Research On The Mechanism Of OTUD1 In Pathological Cardiac Hypertrophy

BackgroundPathological cardiac hypertrophy is a cardiac disease characterized by an increase in the volume of cardiomyocytes,leading to an augmented thickness of the heart walls.Unlike physiological hypertrophy,such as seen in athlete’s hearts,this thickening does not accompany an enhancement in heart function.On the contrary,pathological cardiac hypertrophy often results in a decline in cardiac performance,potentially progressing to congestive heart failure.Unlike physiological hypertrophy,pathological hypertrophy is induced by adverse cardiac loading conditions such as hypertension,valvular heart disease,genetic factors,or prolonged excessive load.Treatment approaches for pathological heart enlargement focus on decreasing the heart’s workload,enhancing its performance,and averting potential complications.Treatment modalities include pharmacological therapy,utilizing ACE inhibitors,ARBs(angiotensin II receptor blockers),β-blockers,and calcium channel blockers to control hypertension and reduce cardiac load.For certain patients,such as those with valvular heart disease-induced hypertrophy,surgical intervention may be necessary to repair or replace damaged valves.Lifestyle adjustments,including weight reduction,salt intake restriction,moderate exercise,and smoking cessation,are also advised.Ubiquitination is a crucial intracellular protein modification process that affects the stability,localization,or activity of proteins by attaching ubiquitin proteins to target proteins.In cardiac diseases,particularly in the development of pathological cardiac hypertrophy,the regulation of ubiquitination plays a key role.Recent studies suggest that modulating the ubiquitination process in cardiomyocytes could offer new strategies for treating pathological cardiac hypertrophy.Interventions,such as regulating the activity of specific ubiquitinases or blocking certain ubiquitination pathways,may help halt or reverse the progression of myocardial hypertrophy.These interventions act by altering intracellular signaling pathways,thus affecting the growth and death processes of cardiomyocytes,offering a new perspective for the treatment of pathological cardiac hypertrophy.OTUD1(Ovarian Tumor Domain Containing 1),a member of the OTU(Ovarian Tumor)enzyme family,plays a significant role in various physiological processes,including cell signaling,protein homeostasis maintenance,cell cycle control,and immune responses.Recent research has revealed OTUD1’s role in regulating the immune system and inflammatory responses.For instance,a study found that loss-of-function mutations in OTUD1 are associated with various autoimmune diseases,indicating its role as an immune checkpoint in inflammatory conditions by regulating RIG-I-like receptor signaling to block immune responses.OTUD1 directly interacts with the transcription factor IRF3,removing K63-linked polyubiquitin chains from its Lysine 98 site,thereby inhibiting IRF3’s nuclear translocation and transcriptional activity,highlighting its importance in maintaining immune homeostasis.Additionally,OTUD1 is involved in regulating various cell death pathways,including apoptosis and ferroptosis.Through its specific deubiquitinating activity,OTUD1 can regulate several key signaling molecules,influencing their function and cell fate.Furthermore,OTUD1’s role in iron transport regulation is a potential mechanism in its anti-tumor immune function.By deubiquitinating and stabilizing Iron-Responsive Element Binding Protein 2(IREB2),OTUD1 promotes transferrin receptor protein 1(TFRC)mediated iron transport,thereby increasing reactive oxygen species(ROS)production and ferroptosis,which in turn promotes the release of damage-associated molecular patterns(DAMPs),enhancing the host’s immune response against tumors.The involvement of OTUD1 in pathological heart enlargement and the precise proteins it targets have yet to be documented.Therefore,this research is dedicated to investigating OTUD1’s role in this condition and understanding the underlying molecular processes,with the goal of identifying novel and effective therapeutic targets for this critical heart ailment.Part One:Expression of OTUD1 in Pathological Cardiac HypertrophyObjectiveTo examine the varied expression levels of OTUD1 within a model of pathological heart enlargement.MethodsCellular Approach:To simulate hypertrophy,neonatal rat cardiac myocytes(NRCMs)were treated with Angiotensin Ⅱ(Ang Ⅱ),using NRCMs treated with phosphate buffer saline(PBS)as the baseline comparison group.Animal Strategy:A model of pathological heart enlargement in mice was created using Transverse aortic constriction(TAC),with mice undergoing sham operations serving as the comparison group.Molecular Level Assessment:The expression levels of OTUD1,atrial natriuretic peptide(ANP),and β-myosin heavy chain(Myh7)in both NRCMs and mouse heart tissue were evaluated using quantitative RT-PCR(qRT-PCR)and Western blot techniques.Immunofluorescence:The expression of OTUD1 in mouse cardiac tissue was assessed through immunofluorescence staining.ResultsCompared to the PBS control group,both mRNA and protein levels of OTUD1 were upregulated in the Ang Ⅱ-induced NRCMs hypertrophy model.Similarly,OTUD1 mRNA and protein expression were also elevated in the cardiac tissue of mice from the TAC group compared to the sham-operated group.Immunofluorescence experiments in mouse cardiac tissue further confirmed the increase in OTUD1 protein expression in the pathological cardiac hypertrophy model.ConclusionOTUD1 protein levels are increased in models of cardiomyocyte hypertrophy as well as in models of cardiac tissue enlargement.Part Two:The Impact of OTUD1 on Pathological Cardiac HypertrophyObjectiveInvestigating the influence of OTUD1 on the development of pathological heart enlargement through the use of OTUD1 gene knockout mice,AAV9-Otudl overexpression adenovirus,and cardiomyocyte models subjected to OTUD1 enhancement and suppression.MethodsCellular Experiments:Adenoviruses carrying short hairpin RNA(Ad-shOtudl)were used to construct Ang Ⅱ-induced hypertrophic cardiomyocyte models,divided into four groups:Ad-Vector PBS,Ad-shOtud1 PBS,Ad-Vector Ang Ⅱ,and Ad-shOtudl AngⅡ.Additionally,adenoviruses overexpressing Otudl(Ad-Otudl)were used to infect NRCMs,divided into four groups:Ad-GFP PBS,Ad-Otud1 PBS,Ad-GFP Ang Ⅱ,and Ad-Otudl Ang Ⅱ.Immunofluorescence assays confirmed cellular dimensions,while Western blot analysis quantified the expression of hypertrophic indicators.Animal Experimentation:Mice were subjected to TAC to create a model of heart enlargement,categorized into four segments according to whether OTUD1 was knocked out or overexpressed.The weights of the body,heart,and lungs,along with the length of the tibia,were recorded for mice across the various groups.Echocardiography:Cardiac function of mice in each group was evaluated using echocardi ography.Histology:H&E staining assessed gross cross-sectional area and hypertrophy of the heart.Marker Expression:Western blot and quantitative qRT-PCR techniques were utilized to evaluate the levels of markers indicative of cardiac hypertrophy,including MYH7 and BNP.Fibrosis Assessment:Masson’s trichrome and Sirius Red staining assessed the degree of fibrosis in cardiac tissue.Inflammation Markers:ELISA measured the expression of inflammatory factors TNF and IL6 in plasma.ResultsCellular experiments showed that overexpressing OTUD1 increased the cell surface area of Ang II-induced hypertrophic cardiomyocytes,whereas knocking down OTUD1 significantly reduced the cell surface area.Overexpression of OTUD1 significantly increased Anp protein expression,while knockdown of OTUD1 reduced Anp expression.In the TAC-induced mouse model,depletion of OTUD1 led to a decrease in body weight,heart weight,lung to body weight ratio,and heart weight relative to tibia length in comparison to wild-type counterparts.Echocardiographic analysis showed that the absence of OTUD1 alleviated the decline in ejection fraction(EF)and fractional shortening(FS)associated with cardiac hypertrophy,as well as diminished left ventricular end-diastolic diameter(LVEDd)and left ventricular end-systolic diameter(LVEDs)in mice with heart enlargement.H&E staining showed that OTUD1 knockout reduced myocardial cross-sectional area in hypertrophic mice.Western blot and qRT-PCR indicated that OTUD1 knockout alleviated the increase in BNP and MYH7 expression caused by pressure overload.Sirius Red and Masson’s trichrome staining showed that OTUD1 knockout alleviated interstitial and perivascular fibrosis in the TAC-induced cardiac hypertrophy model.ELISA results indicated that OTUD1 knockout reduced the expression of inflammatory factors IL6 and TNF in plasma due to pathological cardiac hypertrophy.ConclusionElevating OTUD1 levels can intensify both pathological heart enlargement and cardiomyocyte hypertrophy in mice,while reducing or eliminating OTUD1 expression can alleviate these conditions.Part Three:Mechanism of OTUD1 in Pathological Cardiac HypertrophyObjectiveTo investigate the specific molecular mechanisms by which OTUD1 promotes pathological cardiac hypertrophy.MethodsTranscriptome Sequencing:Cardiomyocytes overexpressing OTUD1 and control group cardiomyocytes were subjected to mRNA sequencing to analyze the impact of OTUD1 overexpression on the transcriptome level associated with cardiac hypertrophy.Protein Immunoprecipitation and Mass Spectrometry:To identify proteins interacting with OTUD1.Protein Co-immunoprecipitation(Co-IP):To confirm the interaction between OTUD1 and PGAM5.Ubiquitination Assay:To explore whether OTUD1 regulates PGAM5 through deubiquitination.Rescue Experiment:To further validate the role of OTUD1 in regulating ASK1 in cardiac hypertrophy.ResultsTranscriptome Sequencing Analysis:Revealed that OTUD1 overexpression activates pathways involved in cardiac hypertrophy,apoptosis,inflammatory responses,and protein processing,with the ASK1-p38/JNK MAPK signaling pathway being a major enriched pathway.Regulation of ASK1-p38/JNK Signaling Pathway:Knockdown of OTUD1 inhibited,whereas overexpression of OTUD1 activated the ASK1-p38/JNK signaling pathway.Protein Interaction:Proteomic analysis and Co-IP experiments indicated an interaction between OTUD1 and PGAM5.Ubiquitination Assay:Demonstrated that OTUD1 regulates PGAM5 through deubiquitination mediated by K63-linked ubiquitin chains.Rescue Experiment:Confirmed that the OTUD1-ASK1 signaling axis promotes pathological cardiac hypertrophy.ConclusionOTUD1 promotes pathological cardiac hypertrophy by deubiquitinating PGAM5 through K63-linked ubiquitin chains,thereby activating the ASK1-p38/JNK MAPK pathway.This study unveils a novel mechanism of OTUD1 in the regulation of cardiac hypertrophy,highlighting its potential as a therapeutic target in the treatment of pathological cardiac conditions.Part Four:m6A Modification Promotes High Expression of OTUD1 During Cardiac HypertrophyObjectiveTo identify m6A modification sites on OTUD1 transcript,methyltransferases,and m6A-binding proteins involved in the process.MethodsOverall m6A Expression Assessment:Immunofluorescence staining and m6A RNA immunoprecipitation(RIP)were used to examine the global expression of m6A during cardiac hypertrophy.Prediction of m6A Sites:OTUD1 transcripts were analyzed using the cuilab prediction website(https://www.cuilab.cn/sramp)to identify potential m6A modification sites.RIP Experiments:These were conducted to ascertain the modification sites on OTUD1,and the interactions between OTUD1 transcripts,methyltransferases,and m6Abinding proteins.Quantitative Analysis of m6A Modification:m6A levels on OTUD1 transcripts under various conditions were determined using methylation-specific quantitative assays.Verification in TAC Mouse Model:The necessity of the METTL3-OTUD1 signaling axis was tested in four groups:AAV9-GFP PBS,AAV9-GFP METTL3 inhibitor group,AAV9-Otud1 PBS group,and AAV9-Otud1 METTL3 inhibitor group.Measurements:Body weight,heart weight,lung weight,and tibia length of mice in different groups were measured.Echocardiography:Cardiac function of each mouse group was evaluated using echocardi ography.H&E Staining:Gross cross-sectional area and hypertrophy of the heart were assessed.Expression of Hypertrophy Markers:Western blot and qRT-PCR analyzed the expression of cardiac hypertrophy markers(MYH7,BNP).Fibrosis Assessment:Masson’s trichrome and Sirius Red staining were performed to determine the degree of fibrosis in cardiac tissue.Inflammation Markers:ELISA measured the expression of inflammatory factors TNF and IL6 in plasma.ResultsImmunofluorescence and m6A FISH:Showed an increase in global m6A and METTL3 expression levels in myocardial tissue during pathological cardiac hypertrophy.Prediction and RIP Experiments:Predicted 6 potential m6A sites on OTUD1 transcripts,with 3’-UTR identified as the primary m6A modification site.METTL3 Interaction:Overexpression of METTL3 increased OTUD1 protein expression and mRNA stability without altering mRNA levels;knocking down METTL3 reduced OTUD1 protein expression and mRNA stability.YTHDF1 Modulation:Knockdown of YTHDF1 reduced OTUD1 protein expression,while overexpression increased it.METTL3 and YTHDF1 Interaction:Knockdown of METTL3 decreased the binding of YTHDF1 to OTUD1 mRNA;the increase in OTUD1 protein expression caused by METTL3 overexpression was reversed by YTHDF1 knockdown.METTL3 Inhibitor STM2457:In the TAC mouse model,STM2457 mitigated the increase in body weight,heart weight,lung/body weight ratio,and heart weight/tibia length.These effects were nullified by AAV9-Otudl administration.Cardiac Function:STM2457 alleviated the decrease in ejection fraction(EF)and fractional shortening(FS)caused by cardiac hypertrophy,which was reversed upon AAV9-Otudl administration.H&E Staining:STM2457 reduced myocardial cross-sectional area in hypertrophic mice,an effect nullified by OTUD1 overexpression.Expression of Hypertrophy Markers:STM2457 alleviated the increase in BNP and MYH7 expression induced by pressure overload,an effect reversed upon AAV9-Otudl administration.Fibrosis:STM2457 mitigated myocardial and perivascular fibrosis in TAC-induced hypertrophic mice,an effect nullified by OTUD1 overexpression.Inflammation:STM2457 reduced the expression of inflammatory factors IL6 and TNF in plasma due to pathological cardiac hypertrophy,an effect reversed upon OTUD1 overexpression.ConclusionThe METTL3-YTHDF1-OTUD1 pathway increases the stability of OTUD1 mRNA,leading to elevated OTUD1 expression in the context of pathological heart enlargement.