The Role And Mechanisms Of MAP4 Phosphorylation In Pathological Cardiac Remodeling

Background:Our previous studies indicated that early serious burn injury led to hypoxia/ischemia of CMs（cardiomyocytes）and elevated microtubule associated protein 4（MAP4）phosphorylation was deemed an important mechanism of hypoxia/ischemia-related cardiac injury,however,the possible cardiac phenotype was still unknown under persistent MAP4phosphorylation.Thus,exploring the pathophysiological significance of constant MAP4phosphorylation might help us unravel the possible cardiac injury after persistent hypoxia/ischemia,and provide the therapeutic potential to ameliorate cardiac injury after serious burn injury.Meanwhile,persistent hypoxia/ischemia-related diseases,such as myocardial infarction（MI）or chronic heart failure,always led to pathological cardiac remodeling accompanied by cardiac hypertrophy,fibrosis,apoptosis or dysfunction,which was the common trigger of various heart diseases,but the detailed mechanisms that led to pathological cardiac remodeling was still unclear and how individual CM sensed the force pattern imposed by hypoxia/ischemia and subsequently transduced the signaling into sarcomere that resulted in cell thickening and lengthening were remained elusive.There were still no effective interventions for pathological cardiac remodeling up to now,therefore,investigating the potential role of MAP4 phosphorylation in cardiac remodeling was essential.As the key regulators of cytoskeletal dynamics,microtubule associated proteins（MAPs）could bind to tubulin and promote microtubule（MT）polymerization.The assembly-promoting MAPs include MAP2,tau and MAP4.MAP4 is ubiquitously expressed in non-neural cells and possesses an important role in MT dynamics.In addition,MAP-MT binding is regulated through MAPs phosphorylation,which leads to the detachment of MAPs from MT and subsequent destabilization of the MT.We and others have reported that the sites S696,S768 and S787 in the proline-rich region of MT-binding domain of human MAP4 are critical sites for phosphorylation governing its detachment from MTs,and that mitogen-activated protein kinase kinase 6（MKK6）/p38mitogen-activated protein kinase（p38/MAPK）is the upstream signaling for hypoxia-induced MAP4 phosphorylation with subsequent MT disassembly.There is also in vitro evidence for a crucial role for phosphorylated MAP4 in the pathophysiological process of cardiac mitochondrial dysfunction and apoptosis.Phosphorylated MAP4detached from MTs is found to be translocated to mitochondria from cytosol,leading to mitochondrial permeability transition pore opening and apoptosis.However,the clinical and in vivo implication of MAP4 phosphorylation is still debatable as that seen in CMs.To explore the widely significance of MAP4 phosphorylation in various heart diseases,we collected heart specimen of tetralogy of Fallot（TOF）and constructed MI and transverse aortic constriction（TAC）mouse models to obtain different pattern of heart samples.Subsequently,to investigate the effect of aberrant MAP4 phosphorylation in vivo,we generated mice that mimicked MAP4 hyperphosphorylation at specific sites（S737 and S760）.The purpose of this study was to provide therapeutic potential in the prevention and treatment of cardiac remodeling and related diseases.Materials and Methods:1.Fresh right ventricular tissues were collected from 16 TOF patients with hypoxemia（arterial oxygen saturation（SaO₂）<90%;n=8）or normal blood oxygen（SaO₂≥90%;n=8）in the Department of Cardiovascular Surgery,Xinqiao Hospital,Chongqing,China.Myocardial specimens were divided into two groups depending on the levels of SaO₂ upon admission.In addition,MI and TAC mouse models were induced by open-chest surgery in C57BL/6J mice and the left ventricular（LV）tissues of heart samples were obtained.All of these heart tissues were used to detect changes of MAP4（S696,S768 and S787,corresponding to the S667,S737 and S760 in mice）phosphorylation and p38/MAPK activation.2.Clustered regularly interspaced short palindromic repeats-associated protein 9（CRISPR/Cas9）technique was used to generate mouse strain with mutated MAP4 by knocking in the coding sequence（S667A,S737E and S760E）in the ninth exon of MAP4gene by homologous recombination.The MAP4（S667A,S737E,S760E）knock in（MAP4KI）mice were constructed and identified by the Shanghai Biomodel Organism Science&Technology Development Co.,Ltd.We explored the cardiac phenotypes of wild type（WT）and MAP4 KI mice using different approaches,including echocardiography and blood pressure analysis,commercial kits detection,immunoblotting and immunofluorescence examination,pathological sections staining,transmission electron microscope（TEM）and general observation,to elucidate the role of MAP4 phosphorylation in cardiac hypertrophy and fibrosis.In addition,to further assess the effects of MAP4 phosphorylation on cardiac remodeling in vitro,an overexpression adenovirus encoding human MAP4（alanine,Ala（A））,in which the serine at 768 and 787 was altered to Ala to mimick the nonphosphorylated form of MAP4,was generated and transfected to primary neonatal mouse CMs or fibroblasts with or without isoprenaline（ISO）stimulation.3.To detect CM apoptosis,necrosis,necroptosis,key enzyme of mitochondrial apoptosis and mitochondrial ultrastructure,LV heart tissues of WT and MAP4 KI mice were obtained at the 10-14,30-34 or 70-74 weeks of age.Additionally,we prepared mitochondrial and non-mitochondrial fractions,and examined mitochondrial cytochrome c release using immunoblotting;furthermore,we observed CM apoptosis and mitochondrial damage of heart tissues of TOF patients by pathological sections staining,TEM observation and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling（TUNEL）staining analysis.While in vitro experiments,to further demonstrate the effect of MAP4phosphorylation on cardiac apoptosis,we treated primary cultured CM under ISO stimulation with or without MAP4（Ala）transfection,and analyzed the above parameters with same methods.4.To explore the potential mechanism of MAP4 phosphorylation-induced pathological cardiac remodeling,we investigated the interaction between MAP4 and microtubule,the expression of polymerized（poly）tubulin and free tubulin and mitochondrial translocation of MAP4 phosphorylation.We obtained heart tissues of WT and MAP4 KI mice at 10-14and 70-74 weeks of age,and examined above parameters by immunoblotting and immunoprecipitation.Besides,we cultured primary mouse CM or fibroblast and detected CM apoptosis,cardiac remodeling,mitochondrial damage or mitochondrial cytochrome c release by immunoblotting,immunofluorescence or TEM observation under ISO stimulation with or without MAP4（Ala）or MKK6（glutamic acid,Glu）transfection or SB203580 pretreatment.Results:1.Using immunoblotting analysis,we observed markedly increased phosphorylation levels of MAP4 at S768 and S787 but little distinction at S696 in cardiac tissues from hypoxemic patients when compared with those in patients with normoxemia.Considering the co-existence of hypoxia and hypertrophy in these specimens,cardiac MAP4phosphorylation（S667,S737 and S760,corresponding to the S696,S768 and S787respectively in human）was detected in mice with MI or TAC.In both mouse models,MAP4 phosphorylation（S737 and S760）displayed a robust rise while phosphorylation of S667 just increased in the MI models,accompanied with intense activation of p38/MAPK pathway.Thus,the changes under MI and TAC pathological conditions further revealed a universal rise in cardiac MAP4 phosphorylation（S737 and S760）.2.To elucidate the physiological function of those phosphorylation sites of MAP4 in vivo,we generated mouse strain that mimicked the phosphorylated form of MAP4 at S737and S760,with unphosphorylated S667.We found elevated MAP4（S737 and S760）phosphorylation but little change of MAP4（S667）in heart,which indicated a successful mouse model.Then we found that there was an age-dependent increase in absolute whole heart weight（HW）or heart weight normalized by body weight（HW/BW）or by tibia length（HW/TL）in MAP4 KI mice by 22%,19%and 23%,respectively at 30-34 weeks of age,and by 32%,31%and 33%at 70-74 weeks of age while being comparable between the genotypes at 10-14 weeks of age.A pronounced thickening of the interventricular septum（IVS）and the ventricular wall was observed from 30 to 74 weeks with histological analysis.Cardiac morphology and function were analyzed using echocardiography.Thickness of end-diastolic and end-systolic IVS and the LV posterior wall as well as LV myocardial mass,were significantly and age-dependently greater in MAP4 KI mice than in WT littermates.Cardiac systolic and diastolic dysfunctions were showed age-dependent aggravation and diastolic dysfunction occurred earlier than systolic dysfunction.Besides,LV CM size,markers of myocardial hypertrophy,including atrial natriuretic peptide（ANP）and myosin heavy chain 7（MYH7）were all increased in MAP4 KI mice at 30-34 and 70-74 weeks of age,but not at 10-14 weeks versus respective WT littermates.The ultrastructure of heart was further observed by TEM.At 10-14 weeks of age,myofilament exhibited similar appearance between genotypes,while the myofilament dissolution and sparseness were evident in MAP4 KI mice aged at 30-34 and 70-74 weeks.For cardiac fibrosis,increased collagen（COL）deposition occurred in the perivascular area could be seen under TEM at the age of 30-34 and 70-74 weeks.Myocardial fibrotic area,detected by Masson’s trichrome staining,was significantly increased in MAP4 KI mice at the age of 70-74 weeks.Protein levels of COL1A1,COL1A2,COL3A1 and fibronectin,the primary extracellular matrix（ECM）components in LV heart tissue,were robustly increased in MAP4 KI mice at the age of 30-34 and 70-74 weeks compared with WT littermates,while no change was observed at the age of 10-14 weeks.Alpha-smooth muscle actin（α-SMA）,a marker of fibrosis in non-CM,was comparable between WT and MAP4 KI mice of 10-14 week-old,but increased in MAP4 KI mice at 30-34 and 70-74 weeks.In vitro experiments,using the primary cultured neonatal mouse cardiac myocytes or fibroblasts stimulated with ISO,we observed that MAP4（Ala）transfectants significantly reduced cardiac hypertrophy and fibrosis in MAP4 KI and WT mice,as reflected by decreased expression levels of ANP,brain natriuretic peptide（BNP）,MYH7 and ECM proteins secretion,as well as reduced CM cross sectional area andα-SMA expression.And the protective effects were more obvious in WT mice than MAP4 KI littermates.3.LV heart tissues of 10-14 and 70-74 week-old WT and MAP4 KI mice were examined.We found that there was an increase in cleaved caspase-3 protein levels,mitochondrial cytochrome c release,mitochondrial ultrastructural damage and apoptosis in hearts of 10-14 week-old MAP4 KI versus WT littermates,which was progressed to 70-74weeks.However,little changes of necrotic and necroptotic markers were detected between WT and MAP4 KI mice at different ages.In addition,we collected right ventricular tissues of TOF patients and demonstrated a robust elevation of CM apoptosis in hypoxic group than normoxic group.Furthermore,the in vitro experiments indicated that MAP4（Ala）transfection could suppress ISO-induced elevated cleaved caspase-3,mitochondrial cytochrome c release,TUNEL-positive CMs and mitochondrial ultrastructure disruption in WT and MAP4 KI mice.4.We have collected heart tissues of 10-14 and 70-74 week-old WT and MAP4 KI mice,and found that there was an reduction of poly-tubulin in parallel with an elevation of free tubulin in MAP4 KI mice as compared to WT littermates at the age of 10-14 weeks,in addition,mitochondrial translocation of MAP4 phosphorylation was still increase in MAP4KI mice compared with WT littermates,and this notion was progressed to 70-74 weeks of age.In vitro studies,pretreatment with p38/MAPK inhibitor SB203580 or transfection with MAP4（Ala）could inhibit ISO-mediated microtubule disassembly,increased mitochondrial translocation of phosphorylated MAP4 and mitochondrial ultrastructure disruption,CM apoptosis or ECM secretion in primary cultured neonatal mouse CMs or fibroblasts.Furthermore,MAP4（Ala）transfection could also suppress p38/MAPK activation-mediated CM apoptosis,microtubule depolymerization and mitochondrial damage.Conclusions:In summary,findings reported in our study reveal that MAP4 phosphorylation may serve as a springboard for the pathogenesis of multiple cardiomyopathies including cardiac hypertrophy,fibrosis,diastolic and systolic dysfunction,suggesting its therapeutic potentials in these comorbidities.Increased CM apoptosis together with microtubule disassembly and mitochondrial translocation of phosphorylated MAP4 was detected prior to the onset of cardiac remodeling and deemed possible mechanisms in MAP4phosphorylation-induced pathological cardiac remodeling,and p38/MAPK was demonstrated to be the possible signaling pathway that mediated MAP4（S737 and S760）phosphorylation.These findings help us recognize the pathophysiological effect of MAP4phosphorylation and bear the therapeutic potential to ameliorate persistent cardiac hypoxia/ischemia-induced pathological cardiac remodeling by attenuating MAP4phosphorylation.