737 abstract "Mitochondrial Sirtuin 5 (SIRT5) is an NAD+-dependent demalonylase, desuccinylase, and deglutarylase that controls several metabolic pathways. A number of recent studies point to SIRT5 desuccinylase activity being important in maintaining cardiac function and metabolism under stress. Previously, we described a phenotype of increased mortality in whole-body SIRT5KO mice exposed to chronic pressure overload compared with their littermate WT controls. To determine whether the survival phenotype we reported was due to a cardiac-intrinsic or cardiac-extrinsic effect of SIRT5, we developed a tamoxifen-inducible, heart-specific SIRT5 knockout (SIRT5KO) mouse model. Using our new animal model, we discovered that postnatal cardiac ablation of Sirt5 resulted in persistent accumulation of protein succinylation up to 30 weeks after SIRT5 depletion. Succinyl proteomics revealed that succinylation increased on proteins of oxidative metabolism between 15 and 31 weeks after ablation. Heart-specific SIRT5KO mice were exposed to chronic pressure overload to induce cardiac hypertrophy. We found that, in contrast to whole-body SIRT5KO mice, there was no difference in survival between heart-specific SIRT5KO mice and their littermate controls. Overall, the data presented here suggest that survival of SIRT5KO mice may be dictated by a multitissue or prenatal effect of SIRT5. Mitochondrial Sirtuin 5 (SIRT5) is an NAD+-dependent demalonylase, desuccinylase, and deglutarylase that controls several metabolic pathways. A number of recent studies point to SIRT5 desuccinylase activity being important in maintaining cardiac function and metabolism under stress. Previously, we described a phenotype of increased mortality in whole-body SIRT5KO mice exposed to chronic pressure overload compared with their littermate WT controls. To determine whether the survival phenotype we reported was due to a cardiac-intrinsic or cardiac-extrinsic effect of SIRT5, we developed a tamoxifen-inducible, heart-specific SIRT5 knockout (SIRT5KO) mouse model. Using our new animal model, we discovered that postnatal cardiac ablation of Sirt5 resulted in persistent accumulation of protein succinylation up to 30 weeks after SIRT5 depletion. Succinyl proteomics revealed that succinylation increased on proteins of oxidative metabolism between 15 and 31 weeks after ablation. Heart-specific SIRT5KO mice were exposed to chronic pressure overload to induce cardiac hypertrophy. We found that, in contrast to whole-body SIRT5KO mice, there was no difference in survival between heart-specific SIRT5KO mice and their littermate controls. Overall, the data presented here suggest that survival of SIRT5KO mice may be dictated by a multitissue or prenatal effect of SIRT5. Sirtuin 5 (SIRT5) is an NAD+-dependent demalonylase (1Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M.M. Tishkoff D. Ho L. Lombard D. He T.C. et al.The first identification of lysine malonylation substrates and its regulatory enzyme.Mol Cell Proteomics. 2011; 10M111.012658Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 2Du J. Zhou Y. Su X. Yu J.J. Khan S. Jiang H. Kim J. Woo J. Kim J.H. Choi B.H. He B. Chen W. Zhang S. Cerione R.A. Auwerx J. et al.Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.Science. 2011; 334 (22076378): 806-80910.1126/science.1207861Crossref PubMed Scopus (962) Google Scholar), desuccinylase (2Du J. Zhou Y. Su X. Yu J.J. Khan S. Jiang H. Kim J. Woo J. Kim J.H. Choi B.H. He B. Chen W. Zhang S. Cerione R.A. Auwerx J. et al.Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.Science. 2011; 334 (22076378): 806-80910.1126/science.1207861Crossref PubMed Scopus (962) Google Scholar), and deglutarylase (3Tan M. Peng C. Anderson K.A. Chhoy P. Xie Z. Dai L. Park J. Chen Y. Huang H. Zhang Y. Ro J. Wagner G.R. Green M.F. Madsen A.S. Schmiesing J. et al.Lysine glutarylation is a protein posttranslational modification regulated by SIRT5.Cell Metab. 2014; 19 (24703693): 605-61710.1016/j.cmet.2014.03.014Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar) that resides primarily in mitochondria. Previous studies demonstrated that SIRT5 desuccinylase activity is important in maintaining cardiac function and metabolism in response to stress. In a model of ischemia–reperfusion injury, infarct size in SIRT5KO 4The abbreviations used are: KOknockoutMHCmyosin heavy chainTMTtandem mass tagFDRfalse discovery rateFC-fold changeRTroom temperatureTFAtrifluoroacetic acidACNacetonitrileFAformic acidAGCautomated gain controlPSMpeptide spectral matchcDNAcomplementary DNAANOVAanalysis of varianceTCAtricarboxylic acidTACtransverse aortic constrictionRT-qPCRquantitative real-time PCRSDHAsuccinate dehydrogenase complex, subunit ASPEsolid phase extractionPCrphosphocreatine. mouse hearts was greater than in WT littermates (4Boylston J.A. Sun J. Chen Y. Gucek M. Sack M.N. Murphy E. Characterization of the cardiac succinylome and its role in ischemia-reperfusion injury.J. Mol. Cell. Cardiol. 2015; 88 (26388266): 73-8110.1016/j.yjmcc.2015.09.005Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Additionally, SIRT5 depletion was detrimental to cardiac function at 39 weeks of age, with defects in cardiac fatty acid oxidation (5Sadhukhan S. Liu X. Ryu D. Nelson O.D. Stupinski J.A. Li Z. Chen W. Zhang S. Weiss R.S. Locasale J.W. Auwerx J. Lin H. Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function.Proc. Natl. Acad. Sci. U.S.A. 2016; 10.1073/pnas.1519858113Crossref PubMed Scopus (188) Google Scholar). Finally, we recently observed increased mortality and impaired oxidative metabolism in whole-body SIRT5KO mice, compared with WT littermates, with the stress of chronic pressure overload–induced cardiac hypertrophy (6Hershberger K.A. Abraham D.M. Martin A.S. Mao L. Liu J. Gu H. Locasale J.W. Hirschey M.D. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.J. Biol. Chem. 2017; 10.1074/jbc.M117.809897Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Each of these three studies on the role of SIRT5 in the heart performed succinyl proteomics measurements in WT and SIRT5KO heart tissue to identify enzymes and pathways potentially regulated by SIRT5. In all three studies, key pathways in mitochondrial metabolism, including oxidative phosphorylation, TCA cycle, and fatty acid oxidation, were identified as being regulated by SIRT5, given the increase in succinylation on many enzymes in these pathways with SIRT5 depletion (6Hershberger K.A. Abraham D.M. Martin A.S. Mao L. Liu J. Gu H. Locasale J.W. Hirschey M.D. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.J. Biol. Chem. 2017; 10.1074/jbc.M117.809897Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Together, these data suggest that SIRT5 is important in maintaining cardiac function by desuccinylating key enzymes in oxidative metabolism. Although these studies suggest that SIRT5 has a cardioprotective role, whether the effect of SIRT5 is cardiomyocyte-specific or due to an effect of SIRT5 outside of the heart remains unknown. knockout myosin heavy chain tandem mass tag false discovery rate -fold change room temperature trifluoroacetic acid acetonitrile formic acid automated gain control peptide spectral match complementary DNA analysis of variance tricarboxylic acid transverse aortic constriction quantitative real-time PCR succinate dehydrogenase complex, subunit A solid phase extraction phosphocreatine. Most characterizations of the physiological roles of mitochondrial sirtuins have been conducted in whole-body sirtuin KO mice, including our previous work on the role of SIRT5 in cardiac function (6Hershberger K.A. Abraham D.M. Martin A.S. Mao L. Liu J. Gu H. Locasale J.W. Hirschey M.D. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.J. Biol. Chem. 2017; 10.1074/jbc.M117.809897Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, differing degrees of acetylation in SIRT3KO tissues (7Dittenhafer-Reed K.E. Richards A.L. Fan J. Smallegan M.J. Fotuhi Siahpirani A. Kemmerer Z.A. Prolla T.A. Roy S. Coon J.J. Denu J.M. SIRT3 mediates multi-tissue coupling for metabolic fuel switching.Cell Metab. 2015; 21 (25863253): 637-64610.1016/j.cmet.2015.03.007Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and malonylation and succinylation in SIRT5KO tissues (8Nishida Y. Rardin M.J. Carrico C. He W. Sahu A.K. Gut P. Najjar R. Fitch M. Hellerstein M. Gibson B.W. Verdin E. SIRT5 regulates both cytosolic and mitochondrial protein malonylation with glycolysis as a major target.Mol. Cell. 2015; 10.1016/j.molcel.2015.05.022Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) suggest that the roles of sirtuins may differ between tissues. More recently, understanding the tissue-specific effects of mitochondrial sirtuins has emerged as an important area of investigation to determine the contribution of individual tissues to the phenotypes described in germline depletion models. For example, whole-body SIRT3KO mice on a high-fat diet have accelerated development of obesity, insulin resistance, hyperlipidemia, and steatohepatitis (9Hirschey M.D. Shimazu T. Jing E. Grueter C.A. Collins A.M. Aouizerat B. Stančáková A. Goetzman E. Lam M.M. Schwer B. Stevens R.D. Muehlbauer M.J. Kakar S. Bass N.M. Kuusisto J. et al.SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome.Mol. Cell. 2011; 44 (21856199): 177-19010.1016/j.molcel.2011.07.019Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). However, in a liver- or skeletal muscle–specific SIRT3KO model (two tissues with a strong influence on whole-body metabolism), no metabolic differences compared with WT controls were observed despite hyperacetylation profiles similar to the whole-body SIRT3KO model (10Fernandez-Marcos P.J. Jeninga E.H. Canto C. Harach T. de Boer V.C.J. Andreux P. Moullan N. Pirinen E. Yamamoto H. Houten S.M. Schoonjans K. Auwerx J. Muscle or liver-specific Sirt3 deficiency induces hyperacetylation of mitochondrial proteins without affecting global metabolic homeostasis.Sci. Rep. 2012; 10.1038/srep00425Crossref PubMed Scopus (114) Google Scholar). Either a tissue beyond liver/skeletal muscle or a coordinated tissue response was responsible for the phenotypes observed in the whole-body SIRT3KO mouse. Another major phenotype in the whole-body SIRT3KO mouse is the development of spontaneous cardiac hypertrophy (11Pillai V.B. Sundaresan N.R. Kim G. Gupta M. Rajamohan S.B. Pillai J.B. Samant S. Ravindra P.V. Isbatan A. Gupta M.P. Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway.J. Biol. Chem. 2010; 285 (19940131): 3133-314410.1074/jbc.M109.077271Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 12Hafner A.V. Dai J. Gomes A.P. Xiao C.-Y. Palmeira C.M. Rosenzweig A. Sinclair D.A. Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy.Aging. 2010; 2 (21212461): 914-92310.18632/aging.100252Crossref PubMed Scopus (432) Google Scholar). A recent study found that this phenotype is recapitulated in a skeletal muscle- and heart-specific SIRT3KO model (13Martin A.S. Abraham D.M. Hershberger K.A. Bhatt D.P. Mao L. Cui H. Liu J. Liu X. Muehlbauer M.J. Grimsrud P.A. Locasale J.W. Payne R.M. Hirschey M.D. Nicotinamide mononucleotide requires SIRT3 to improve cardiac function and bioenergetics in a Friedreich's ataxia cardiomyopathy model.JCI Insight. 2017; 10.1172/jci.insight.93885Crossref Scopus (71) Google Scholar). However, it is not yet known whether this is exclusively an effect of SIRT3 in cardiomyocytes; indeed, SIRT3 has also been shown to play a direct role in fibrosis (14Sundaresan N.R. Bindu S. Pillai V.B. Samant S. Pan Y. Huang J.-Y. Gupta M. Nagalingam R.S. Wolfgeher D. Verdin E. Gupta M.P. SIRT3 Blocks aging-associated tissue fibrosis in mice by deacetylating and activating glycogen synthase kinase 3β.Mol. Cell. Biol. 2015; 36 (26667039): 678-692Crossref PubMed Scopus (138) Google Scholar), a key component in the development of cardiac hypertrophy. So far, no studies have been published on the tissue-specific roles of SIRT4 or SIRT5. Thus, there is a need to develop tissue-specific sirtuin KO models to better understand the tissues contributing to the phenotypes revealed in whole-body KO models. To this end, we set out to determine whether the phenotypes described in the whole-body SIRT5KO mouse under chronic pressure overload–induced hypertrophy (6Hershberger K.A. Abraham D.M. Martin A.S. Mao L. Liu J. Gu H. Locasale J.W. Hirschey M.D. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.J. Biol. Chem. 2017; 10.1074/jbc.M117.809897Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) were recapitulated in a heart-specific SIRT5KO mouse model. A well-established experimental tool to knock out genes in an inducible and cardiomyocyte-specific manner is the α-MHC-MerCreMer mouse model. In this model, two mutated estrogen receptors (Mer) flank a Cre transgene located upstream of the cardiomyocyte-specific myosin heavy chain α (α-MHC) promoter (15Sohal D.S. Nghiem M. Crackower M.A. Witt S.A. Kimball T.R. Tymitz K.M. Penninger J.M. Molkentin J.D. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein.Circ. Res. 2001; 89 (11440973): 20-2510.1161/hh1301.092687Crossref PubMed Scopus (474) Google Scholar). This system requires delivery of tamoxifen to bind to the mutated estrogen receptors, translocate to the nucleus, and induce expression of Cre. One benefit of using an inducible system is that the gene can be depleted postnatally, circumventing problems with embryonic lethality or compensation during early development. However, a major caveat of this mouse model is Cre toxicity in the heart, as evidenced by heart failure with high amounts of tamoxifen (16Bersell K. Choudhury S. Mollova M. Polizzotti B.D. Ganapathy B. Walsh S. Wadugu B. Arab S. Kühn B. Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death.Dis. Model Mech. 2013; 6 (23929941): 1459-146910.1242/dmm.010447Crossref PubMed Scopus (94) Google Scholar) and transient inflammation and hypertrophy with lower doses of tamoxifen (17Lexow J. Poggioli T. Sarathchandra P. Santini M.P. Rosenthal N. Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver.Dis. Model Mech. 2013; 6 (23929940): 1470-147610.1242/dmm.010470Crossref PubMed Scopus (62) Google Scholar). Here we present data on the development and characterization of an inducible cardiomyocyte-specific SIRT5KO mouse model. Further, we analyze the cardiac morphological and functional changes with chronic TAC and survey the succinylation profile in this novel mouse model. To test whether the cardiac phenotypes observed in the whole-body SIRT5KO mouse (6Hershberger K.A. Abraham D.M. Martin A.S. Mao L. Liu J. Gu H. Locasale J.W. Hirschey M.D. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.J. Biol. Chem. 2017; 10.1074/jbc.M117.809897Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) were heart-intrinsic, we developed a heart-specific, tamoxifen-inducible SIRT5KO mouse model. To generate this mouse, we crossed SIRT5fl/fl (18Yu J. Sadhukhan S. Noriega L.G. Moullan N. He B. Weiss R.S. Lin H. Schoonjans K. Auwerx J. Metabolic characterization of a Sirt5 deficient mouse model.Sci. Rep. 2013; 3 (24076663)280610.1038/srep02806Crossref PubMed Scopus (96) Google Scholar) mice with αMHC-MerCreMer mice to generate littermates with the following genotypes: SIRT5fl/fl; αMHC-MerCreMer−/− (hereafter referred to as fl/fl) and SIRT5fl/fl; αMHC-MerCreMer+/− (hereafter referred to as fl/fl;MCM). Additionally, the floxed alleles were crossed out of this line to generate a second line of αMHC-MerCreMer−/− and αMHC-MerCreMer+/− mice to generate αMHC-MerCreMer+/− (hereafter referred to as MCM) mice to control for Cre toxicity. Two methods of delivering tamoxifen were tested to determine optimal SIRT5 depletion in the heart. First, adult mice were fed a commercially available tamoxifen citrate diet (Envigo Tekland Diets, TD.130860) for 3 weeks. In a second method, tamoxifen was dissolved in corn oil and given to adult mice by oral gavage at a dose of 80 mg/kg for 3 consecutive days. We found that with either method of delivery, depletion of SIRT5 took more than 2 weeks (Fig. 1). Feeding tamoxifen citrate for 3 weeks followed by 2 weeks of regular chow resulted in ∼75% depletion of SIRT5 (Fig. 1, A and B), whereas 2 weeks after the oral gavage regimen, we observed about 50% depletion of SIRT5 (Fig. 1, D and E). Because expression of Cre in the heart has been shown to be toxic and results in acute cardiomyopathy (17Lexow J. Poggioli T. Sarathchandra P. Santini M.P. Rosenthal N. Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver.Dis. Model Mech. 2013; 6 (23929940): 1470-147610.1242/dmm.010470Crossref PubMed Scopus (62) Google Scholar), we measured cardiac hypertrophy by calculating the ratio of heart weight to body weight at multiple time points throughout these pilot studies. Although there was only a small sample size at each time point (n = 1 or 2), there were trends of increased hypertrophy in the fl/fl;MCM mice compared with the fl/fl mice after 3 weeks of the tamoxifen diet (Fig. 1C). This hypertrophy appeared to normalize after 2 weeks of regular chow following the tamoxifen diet. No differences in heart weight to body weight were observed between the fl/fl controls and fl/fl;MCM mice in the oral gavage pilot study (Fig. 1F). Finally, we used RT-qPCR to further measure the transcript levels of Sirt5 and markers of Cre toxicity, including Nppa (Anf), Nppb (Bnp), and Il6. Although SIRT5 protein levels were not diminished until weeks after tamoxifen dosing, Sirt5 transcript levels were depleted at earlier time points (Fig. 1G). Maximal depletion of Sirt5 transcript levels occurred after 2 weeks of the tamoxifen diet (Fig. 1G, left) and 1 day after the oral gavage dosing regimen (Fig. 1G, right). The transcript levels of Cre toxicity markers were highest after 3 weeks of tamoxifen diet feeding or 1 day to 1 week after oral gavage dosing (Fig. 1, H–J). In the tamoxifen diet pilot group, transcript levels of Anf, Bnp, and Il6 returned to control levels after 2 weeks of regular chow feeding. Anf and Bnp remained elevated in the oral gavage pilot group 2 weeks after oral gavage dosing (Fig. 1, H and I). Based on these data, we proceeded with optimization of the tamoxifen diet feeding regimen because greater depletion of SIRT5 was achieved, and we predicted that a shorter time on the diet would be sufficient to achieve depletion of SIRT5 and would result in less Cre toxicity. In studies described below, tamoxifen diet was fed for 8–10 days, and we observed decreased evidence of Cre toxicity (data not shown). Given that SIRT5 is a protein lysine desuccinylase and that succinylation in the whole-body SIRT5KO heart is abundant, we looked at succinylation over time after tamoxifen-induced ablation of Sirt5. Adult female mice were fed a tamoxifen citrate diet for 10 days, followed by a regular chow diet. Mice were sacrificed immediately after the tamoxifen diet regimen and 3, 6, 12.5, and 32.5 weeks after the tamoxifen diet regimen. Western blotting was used to analyze succinylation and SIRT5 protein expression in whole-heart lysates. Interestingly, we found that it took several weeks for protein succinylation to accumulate to levels that were comparable with succinylation in the whole-body SIRT5KO mouse (Fig. 2, A and B). Specifically, 32.5 weeks after depletion of Sirt5, succinylation in fl/fl;MCM mouse hearts was about 3-fold greater than succinylation in fl/fl mouse hearts. Although this was the maximum succinylation achieved in this time course, the whole-body SIRT5KO mouse heart has an approximate 5.5-fold increase in succinylation over the WT control (6Hershberger K.A. Abraham D.M. Martin A.S. Mao L. Liu J. Gu H. Locasale J.W. Hirschey M.D. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.J. Biol. Chem. 2017; 10.1074/jbc.M117.809897Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, it is possible that sites of lysine succinylation would continue to accumulate when the time course is taken past 32.5 weeks. Interestingly, SIRT5 protein appeared to be maximally depleted 3 weeks after the tamoxifen diet feeding regimen (Fig. 2C). Together, these data show that, in our heart-specific, inducible SIRT5KO mouse model, SIRT5 is depleted after 3 weeks on the tamoxifen diet, but succinylation continues to increase for at least 30 weeks. This important finding provides a unique tool to assess the effects of SIRT5 ablation with varying degrees of protein lysine hypersuccinylation, which might allow us to disentangle the effects of SIRT5 from effects of protein succinylation. To further explore the increase in protein lysine succinylation after Sirt5 ablation, we performed succinyl proteomics in fl/fl and fl/fl;MCM mice 15 weeks and 31 weeks after tamoxifen diet feeding. From the previous time course experiment, we predicted that we would observe a small increase in protein succinylation in fl/fl;MCM mice 15 weeks after tamoxifen diet feeding and a large increase in protein succinylation in fl/fl;MCMC mice 31 weeks after tamoxifen diet feeding. Investigation of the cardiac succinylome at these time points would allow us to determine how the cardiac succinylome landscape changes with time after Sirt5 depletion. Immediately after weaning, fl/fl and fl/fl;MCM mice were fed tamoxifen for 8 days and then returned to a regular chow diet. One group of mice was sacrificed 15 weeks after tamoxifen-induced ablation of Sirt5 (n = 2 fl/fl and n = 3 fl/fl;MCM), and a second group of mice was sacrificed 31 weeks after tamoxifen-induced ablation of Sirt5 (n = 2 fl/fl and n = 2 fl/fl;MCM) (Fig. 3A). Left ventricles of hearts were pulverized and processed at the same time for succinyl proteomics analysis using a workflow leveraging peptide labeling with tandem mass tag (TMT) 10-plex reagents (Thermo Fisher Scientific), immunoprecipitation with anti-succinyl lysine antibody (Cell Signaling Technology), and nanoflow LC-MS/MS analysis on a Q Exactive Plus Orbitrap. See Table S1 for quantitative data on relative succinylpeptide and protein abundances. First we analyzed changes in the proteome with respect to time and genotype. Using the parameters of log2 FC ≥ 1 and an adjusted p ≤ 0.1 (Benjamini–Hochberg correction, 10% FDR), there existed only one significant change in the proteome. Esr1 (estrogen receptor) was significantly increased in the 15-week SIRT5KO compared with the 15-week WT control (Fig. 3B). Esr1 induction is due to the constitutive transgenic overexpression of mutated estrogen receptors under the control of the MHC promoter and is thus not an effect of Sirt5 ablation. We interrogated the pathways represented in the top 5% (Fig. 3B, red) and bottom 5% (Fig. 3B, blue) of proteins when ranked according to -fold change and found pathways that were significantly overrepresented in these groups. Among the proteins that increased the most in hearts with Sirt5 ablation, the pathways of muscle contraction and response to stress were increased, whereas, among the proteins that decreased the most in hearts with Sirt5 ablation, pathways of muscle organ development, sensory perception of sound (an irrelevant pathway in heart), muscle contraction, and cellular component of morphogenesis decreased. Given that proteins in muscle contraction both increased and decreased, this is likely because a large number of these proteins were identified in heart tissue. These pathways were also represented in a pathway analysis comparing the log2 FC of SIRT5KO/WT 31 weeks after tamoxifen feeding (data not shown). Importantly, proteins in oxidative metabolism (the main targets of protein lysine succinylation) are not significantly changed with ablation of Sirt5 in the heart. Next we compared the succinylome generated in the heart-specific SIRT5KO and the succinylome we previously generated from the hearts of whole-body SIRT5KO mice (6Hershberger K.A. Abraham D.M. Martin A.S. Mao L. Liu J. Gu H. Locasale J.W. Hirschey M.D. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload.J. Biol. Chem. 2017; 10.1074/jbc.M117.809897Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Given the different proteomic techniques and study designs, we expected to identify a smaller number of sites of succinylation in the heart-specific SIRT5KO succinylome compared with the whole-body SIRT5KO heart succinylome. In the heart-specific SIRT5KO succinylome, we identified (at 1% FDR) and quantified 1,631 succinylated peptides (an additional 504 succinylated peptides were identified but not quantified) that mapped to 462 unique proteins (Fig. 3C). Interestingly, only about half of these succinylated peptides were also identified in the whole-body SIRT5KO heart succinylome. We ranked the identified succinylated peptides in descending order in both succinylome datasets and found that, of the top 50 peptides increased in heart-specific SIRT5KO compared with WT hearts 31 weeks after tamoxifen feeding, half were also in the top 50 peptides increased in the whole-body SIRT5KO heart compared with the WT heart (Fig. 3D, light blue columns). Additionally, the top 50 succinylated peptides in the heart-specific dataset were all identified in the whole-body SIRT5KO heart succinylome. This comparison illustrates that the SIRT5 targets identified in these proteomic datasets had strong overlap and that the two models of SIRT5 deficiency have similar effects on the succinylome of the heart relative to their respective controls. The goal of this study was to determine how the protein succinylation landscape changes over time after SIRT5 depletion, specifically in the heart. To begin to answer this question, we performed pathway analyses on a group of peptides that had increased succinylation, compared both with their respective time control (KO/WT at 15 weeks and KO/WT at 31 weeks; Fig. 4, A and B) and with their genotype control (KO 31 weeks/KO 15 weeks and WT 31 weeks/WT 15 weeks, Fig. 4C). We used parameters of log2 FC ≥ 1 and an adjusted p ≤ 0.1 (Benjamini–Hochberg correction, 10% FDR) to make a list of proteins for each given comparison that had increased succinylation. These lists were uploaded to PANTHER (Protein ANalysis THrough Evolutionary Relationships) to perform statistical overrepresentation tests (19Mi H. Muruganujan A. Casagrande J.T. Thomas P.D. Large-scale gene function analysis with the PANTHER classification system.Nat. Protoc. 2013; 8 (23868073): 1551-156610.1038/nprot.2013.092Crossref PubMed Scopus (1690) Google Scholar) to determine pathways that were particularly susceptible to succinylation in this model. The top three significantly overrepresented pathways in each comparison (except WT 31 weeks/WT 15 weeks, where we saw no change in protein succinylation) were fatty acid β-oxidation, tricarboxylic acid cycle, and oxidative phosphorylation or cellular amino acid catabolic process. Together, these data suggest that proteins in the same metabolic pathways are succinylated in the SIRT5KO heart at 15 weeks and 31 weeks after SIRT5 depletion; indeed, these pathways are significantly overrepresented in this comparison (Fig. 4D). There were no peptides with changes in succinylation when comparing the succinylation profile of WT hearts 15 and 31 weeks after tamoxifen feeding (data not shown), demonstrating that few changes in the sites of protein succinylation occur under basal conditions in this time frame. We hypothesized that the increase in succinylation that occurs in the SIRT5KO heart between 15 and 31 weeks after SIRT5 depletion was due to either an increase in the number of unique sites of succinylation or a further increase in succinylation at lysine residues already succinylated. The succinyl proteomics performed here do not allow us to fully address the former possibility because of the multiplexed nature of our workflow (i.e. qualitative identifications are made on a pool of succinylpeptides from nine samples mixed together for which relative quantitative comparisons are made). To explore the latter possibility, we plotted the -fold change of all sites of lysine succinylation at 31 weeks (KO/WT) and 15 weeks (KO/WT) in fatty acid oxidation (Fig. 4E) and the TCA cycle (Fig. 4F). We found that the -fold change of at least 50% of succinylation sites on proteins in fatty acid oxidation and the TCA cycle increased from 15 weeks (Fig. 4F, light red circles) to 31 weeks (Fig. 4F, dark red circles) after SIRT5 depletion. Only a few SIRT5 succinylation targets have been validated in the heart: lysine 179 and 335 on SDHA (4Boylston J.A. Sun J. Chen Y. Gucek M. Sack M.N. Murphy E. Characterization of the cardiac succinylome and its role in ischemia-reperfusion injury.J. Mol. Cell. Cardiol. 2015; 88 (26388266): 73-8110.1016/j.yjmcc.2015.09.005Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and lysine 351 on HDHA (hydroxyacyl-CoA dehydrogenase, subunit A) (5Sadhukhan S. Liu X. Ryu D. Nelson O.D. Stupinski J.A. Li Z. Chen W. Zhang S. Weiss R.S. Locasale J.W. Auwerx J. Lin H. Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regul" @default.
• W2804205737 created "2018-06-01" @default.
• W2804205737 creator A5018876944 @default.
• W2804205737 creator A5019281134 @default.
• W2804205737 creator A5026453164 @default.
• W2804205737 creator A5034931695 @default.
• W2804205737 creator A5076304034 @default.
• W2804205737 creator A5080125836 @default.
• W2804205737 date "2018-07-01" @default.
• W2804205737 modified "2023-10-16" @default.
• W2804205737 title "Ablation of Sirtuin5 in the postnatal mouse heart results in protein succinylation and normal survival in response to chronic pressure overload" @default.
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