Speg Deficiency In Mice Leads To Abnormal Mitochondria In Developing Cardiomyocytes Through PGC-1α Pathway

Part I Expression Profile of Speg and Mitochondria-related Genes in the Early Embryonic Stage in MiceBackground and Aims: Striated preferentially expressed gene(Speg)is a member of the myosin light chain kinase(MLCK)family.Our previous study showed that the deficiency of Speg in mice causes dilated cardiomyopathy,and mutations in the gene encoding Speg have also been found in patients with centronuclear myopathy,a congenital skeletal muscle disorder,who also have dilated cardiomyopathy.Previous studies have reported that Speg acts as a phosphokinase and regulates the phosphorylation of tropomyosin and calcium channel-related proteins,it plays a critical role in myofibrillar structure and calcium homeostasis in the fetal and adult heart,respectively.We also found that compared with the inducible knockout of Speg in adulthood,the embryonic knockout of Speg leads to a more rapid progression of heart failure and more obvious mitochondrial abnormalities.In addition,calcium channels and T tubes are still immature in the embryonic period,indicating that there may be different pathogenic mechanisms of Speg in the embryonic period.This part of the study aims to characterize the expression profile of Speg and mitochondria-related genes in the early embryonic stage and establish the relationship between Speg and mitochondrial development.Methods:(1)The expression of Speg in the heart of wild type(Speg+/+)mice at different developmental stages was measured by quantitative real-time polymerase chain reaction(q RT-PCR).(2)Heterozygote or homozygote of Speg knockout(Speg+/-or Speg-/-)mice were used for β-galactosidase activity staining.Tissue-specific expression and the expression level of Speg were measured.(3)Mitochondria and related genes of Speg+/+ and Speg-/-mice were labeled by immunofluorescence staining,such as PGC-1α,phosphorylated PGC-1α,TOM20,NCLX,etc.The earliest detectable mitochondrial signals between wild type and homozygote were compared and quantified.(4)Through the public database,Mouse Organogenesis Spatiotemporal Transcriptomic Atlas(MOSTA)and Evo-devo mammalian organs(EDMO),the spatial and temporal expression of Speg and Ppargc1α genes were analyzed.Results:(1)The expression of Speg gene was detected as early as embryonic(E)day 7.5 and increased steadily during the embryonic development(E10.5,E16.5,E18.5,P2,10wks).The expression level of Speg gene in adult mice was slightly lower than that in newborn mice.(2)Lac Z,an inserted reporter gene for Speg expression,was detected as early as E7.5 and expressed in both the first and second heart fields.Lac Z the expression was detected in primitive atria and ventricles at E8.5.Lac Z was detected in the outflow tract at E18.5.(3)Lac Z expression was tissue-specific during embryonic development and was not detected in other organs except the heart;(4)PGC-1α and phosphorylated PGC-1αwere positive at E7.5 and E8.5,respectively;(5)The mitochondrial signals,NCLX and TOM20,were not expressed at E7.5,but expressed at E8.5.(6)Immunofluorescence staining at E11.5 showed that the expression of NCLX in the hearts of Speg-/-mice was significantly lower than that of Speg+/+ mice(p < 0.05).(7)Speg and Ppargc1α co-localized at cardiomyocytes during the embryonic development and shared similar expression profiles.Conclusions:(1)Speg expressed in fetal heart as early as E7.5(the gestational age at which heart begins to develop)without detectable mitochondrial signal;(2)Speg expressed only in the heart during development,and Speg deficiency does not cause structural cardiac malformations during development;(3)Mitochondrial dysfunction was observed in Speg-/-mice and the phenotype was earlier than that of heart failure.(4)Speg and Ppargc1α are highly correlated at embryonic stage.Part II The Abnormalities of Mitochondrial Structure and Function in the Heart of Speg Knockout MiceBackground and Aims: Mitochondrial disorders are often associated with high mortality at birth.The density of mitochondria in myocardium is large,and heart is the tissue that consumes the highest amount of ATP per unit weight in the body,but its capacity of energy reserve is relatively limited.Thus,shortly after birth,mitochondria must adapt to the transition from low oxygen level in utero to an aerobic environment and the rapid increase in energy demands during beating of the heart.Mitochondrial oxidative phosphorylation is the most efficient way to produce ATP,and the density and mass of mitochondria increase dramatically during the perinatal period.In part I,we found that the expression of Speg precedes mitochondrial signal and is highly correlated with PGC-1α,a critical regulator of mitochondrial development.However,how Speg affects mitochondrial structure and function remains unclear.In this part of the study,we investigated the structural and functional abnormalities of cardiac mitochondria in Speg knockout mice.Methods: Wild type(Speg+/+)and Speg knockout(Speg-/-)mice were used in this study.(1)Transmission electron microscopy(TEM)and ultra high-resolution laser scanning confocal microscopy(Airy Scan)were used to observe the changes of cardiac mitochondria and their ultrastructure.(2)Compare the differences of metabolism substrates,ATP production rate and ATP concentration of cardiomyocytes by Seahorse and ATP kit;(3)Flow cytometry analysis or fluorescent labeling was used to detect mitochondrial oxidative stress,mitochondrial membrane potential,mitochondrial edema and mitochondrial distribution.(4)Quantitative real-time polymerase chain reaction(q RT-PCR)was used to analyze the differences in the number of mitochondria and mitochondrial fission and fusion signals;(5)Apoptotic signals in cardiac tissue were detected by Western Blotting and fluorescent staining.Results:(1)Mitochondria in Speg+/+ hearts were distributed along myofibrils and filled with dense cristae and matrix,while mitochondria in Speg-/-hearts were irregularly distributed around the nucleus and filled with abnormal cristae and translucent matrix;(2)The percentage of Speg+/+ mitochondria with class III(33.5±1.8%)and class IV(52.3±6.8%)was higher than that of Speg-/-mitochondria with class I(26.4±2.5%)and class II(67.1±2.6%).(3)Compared with Speg+/+ cells,the ATP concentration of Speg-/-cells was significantly decreased(p < 0.05).The ATP production rate of aerobic oxidation was significantly decreased(p < 0.05),while there was no difference in glycolysis.When palmitate acid was used as substrate,the maximum oxygen consumption rate(OCR)decreased significantly(p < 0.05),and it was not sensitive to etomoxir.(4)Compared with Speg+/+ cells,Mito Sox level was increased(p < 0.05),TMRM,JC-1 parameters and mt DNA/n DNA ratio were decreased(p < 0.05)in Speg-/-cells.(5)Compared with Speg+/+ group,Speg-/-group showed no obvious edema,no PARP and caspase 3 activation,no difference in the percentage of TUNEL positive cells,and no difference in mitochondrial fission and fusion signals.Conclusion: Compared with Speg+/+,the mitochondria in Speg-/-myocardium at E18.5 showed(1)structural abnormalities,such as decrease in number and lack of crista;(2)functional abnormalities,such as decreased ATP concentration,decreased ATP production rate,and decreased fatty acid utilization capacity;Increased oxidative stress level;Decreased membrane potential;Decreased biogenesis;(3)Taken together,the mitochondria in Speg-/-was relatively immature.Part III Speg Deficiency Donwregulates Mitochondrial ATP Synthesis through PGC-1α PathwayBackground and Aims: As a member of the MLCK family,Speg contains two tandem serine/threonine kinase domains(SK).Tropomyosin,Serca2 a,Ry R2 and JPH2,are known to interact with Speg and be phosphorylated.The PGC-1 family is central to the regulation of mitochondrial development and function in cardiomyocytes during embryonic development.Its expression is regulated by extracellular and physiological signals.For example,the expression of PGC-1α is suppressed in many models of heart failure;the expression of PGC-1α gradually increases over time during cardiac development.PGC-1α protein enters the nucleus and acts as a transcriptional coactivator to regulate energy metabolism by interacting with nuclear respiratory factors(NRFs),estrogen related receptors(ERRs)and peroxisome proliferator-activated receptors(PPARs).In the first two parts of the study,we found that there is a high spatial and temporal overlap between Speg and PGC-1α(Ppargc1α)during embryonic period,and the deficiency of Speg leads to mitochondrial dysfunction in the fetal heart.In this part of the study,we aimed to clarify whether Speg interacts with PGC-1α and phosphorylates PGC-1α to regulate mitochondrial function.Methods: Speg+/+ and Speg-/-mice were used in this study.(1)The interaction between Speg and PGC-1α,mitochondrial calcium channels such as NCLX,VDAC1 and MCU were verified by co-immunoprecipitation or immunofluorescence staining.(2)q RT-PCR was used to verify the changes in the downstream signaling pathways of PGC-1α.(3)The phosphorylation of PGC-1α and its upstream signaling pathway were verified by Western Blotting.Primary cardiomyocytes from Speg+/+ and Speg-/-mice were used for study.(1)SK1 of Speg and PGC-1α were overexpressed or silenced by plasmid transfection or small interfering RNA(si RNA).(2)The phosphorylation of PGC-1α and restoration of PGC-1α nuclear translocation were detected by immunofluorescence staining;(3)The recovery of mitochondrial function was detected by ATP kit.(4)Airy Scan was used to detect the recovery of mitochondrial structure.Results:(1)Co-immunoprecipitation showed that Speg did not interact with mitochondrial calcium channel-related proteins,such as NCLX,VDAC1 and MCU,but with PGC-1α.Immunofluorescence staining showed that Speg and PGC-1α co-stained in cardiomyocytes.(2)q RT-PCR results showed that NRF1,PPARα and ERRα/β/γ were significantly down-regulated in Speg-/-mice compared with Speg+/+ mice(p < 0.05).(3)Western Blotting showed that the protein and phosphorylation of PGC-1α were significantly down-regulated in Speg-/-mice compared with Speg+/+ mice(p < 0.05).The upstream regulators of PGC-1α,such as p Ca MKII/Ca MKII,p MAPK/MAPK,p AKT/AKT,p GSK3β/GSK3β,p PKA/PKA,SIRT1,AMPK1/2,and Ca N,were not significantly different between the two groups.(4)Overexpression of SK1 in 293 t cells significantly increased the protein level of phosphorylated PGC-1α(p < 0.05).The ATP concentration was significantly increased too(p < 0.05).(5)Overexpression of SK1 in Speg-/-cardiomyocytes significantly increased the proportion of phosphorylated PGC-1α and reached the same level as Speg+/+ cardiomyocytes(p < 0.05).(6)Overexpression of SK1 in Speg-/-primary cardiomyocytes significantly increased ATP concentration and reached the same level as Speg+/+ cells(p < 0.05),while the effects can be abolished by PGC-1α knockdown.Conclusions:(1)Speg and PGC-1α partially colocalize in cardiomyocytes,and there is a direct protein-protein interaction between Speg and PGC-1α.(2)Speg regulated mitochondrial ATP synthesis through PGC-1α phosphorylation.