Congenital heart diseases are the most common birth defects around the world. Emerging evidence suggests that mitochondrial homeostasis is required for normal heart development. In mitochondria, a series of molecular chaperones including heat shock protein 60 (HSP60) are engaged in assisting the import and folding of mitochondrial proteins. However, it remains largely obscure whether and how these mitochondrial chaperones regulate cardiac development. Here, we generated a cardiac-specific Hspd1 deletion mouse model by αMHC-Cre and investigated the role of HSP60 in cardiac development. We observed that deletion of HSP60 in embryonic cardiomyocytes resulted in abnormal heart development and embryonic lethality, characterized by reduced cardiac cell proliferation and thinner ventricular walls, highlighting an essential role of cardiac HSP60 in embryonic heart development and survival. Our results also demonstrated that HSP60 deficiency caused significant downregulation of mitochondrial ETC subunits and induced mitochondrial stress. Analysis of gene expression revealed that P21 that negatively regulates cell proliferation is significantly upregulated in HSP60 knockout hearts. Moreover, HSP60 deficiency induced activation of eIF2α-ATF4 pathway, further indicating the underlying mitochondrial stress in cardiomyocytes after HSP60 deletion. Taken together, our study demonstrated that regular function of mitochondrial chaperones is pivotal for maintaining normal mitochondrial homeostasis and embryonic heart development.

Aim Transcriptional regulation of gene expression plays a crucial role in orchestrating complex morphogenetic and molecular events during heart development and function. Mediator complex is an essential multi-subunit protein complex that governs gene expression in eukaryotic cells. Although Mediator subunits (MEDs) work integrally in the complex, individual MED component displays specialized functions. MED27, categorized as an Upper Tail subunit, possesses an as-yet-uncharacterized function. In this study, we aimed to investigate the physiological role of MED27 in cardiomyocytes. Materials and methods we generated a Med27 floxed mouse line, which was further used to generate constitutive (cKO) and inducible (icKO) cardiomyocyte-specific Med27 knockout mouse models. Morphological, histological analysis and cardiac physiological studies were performed in Med27 cKO and icKO mutants. Transcriptional profiles were determined by RNA sequencing (RNAseq) analysis. Key fundings Ablation of MED27 in developing mouse cardiomyocytes results in embryonic lethality, while its deletion in adult cardiomyocytes leads to heart failure and mortality. Similar to the ablation of another Upper Tail subunit, MED30 in cardiomyocytes, deletion of MED27 leads to decreased protein levels of most MEDs in cardiomyocytes. Interestingly, overexpression of MED30 fails to restore the protein levels of Mediator subunits in MED27-deficient cardiomyocytes, demonstrating that the role of MED27 in maintaining the integrity and stability of the Mediator complex is independent of MED30. Significance Our results revealed an essential role of MED27 in cardiac development and function by maintaining the stability of the Mediator core.

Aims: Lipids are essential cellular components with many important biological functions. Disturbed lipid biosynthesis and metabolism has been shown to cause cardiac developmental abnormality and cardiovascular diseases. In this study, we aimed to investigate the composition and the molecular profiles of lipids in mammalian hearts between embryonic and adult stages and uncover the underlying links between lipid and cardiac development and maturation. Materials and methods: We collected mouse hearts at the embryonic day 11.5 (E11.5), E15.5, and the age of 2 months, 4 months and 10 months, and performed lipidomic analysis to determine the changes of the composition, molecular species, and relative abundance of cardiac lipids between embryonic and adult stages. Additionally, we also performed the electronic microscopy and RNA sequencing in both embryonic and adult mouse hearts. Key findings: The relative abundances of certain phospholipids and sphingolipids including cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and ceramide, are different between embryonic and adult hearts. Such lipidomic changes are accompanied with increased densities of mitochondrial membranes and elevated expression of genes related to mitochondrial formation in adult mouse hearts. We also analyzed individual molecular species of phospholipids and sphingolipids, and revealed that the composition and distribution of lipid molecular species in hearts also change with development.

目的 探讨MC1568对胎牛血清（FBS）诱导的大鼠乳鼠心肌细胞肥大的作用及其机制。方法 本实 验时间为2021年5月至2022年11月。选取出生3 d内的SPF级SD大鼠乳鼠40只，分离并培养其心肌细胞。取原代大鼠乳 鼠心肌细胞，将其随机分为对照组（不进行干预）、FBS组（加入20% FBS培养48 h以诱导心肌细胞肥大）、曲古抑 菌素A（TSA）组（加入20% FBS及200 nmol/L TSA培养48 h）、MC1568组（加入20% FBS及10 μmol/L MC1568培养 48 h）。采用α-辅肌动蛋白染色检测各组心肌细胞表面积，采用RT-qPCR法检测各组心肌肥大标志物〔心房利钠肽 （ANP）、β-肌球蛋白重链（MHC）〕mRNA表达水平，采用Western blot检测各组心肌细胞中P300、组蛋白去乙酰 化酶（HDAC）4、磷酸化HDAC4（pHDAC4）表达水平。结果 FBS组心肌细胞表面积大于对照组、TSA组、MC1568 组（P＜0.05）。FBS组ANP、β-MHC mRNA表达水平高于对照组、TSA组、MC1568组（P＜0.05）。四组心肌细胞 中HDAC4表达水平比较，差异无统计学意义（P＞0.05）；FBS组心肌细胞中P300表达水平高于对照组、TSA组， pHDAC4表达水平高于对照组、TSA组、MC1568组（P＜0.05）。结论 FBS可引起心肌细胞肥大，而MC1568可以抑制 心肌细胞肥大，其保护作用可能与抑制HDAC4/MEF2D信号通路有关。

HomeCirculationVol. 146, No. 13Mitochondrial Stress Induces an HRI-eIF2α Pathway Protective for Cardiomyopathy Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBMitochondrial Stress Induces an HRI-eIF2α Pathway Protective for Cardiomyopathy Siting Zhu, Anh Nguyen, Jing Pang, Jun Zhao, Ze’e Chen, Zhengyu Liang, Yusu Gu, Helen Huynh, Yutong Bao, Sharon Lee, Yuval Kluger, Kunfu Ouyang, Sylvia M. Evans and Xi Fang Siting ZhuSiting Zhu https://orcid.org/0000-0002-6950-9556 Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (S.Z., Z.C., K.O.). Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Anh NguyenAnh Nguyen https://orcid.org/0000-0003-1272-6856 Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Jing PangJing Pang Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Jun ZhaoJun Zhao Department of Pathology, Yale School of Medicine, New Haven, CT (J.Z., Y.K.). , Ze’e ChenZe’e Chen Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (S.Z., Z.C., K.O.). Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Zhengyu LiangZhengyu Liang https://orcid.org/0000-0002-3307-0959 Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Yusu GuYusu Gu Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Helen HuynhHelen Huynh Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Yutong BaoYutong Bao https://orcid.org/0000-0003-4021-2991 Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Sharon LeeSharon Lee Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. , Yuval KlugerYuval Kluger Department of Pathology, Yale School of Medicine, New Haven, CT (J.Z., Y.K.). , Kunfu OuyangKunfu Ouyang Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (S.Z., Z.C., K.O.). , Sylvia M. EvansSylvia M. Evans https://orcid.org/0000-0001-5035-3697 Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. Department of Pharmacology (S.M.E.), University of California San Diego, La Jolla. Skaggs School of Pharmacy and Pharmaceutical Sciences (S.M.E.), University of California San Diego, La Jolla. and Xi FangXi Fang Correspondence to: Xi Fang, PhD, Department of Medicine, University of California San Diego, 9500 Gilman Dr, Mail Code 0613-C, La Jolla, CA 92093. Email E-mail Address: [email protected] https://orcid.org/0000-0001-7816-8821 Department of Medicine (S.Z., A.N., J.P., Z.C., Z.L., Y.G., H.H., Y.B., S.L., S.M.E., X.F.), University of California San Diego, La Jolla. Originally published26 Sep 2022https://doi.org/10.1161/CIRCULATIONAHA.122.059594Circulation. 2022;146:1028–1031Mitochondrial dysfunction elicits a mitochondrial stress response (MSR) through mitochondrial-nuclear communication and activates activating transcription factor 4 (ATF4), a master transcriptional regulator of the cellular stress response.1 Knowledge of consequences of MSR-triggered ATF4 activation in mitochondrial cardiomyopathy is limited yet critical for therapeutic approaches.Mitochondrial phosphatase protein tyrosine phosphatase mitochondrial 1 (Ptpmt1) cKO (PKO) mice2 provide a model to investigate in vivo mechanisms of MSR in cardiomyocytes, displaying key features of fetal mitochondrial cardiomyopathy while evidencing lethality between embryonic day (E) 16.5 and 18.5.2 All mouse protocols were approved by the Institutional Animal Care and Use Committee. Molecular analyses confirmed that loss of PTPMT1 in cardiomyocytes resulted in upregulation of ATF4 and its target genes at E11.5 (Figure [A–D]), similar to other MSR models. The most well-recognized upstream regulator of ATF4 is eukaryotic initiation factor 2α (eIF2α) phosphorylation, which increases ATF4 translation. Atf4 is also a direct target of transcriptional repression by hypoxia-inducible factor-1α (HIF1α) in cardiomyocytes. Hif1α cKO hearts display increased Atf4 mRNA and protein. The mammalian target of rapamycin (mTOR) is another upstream regulator of ATF4. We examined these upstream regulators of ATF4 in PKO hearts and found that phosphorylation of eIF2α was significantly increased in PKO versus control hearts at E11.5, whereas HIF1α and mTOR phosphorylation were not altered (Figure [E]), suggesting that eIF2α phosphorylation induced the expression of ATF4.Download figureDownload PowerPointFigure. HRI-eIF2α pathway benefits mitochondrial cardiomyopathy. A, Volcano plot obtained from DESeq2 analysis of gene expression in Ptpmt1 cardiomyocyte-specific knockout (Ptpmt1f/f; Xml-Cre+; PKO) vs Cre-negative control hearts at embryonic day E 11. 5. Genes with adjusted P<0.05 and log2 (fold change) >1 are considered significantly upregulated or downregulated genes in PKO hearts. Green indicates downregulated genes; red indicates upregulated genes. n=3 per group. RNA-sequencing data were deposited to the GEO database (Accession No. GSE201042). B, Functional clustering analysis using the Reactome database revealed that deletion of PTPMT1 in cardiomyocytes significantly upregulates pathways involved in amino acid (AA) metabolism, which is classically found to be regulated by activating transcription factor 4 (ATF4). C, Motif enrichment analysis at promoter regions (±2 kb from transcription start sites) of the genes that were upregulated in PKO hearts revealed that the top enriched transcription factor binding motifs were ATF4 and its cofactors C/EBP (CCAAT/enhancer-binding protein) or C/EBP homologous protein binding motifs. D, Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) validated the upregulation of classic ATF4 target genes in PKO (red) vs control (Ctrl; blue) hearts at E11.5. n=3 to 4 per group. E, Western blot analysis of ATF4, phosphorylated eukaryotic initiation factor 2α (eIF2α) at serine 51 (Ser51), phosphorylated mammalian target of rapamycin (mTOR) at serine 2448 (Ser2448), and hypoxia-inducible factor-1α (HIF1α) in PKO and Ctrl hearts at E11.5. n=4 per group. F, Model for eIF2α-mediated translational control of ATF4 in response to diverse cellular stress responses. G, Western blot analysis of ATF4 and phosphorylated eIF2α at Ser51 and ATF4 in eIF2αS51A mutant (Ptpmt1f/f; eIF2αm/m; Xml-Cre−; S51A), PKO (Ptpmt1f/f; eIF2α+/+; Xml-Cre+), PKO/eIF2αS51A double-mutant (dMut; Ptpmt1f/f; eIF2αm/m; Xml-Cre+), and control (Ctrl; Ptpmt1f/f; eIF2α+/+; Xml-Cre−) hearts at E11.5. n=4 per group. H, qRT-PCR analysis of ATF4 target genes in S51A, PKO, dMut, and Ctrl hearts at E11.5. n=3 to 4 per group. I, Heat map representation of transcript levels of selected ATF4 target genes in S51A, PKO, dMut, and Ctrl hearts at E11.5. J, Heat map representation of transcript levels of selected endoplasmic reticulum stress–induced genes, including the target genes of ATF4, and XBP1 and ATF6 in PKO and Ctrl hearts at E11.5. K, Heat map representation of transcript levels of selected ATF4 target genes in PKO/GCN2 double-knockout (dKO; Ptpmt1f/f-Eif2ak4−/−; Xml-Cre+) and wild-type (WT) control (Ptpmt1+/+-Eif2ak4+/+; Xml-Cre−) hearts (left) and HRI-null (HKO; Ptpmt1f/f; Eif2ak4−/−; Xml-Cre−), PKO (Ptpmt1f/f; Eif2ak4+/+; Xml-Cre+), PKO/HRI dKO (Ptpmt1f/f; Eif2ak4−/−; Xml-Cre+), and Ctrl (Ptpmt1f/f; Eif2ak4+/+; Xml-Cre−) hearts (right). Note that the genes that encode GCN2 and PTPMT1 are at the same allele, so we could not obtain single-knockout littermates. L, qRT-PCR analysis of ATF4 target genes in PKO/GCN2 dKO and WT hearts at E11.5. n=5 to 7 per group. M, Western blot analysis of ATF4 and phosphorylated eIF2a at Ser51 in PKO/GCN2 dKO and WT hearts. n=4 per group. N, qRT-PCR analysis of ATF4 target genes in HKO, PKO, PKO/HRI dKO, and Ctrl hearts at E11.5. n=3 to 4 per group. O, Western blot analysis of ATF4 and phosphorylated eIF2a at Ser51 in HKO, PKO, PKO/HRI dKO, and Ctrl hearts. n=4 per group. P, Whole embryonic (top) and heart (bottom) morphology of S51A, PKO, dMut, and Ctrl hearts at E13.5. Scale bar, 1 mm. Q, Whole embryonic (top) and heart (bottom) morphology of HKO, PKO, PKO/HRI dKO, and Ctrl hearts at E14.5. Scale bar, 1 mm. R, qRT-PCR analysis of ATF4 target genes in HKO (Tazf/Y; Eif2ak4−/−; Xml-Cre−), Tafazzin cardiomyocyte-specific knockout (TKO; Tazf/Y; Eif2ak4+/+; Xml-Cre+), TKO/HRI dKO (Tazf/Y; Eif2ak4−/−; Xml-Cre+), and Ctrl (Tazf/Y; Eif2ak4+/+; Xml-Cre−) hearts at postnatal day (P) 7. n=3 to 4 per group. S, Western blot analysis of ATF4 and phosphorylated eIF2α at Ser51 in HKO, TKO, TKO/HRI dKO, and Ctrl hearts at P7. n=4 per group. T, Cardiac ventricular weight to body weight ratio for HKO, TKO, TKO/HRI dKO, and Ctrl hearts at P7. n= 5 to 7 per group. U, Whole-mount (top) and hematoxylin and eosin–stained sections (bottom) for HKO, TKO, TKO/HRI dKO, and Ctrl hearts at P8. Scale bar, 1 mm. V, Echocardiographic measurements of left ventricular percentage of fractional shortening (left), end-diastolic left ventricular internal diameter (LVIDd; middle), and end-systolic left ventricular internal diameter (LVIDs; right) for HKO, TKO, TKO/HRI dKO, and Ctrl mice at P7. n=4 to 6 mice per group. GAPDH was used as a loading control for Western blots. qRT-PCR data were normalized to corresponding 18S levels, and levels in mutants are expressed as the fold change vs Ctrl. Data are represented as mean±SEM. *P<0.05, PKO, TKO, or dMut or dKO vs Ctrl or as indicated. #P<0.05, dMut or dKO vs PKO or TKO or as indicated by 2-tailed Student t test (2-group comparison) or 2-way ANOVA (4-group comparison). AA indicates amino acid; DEG, indicates differentially expressed gene; His, histidine; Lys, lysine; Phe, phenylalanine; SLC, solute carrier superfamily; and Trp, tryptophan.Phosphorylation of eIF2α at serine 51 integrates signals from diverse cellular stress responses (Figure [F]).1 We generated a phosphorylation-resistant eIF2α-mutant allele1 in which the serine 51 phosphorylation site was mutated to alanine (eIF2αS51A) and crossed this allele into PKO mice to generate PKO/eIF2αS51A double-mutant (dMut) mice. The absence of eIF2α phosphorylation in eIF2αS51A and dMut hearts was validated (Figure [G]). ATF4 protein and downstream targets were significantly decreased to baseline levels in dMut hearts (Figure [G–I]), confirming that eIF2α phosphorylation was essential for MSR-triggered ATF4 activation in PKO hearts.Four eIF2α kinases are activated by distinct forms of stress (Figure [F]).1 RNA-sequencing analysis from PKOs indicated increased expression of ATF4 targets but no increase in XBP1 or ATF6 (Figure [J]), the latter negating the likelihood that a eukaryotic translation initiation factor 2 alpha kinase 3 (PERK)-mediated endoplasmic reticulum (ER) stress response was involved.1 Next, we generated null alleles for eukaryotic translation initiation factor 2 alpha kinase 4 (GCN2) or heme-regulated eIF-2alpha kinase (HRI) and generated PKO/GCN2 and PKO/HRI double-knockout (dKO) mice. Loss of HRI abolished activation of eIF2α-ATF4 signaling in PKOs, whereas eIF2α-ATF4 remained activated in PKO/GCN2 dKO hearts (Figure [K–O]). Thus, MSR-induced eIF2α-ATF4 signaling was dependent on HRI, and other eIF2α kinases were not involved. Our results are consistent with recent in vitro findings in HeLa cells,3,4 providing the first demonstration of the in vivo relevance of this pathway in mitochondrial cardiomyopathy. It is not clear whether activation of HRI depends on the amount of heme in cardiomyocytes.Whether the eIF2α pathway was adaptive or maladaptive remained to be addressed. Although global suppression of protein synthesis by eIF2α phosphorylation conserves energy and increasing ATF4 translation allows cells to survive periods of stress, persistent ATF4 and suppression of protein synthesis may also be detrimental. Homozygous eIF2αS51A mutants die within 18 hours after birth.1 However, constitutive activation of eIF2α by deleting both eIF2α phosphatases Ppp1r15a and Ppp1r15b results in embryonic lethality.1 Thus far, in vivo physiological consequences of eIF2α phosphorylation in response to stress have not been addressed. We analyzed the morphology and survival of PKO/eIF2αS51A dMut mice. PKO/eIF2αS51A dMut mice died at E13.5, whereas PKO mice survived at this stage with abnormal heart morphology, demonstrating that blocking eIF2α phosphorylation negatively impacted survival (Figure [P]).Although HRI-null mice were viable1 and displayed normal cardiac development and function at baseline, the role of HRI activation in mitochondrial cardiomyopathy remained to be addressed. We found that PKO/HRI dKO mice died between E14.5 and E16.5, whereas PKO littermates survived with abnormal cardiac morphology (Figure [Q]). Thus, although milder than effects of the eIF2α S51A mutant, deletion of HRI was also detrimental to survival. Thus HRI-eIF2α activation was protective for fetal mitochondrial cardiomyopathy.To determine the role of HRI in adult mitochondrial cardiomyopathy, we crossed HRI-knockout mice with Tafazzin cKO (TKO) mice. TKO mice display mitochondrial dysfunction at 2 months and dilated cardiomyopathy at 4 months, but they survive >1 year with impaired cardiac function.5 Western blot and qRT-PCR analysis confirmed that eIF2α-ATF4 signaling was activated in TKO hearts but abolished in TKO/HRI dKO hearts (Figure [R and S]). TKO/HRI dKO mice died between postnatal days 7 and 10 with enlarged hearts, compared with TKO mice that survived >1 year.5 We also observed increased ratios of ventricular weight to body weight in dKO mice (Figure [T and U]). Echocardiographic analysis revealed severe cardiac dysfunction in dKO mice (Figure [V]). Thus, MSR-triggered HRI-eIF2α was also protective for adult mitochondrial cardiomyopathy.Overall, we demonstrated that an HRI-eIF2α pathway mediated mitochondrial-nuclear communication and MSR-triggered ATF4 activation in both embryonic and adult heart. Our results uncovered a protective role for MSR-triggered HRI-eIF2α-ATF4 signaling in both fetal and adult mitochondrial cardiomyopathy. Therefore, intention to inhibit the HRI-eIF2α-ATF4 pathway in mitochondrial cardiomyopathy could be detrimental, rather than beneficial, for cardiac dysfunction.The data, analytical methods, and study materials that support the findings of this study will be available to other researchers from the corresponding authors on reasonable request. RNA-sequencing data were deposited to the GEO database (Accession No. GSE201042).Article InformationSources of FundingDr Fang is supported by National Institutes of Health grants. Dr Evans is supported by National Institutes of Health grants and the Foundation Leducq (16 CVD 03).Nonstandard Abbreviations and AcronymsATF4activating transcription factor 4dKOdouble-knockoutdMutdouble-mutanteIF2αeukaryotic initiation factor 2αHRIheme-regulated inhibitorMSRmitochondrial stress responsePKOphosphatase Ptpmt1 cKO micePTPMT1Protein Tyrosine Phosphatase Mitochondrial 1qRT-PCRQuantitative reverse transcriptase -polymerase chain reactionTKOTafazzin cKODisclosures Dr Zhao is currently employed by Guardant Health. This work is not related to her employment at Guardant Health. The other authors report no conflicts.FootnotesCirculation is available at www.ahajournals.org/journal/circ*S. Zhu, A. Nguyen, and J. Pang contributed equally.For Sources of Funding and Disclosures, see page 1031.Correspondence to: Xi Fang, PhD, Department of Medicine, University of California San Diego, 9500 Gilman Dr, Mail Code 0613-C, La Jolla, CA 92093. Email [email protected]eduReferences1. Costa-Mattioli M, Walter P. The integrated stress response: from mechanism to disease.Science. 2020; 368:eaat5314.CrossrefMedlineGoogle Scholar2. Chen Z, Zhu S, Wang H, Wang L, Zhang J, Gu Y, Tan C, Dhanani M, Wever E, Wang X, et al. PTPMT1 is required for embryonic cardiac cardiolipin biosynthesis to regulate mitochondrial morphogenesis and heart development.Circulation. 2021; 144:403–406. doi: 10.1161/CIRCULATIONAHA.121.054768LinkGoogle Scholar3. Fessler E, Eckl EM, Schmitt S, Mancilla IA, Meyer-Bender MF, Hanf M, Philippou-Massier J, Krebs S, Zischka H, Jae LT. A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol.Nature. 2020; 579:433–437. doi: 10.1038/s41586-020-2076-4CrossrefMedlineGoogle Scholar4. Guo X, Aviles G, Liu Y, Tian R, Unger BA, Lin YT, Wiita AP, Xu K, Correia MA, Kampmann M. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.Nature. 2020; 579:427–432. doi: 10.1038/s41586-020-2078-2CrossrefMedlineGoogle Scholar5. Zhu S, Chen Z, Zhu M, Shen Y, Leon LJ, Chi L, Spinozzi S, Tan C, Gu Y, Nguyen A, et al. Cardiolipin remodeling defects impair mitochondrial architecture and function in a murine model of Barth syndrome cardiomyopathy.Circ Heart Fail. 2021; 14:e008289. doi: 10.1161/CIRCHEARTFAILURE.121.008289LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Huynh H, Zhu S, Lee S, Bao Y, Pang J, Nguyen A, Gu Y, Chen C, Ouyang K, Evans S and Fang X (2023) DELE1 is protective for mitochondrial cardiomyopathy, Journal of Molecular and Cellular Cardiology, 10.1016/j.yjmcc.2022.12.003, 175, (44-48), Online publication date: 1-Feb-2023. September 27, 2022Vol 146, Issue 13 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.122.059594PMID: 36154620 Originally publishedSeptember 26, 2022 Keywordsmitochondriaheme-regulated eIF-2alpha kinasecardiomyopathyactivating transcription factor 4PDF download Advertisement SubjectsCardiomyopathyCell Signaling/Signal TransductionPhysiology

Background: Cardiomyopathy is a major clinical feature in Barth syndrome (BTHS), an X-linked mitochondrial lipid disorder caused by mutations in Tafazzin ( TAZ ), encoding a mitochondrial acyltransferase required for cardiolipin remodeling. Despite recent description of a mouse model of BTHS cardiomyopathy, an in-depth analysis of specific lipid abnormalities and mitochondrial form and function in an in vivo BTHS cardiomyopathy model is lacking. Methods: We performed in-depth assessment of cardiac function, cardiolipin species profiles, and mitochondrial structure and function in our newly generated Taz cardiomyocyte-specific knockout mice and Cre-negative control mice (n≥3 per group). Results: Taz cardiomyocyte-specific knockout mice recapitulate typical features of BTHS and mitochondrial cardiomyopathy. Fewer than 5% of cardiomyocyte-specific knockout mice exhibited lethality before 2 months of age, with significantly enlarged hearts. More than 80% of cardiomyocyte-specific knockout displayed ventricular dilation at 16 weeks of age and survived until 50 weeks of age. Full parameter analysis of cardiac cardiolipin profiles demonstrated lower total cardiolipin concentration, abnormal cardiolipin fatty acyl composition, and elevated monolysocardiolipin to cardiolipin ratios in Taz cardiomyocyte-specific knockout, relative to controls. Mitochondrial contact site and cristae organizing system and F1F0-ATP synthase complexes, required for cristae morphogenesis, were abnormal, resulting in onion-shaped mitochondria. Organization of high molecular weight respiratory chain supercomplexes was also impaired. In keeping with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity. Conclusions: Our mouse model mirrors multiple physiological and biochemical aspects of BTHS cardiomyopathy. Our results give important insights into the underlying cause of BTHS cardiomyopathy and provide a framework for testing therapeutic approaches to BTHS cardiomyopathy, or other mitochondrial-related cardiomyopathies.

HomeCirculationVol. 144, No. 5PTPMT1 Is Required for Embryonic Cardiac Cardiolipin Biosynthesis to Regulate Mitochondrial Morphogenesis and Heart Development Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBPTPMT1 Is Required for Embryonic Cardiac Cardiolipin Biosynthesis to Regulate Mitochondrial Morphogenesis and Heart Development Ze’e Chen, PhD Siting Zhu, BS Hong Wang, MS Li Wang, BS Jianlin Zhang, PhD Yusu Gu, MD Changming Tan, MD Mehul Dhanani, BS Eric Wever, MS Xinru Wang, BS Boyu Xie, BS Shijia Wang, MD Lei Huang, MD Antoine H.C. van Kampen, PhD Jie Liu, PhD Zhen Han, MD Hemal H. Patel, PhD Frédéric M. Vaz, PhD Xi Fang, PhD Ju Chen, PhD Kunfu OuyangPhD Ze’e ChenZe’e Chen Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) , Siting ZhuSiting Zhu https://orcid.org/0000-0002-6950-9556 Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) , Hong WangHong Wang Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). , Li WangLi Wang Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) , Jianlin ZhangJianlin Zhang Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) , Yusu GuYusu Gu Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) , Changming TanChangming Tan Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) Department of Cardiothoracic Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (C.T.). , Mehul DhananiMehul Dhanani Anesthesiology (M.D., H.H.P.), School of Medicine, University of California San Diego, La Jolla. Veterans Administration San Diego Healthcare System, CA (M.D., H.H.P.). , Eric WeverEric Wever Departments of Clinical Chemistry and Pediatrics, Amsterdam Gastroenterology Endocrinology Metabolism (E.W., F.M.V.) Core Facility Metabolomics (E.W., F.M.V.), Amsterdam Universitair Medische Centra Laboratory Genetic Metabolic Diseases; Bioinformatics Laboratory, Department of Epidemiology and Data Science, Amsterdam Public Health Research Institute (E.W., A.H.C.v.K.), Amsterdam Universitair Medische Centra , Xinru WangXinru Wang Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). , Boyu XieBoyu Xie Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). , Shijia WangShijia Wang https://orcid.org/0000-0001-5063-6224 Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). , Lei HuangLei Huang Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). , Antoine H.C. van KampenAntoine H.C. van Kampen Laboratory Genetic Metabolic Diseases; Bioinformatics Laboratory, Department of Epidemiology and Data Science, Amsterdam Public Health Research Institute (E.W., A.H.C.v.K.), Amsterdam Universitair Medische Centra Biosystems Data Analysis, Swammerdam Institute for Life Sciences (A.H.C.v.K.), University of Amsterdam, The Netherlands. , Jie LiuJie Liu Department of Pathophysiology, School of Medicine, Shenzhen University, Shenzhen, China (J.L.). , Zhen HanZhen Han Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). , Hemal H. PatelHemal H. Patel Anesthesiology (M.D., H.H.P.), School of Medicine, University of California San Diego, La Jolla. Veterans Administration San Diego Healthcare System, CA (M.D., H.H.P.). , Frédéric M. VazFrédéric M. Vaz https://orcid.org/0000-0002-9048-1041 Departments of Clinical Chemistry and Pediatrics, Amsterdam Gastroenterology Endocrinology Metabolism (E.W., F.M.V.) Core Facility Metabolomics (E.W., F.M.V.), Amsterdam Universitair Medische Centra , Xi FangXi Fang Xi Fang, PhD, Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093; Email E-mail Address: [email protected] https://orcid.org/0000-0001-7816-8821 Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) , Ju ChenJu Chen Correspondence to: Ju Chen, PhD, Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093; Email E-mail Address: [email protected] https://orcid.org/0000-0001-7674-4776 Departments of Medicine (Z.C., S.Z., L.W., J.Z., Y.G., C.T., X.F., J.C.) and Kunfu OuyangKunfu Ouyang Kunfu Ouyang, PhD, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China. Email E-mail Address: [email protected] https://orcid.org/0000-0003-0292-375X Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China (Z.C., S.Z., H.W., X.W., B.X., S.W., L.H., Z.H., K.O.). Originally published2 Aug 2021https://doi.org/10.1161/CIRCULATIONAHA.121.054768Circulation. 2021;144:403–406Cardiolipin is a unique glycerol-bridged dimeric phospholipid representing up to 20% of total lipids in mitochondrial membranes in cardiomyocytes. Abnormal cardiolipin metabolism is linked to heart diseases, including Barth syndrome, myocardial ischemia-reperfusion injury, and heart failure.1 However, cardiolipin profiles and specific roles of cardiolipin in cardiac mitochondria remain largely obscure.We first performed quantitative lipidomic analysis on mouse hearts at different stages and revealed a strong discrepancy in molecular compositions of cardiolipin and cardiolipin-related metabolites between embryonic and adult mouse hearts (Figure [A]). In particular, cardiolipin in embryonic hearts displayed more diverse acyl compositions, while the predominant form of cardiolipin in the adult heart was tetralinoleoyl cardiolipin, implicating that the pathways involved in cardiolipin biosynthesis and metabolism may have different functions between embryonic and adult hearts.Download figureDownload PowerPointFigure. PTPMT1 is required for embryonic cardiac cardiolipin biosynthesis to regulate mitochondrial morphogenesis and heart development. A, Quantitative lipidomic analysis was applied to identify individual species of cardiolipin and cardiolipin-related metabolites, including PA, PGP, PG, CL, MLCL, and DLCL in the ventricles of control mice at E11.5, E15.5, 2 months, and 10 months of age, respectively. All the species of each metabolite, or the 10 most abundant species if more than 10 species were observed, were selected and further analyzed at each stage. The abundance (%) of individual species was calculated according to its total level and is represented by the area of the circle (n = 3, 3, 6, and 6, respectively). B, The levels of total PA, PGP, PG, CL, MLCL, and DLCL in CKO ventricles at E11.5 (n = 3) and E15.5 (n = 3), and in iCKO ventricles 10 months post–tamoxifen-induced gene deletion (n = 5) were analyzed and normalized to their control ventricles (n = 3, 3, and 5, respectively). Twenty-five and 6 ventricles with the same genotype were pooled as 1 sample at E11.5 and E15.5, respectively. C, The levels of every species, or the levels of the 10 most abundant species if more than 10 species were observed, of PA, PGP, PG, CL, MLCL, and DLCL in CKO and iCKO ventricles were further analyzed and normalized to their control ventricles, respectively. D through H, Representative images of embryonic hearts (D), sections stained with hematoxylin and eosin (E), and images of EdU labeling (G) of control and CKO mice at indicated stages. Cardiac cells were costained with α-actinin. Scale bar = 0.5 mm, 100 μm, and 50 μm, respectively. The thickness of left and right ventricular walls (F) of control (n = 4 at both stages) and CKO (n = 3 and 4, respectively) hearts, and the ratios of EdU-positive cardiomyocytes in ventricular compaction zone and trabecular (H) of control (n = 3, 4, and 4, respectively) and CKO (n = 3, 5, and 5, respectively) hearts were measured at indicated stages. I, Oxygen flux representing the respiratory function of CI and CII, mOX, and mUC were measured in control and CKO hearts by high-resolution respirometry at E11.5 and E12.5, respectively. The measurement was performed on 2 hearts with the same phenotype at E11.5 or 1 heart at E12.5 in one chamber (n = 8 per group at E11.5; n = 6 per group at E12.5). (J) Immunoblot analysis on the expression of mitochondrial oxidative phosphorylation subunits including NDUFB8 (complex I), SDHB (complex II), UQCRC2 (complex III), MTCO1 (complex IV), and ATP5A (complex V) in control and CKO embryonic hearts at E11.5 and E12.5. GAPDH was used as the loading control. K, Representative transmission electron microscopic images of mitochondria in control and CKO cardiomyocytes at E11.5 and E12.5. Red arrows indicate the mitochondria with bubble-like cristae in CKO cardiomyocytes. Scale bar = 0.5 μm. L through O, Quantitative analysis of length of cristae (L), mitochondrial diameter in the short axis (M), the percentage of mitochondria with lamellar cristae (N), and the number of lamellar cristae along the mitochondrial long axis (O) in control and CKO cardiomyocytes at E11.5 (n = 4 per group) and E12.5 (n = 3 per group). At least 160 mitochondria were measured for each embryonic heart. P through T, Immunoblot analysis of F1F0-ATP synthase subunits (P) including ATP5A, ATP5H, ATP5F1, ATP5I, and ATP5L, MICOS complex subunits (R) including MIC60, MIC27, MIC25, MIC19, MIC13, and MIC10, as well as mitochondrial dynamics–related proteins (T) including MFN1, MFN2, OPA1, DRP1, and FIS1 and prohibitin proteins including PHB and PHB2 at E11.5. GAPDH was used as the loading control. Blue Native-PAGE and immunoblot analysis of F1F0-ATP synthase complex using the antibodies against ATP5F1 and ATP5H (Q), and MICOS complex assembly using the antibodies against MIC10 and MIC27 (S). To note, the deletion of PTPMT1 reduced the formation of F1F0-ATP synthase dimers, whereas monomers were not affected. The reduction of the MICOS complex assembly in CKO mitochondria was also observed. Each mitochondrial sample was prepared from more than 50 embryonic ventricular tissues of the same genotype in 1% digitonin-containing extraction buffer, and separated by Blue Native-PAGE. CB-stained membranes were scanned for loading control. U, Representative hearts of control and iCKO mice at 10 months post–tamoxifen injection. Scale bar = 1 mm. V, Ratios of ventricle weight to body weight in control and iCKO mice at 2 months and 10 months post–tamoxifen injection (n = 3 to 7 mice per group). W, Echocardiographic assessment in control and iCKO mice at 2 months and 10 months post–tamoxifen injection (n = 6 to 10 mice per group). All data represent mean±SEM. Significance was determined by 2-tailed, unpaired Student t test. *P<0.05, **P<0.01, ***P<0.001 vs control. ATP5A indicates F1 complex subunit α; ATP5F1, F0 complex subunit b; ATP5H, F0 complex subunit d; ATP5I, F0 complex subunit e; ATP5L F0 complex subunit g; CB, Coomassie blue; CI, complex I; CII, complex II; CKO, TnT-Cre-mediated cardiac-specific PTPMT1 knockout; CL, cardiolipin; DLCL, dilysocardiolipin; DRP1, dynamin-related protein 1; E11.5, embryonic day 11.5; E12.5, embryonic day 12.5; E15.5, embryonic day 15.5; FS, fractional shortening; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iCKO, αMHC-CreER–mediated PTPMT1 knockout; KD, kilodaltons; LV, left ventricle; LVIDd, left ventricular internal diameter end diastole; LVIDs, left ventricular internal diameter end systole; mOX, maximum oxidative phosphorylation capacity; mUC, maximum uncoupled capacity; MLCL, monolysocardiolipin; MTCO1, mitochondrially encoded cytochrome C oxidase I; MICOS, mitochondrial contact site and cristae organizing system; MIC60, MICOS complex subunit Mic60; MIC27, MICOS complex subunit Mic27; MFN1, mitofusin 1; MFN2, mitofusin 2; NDUFB8, 1,4-dihydronicotinamide adenine dinucleotide:ubiquinone oxidoreductase subunit B8; OPA1, optic atrophy protein 1 mitochondrial dynamin-like guanosine triphosphatase; PA, phosphatidic acid; PG, phosphatidylglycerol; PGP, phosphatidylglycerophosphate; PHB, prohibitin; PHB2, prohibitin 2; PTPMT1, protein tyrosine phosphatase mitochondrial 1; RV, right ventricle; SDHB, succinate dehydrogenase [ubiquinone] iron-sulfur subunit B; UQCRC2, cytochrome b-c1 complex subunit 2, mitochondrial; Vd, F1F0-ATP synthase dimer; Vm, F1F0-ATP synthase monomer; and VW/BW, ventricle weight/body weight.To investigate the role of cardiolipin biosynthesis in cardiac development, we used cardiac Troponin T (TnT-Cre) to generate a mouse model with cardiac-specific deletion of PTPMT1 ([protein tyrosine phosphatase mitochondrial 1] CKO), a mitochondrial phosphatase that removes the terminal phosphate group from phosphatidylglycerophosphate to form phosphatidylglycerol,2 to disrupt cardiolipin biosynthesis in embryonic hearts. PTPMT1 deficiency indeed reduced the contents of both total cardiolipin and most of the abundantly expressed cardiolipin species in embryonic hearts at both embryonic day 11.5 (E11.5) and embryonic day 15.5, accompanied with a broad alteration in levels of total and individual cardiolipin-related metabolites (Figure [B and C]), demonstrating that PTPMT1 is required for cardiolipin biosynthesis in embryonic cardiomyocytes. All mouse protocols were approved by the Institutional Animal Care and Use Committee.Deletion of PTPMT1 in cardiomyocytes caused abnormal cardiac development and embryonic lethality between embryonic day 16.5 and embryonic day 18.5. Morphological changes started in CKO hearts at embryonic day 12.5 (Figure [D]), accompanied with decreased thicknesses of ventricular walls at the same stage (Figure [E and F]), which could be a consequence of defects in cardiac cell proliferation first observed in compact zone of CKO hearts at E11.5 (Figure [G and H]).Cardiolipin has been proposed to participate in regulating both mitochondrial function and structure.1 We then evaluated mitochondrial respiration in permeabilized embryonic hearts by measuring oxygen consumption. Decreases in respiratory function of complex I and maximum oxidative phosphorylation capacity were observed in CKO hearts as early as E11.5 (Figure [I]). At embryonic day 12.5, respiratory functions were further impaired, and changes in expression of mitochondrial complex proteins were observed in CKO hearts (Figure [I and J]).In developing embryonic cardiomyocytes, mitochondria undergo a maturation process with increased mitochondria number and more organized lamellar cristae at later stages.3 The function of cardiolipin in regulating mitochondrial morphology and ultrastructure in embryonic hearts remains unclear. We performed transmission electron microscopy analysis, and found that membrane invagination was impaired and a bubble-like inner membrane structure—instead of lamellar cristae—could be easily observed in CKO mitochondria at E11.5, and became more severe at embryonic day 12.5 (Figure [K]). PTPMT1 deletion also altered mitochondrial diameter, percentages of mitochondria with lamellar cristae, cristae lengths, and cristae numbers in embryonic cardiomyocytes (Figure [L–O]), suggesting that PTPMT1-mediated cardiolipin biosynthesis is required for normal mitochondrial morphogenesis and cristae biogenesis in developing cardiomyocytes. Because cristae are recognized as fundamental structures to provide a sufficient area and proper spatial organization for oxidative phosphorylation and other membrane proteins in mitochondria,4 abnormalities in cristae biogenesis could account for dysfunctional mitochondrial respiration in E11.5 CKO hearts.The F1F0-ATP synthase dimers, MICOS (mitochondrial contact site and cristae organizing system) complex, OPA1 (optic atrophy protein 1 mitochondrial dynamin-like guanosine triphosphatase), and prohibitin proteins, have been proposed to participate in regulating mitochondrial cristae biogenesis.5 ATP5I (ATP synthase, H+ transporting, mitochondrial F0 complex, subunit E) and ATP5L (ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G), 2 components of F1F0-ATP synthase, were downregulated in CKO hearts at E11.5. The dimerization of F1F0-ATP synthase complex was also impaired, while the assembly of complex monomer remained unaffected in CKO hearts at the same stage (Figure [P and Q]). It is interesting that deletion of PTPMT1 in cardiomyocytes impaired the assembly of MICOS complex in CKO hearts at E11.5 but did not alter the expressions of individual MICOS components (Figure [R and S]). Furthermore, we did not observe significant differences in the expressions of MFN1 (mitofusin 1), MFN2 (mitofusin 2), OPA1 (optic atrophy 1 mitochondrial dynamin-like guanosine triphosphatase), DRP1 (dynamin-related protein 1), FIS1 (mitochondrial fission 1 protein), and prohibitin proteins between control and CKO hearts at E11.5 (Figure [T]).We also generated an inducible cardiac-specific Ptpmt1 knockout mouse model using αMHC-CreER. Induced deletion of PTPMT1 in adult mouse cardiomyocytes by tamoxifen reduced phosphatidylglycerol (34:1), the most abundant phosphatidylglycerol species, and increased phosphatidylglycerophosphate (34:1), the most abundant phosphatidylglycerophosphate species (Figure [A and C]), indicating that PTPMT1 is also responsible for catalyzing the conversion from phosphatidylglycerophosphate to phosphatidylglycerol in adult hearts. Although PTPMT1 deficiency also altered the levels of monolysocardiolipin and dilysocardiolipin (Figure [B and C]), the levels of total cardiolipin and individual cardiolipin species remained intriguingly unchanged (Figure [B and C]), and no morphological and functional changes were observed in the cardiomyocytes of adult mice with induced deletion of PTPMT1 (Figure [U–W]).Taken together, our results demonstrated an essential role of PTPMT1-mediated cardiolipin biosynthesis in regulating mitochondrial cristae morphogenesis in embryonic mouse cardiomyocytes and heart development. Our results also revealed a difference in not only the composition but also the metabolism of cardiolipin and cardiolipin-related metabolites between embryonic and adult hearts.Nonstandard Abbreviations and AcronymsCKOTnT-Cre-mediated cardiac-specific PTPMT1 knockoutE11.5embryonic day 11.5MICOSmitochondrial contact site and cristae organizing systemPTPMT1protein tyrosine phosphatase mitochondrial 1Sources of FundingDrs J. Chen, Fang, and Patel are funded by grants from the National Heart, Lung, and Blood Institute of the US National Institutes of Health. Dr J. Chen holds an American Heart Association Endowed Chair in Cardiovascular Research. This work was also supported by the National Science Foundation of China (81970421 to K.O.), the Shenzhen Basic Research Foundation (JCYJ20190808174001746 to K.O.), the Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions (2019SHIBS0004), the National Institutes of Health (HL091071 to H.H.P.), and the Veterans Administration (BX001963 and BX005229 to H.H.P.).Disclosures Dr J. Chen has consulted for and receives research funding from MyoKardia Inc.Footnoteshttps://www.ahajournals.org/journal/circFor Sources of Funding and Disclosures, see page 404.The data, analytical methods, and study materials that support the findings of this study will be available to other researchers from the corresponding authors on reasonable request.Correspondence to: Ju Chen, PhD, Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093; Email [email protected]ucsd.eduXi Fang, PhD, Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093; Email [email protected]ucsd.eduKunfu Ouyang, PhD, School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China. Email [email protected]edu.cnRefernces1. Dudek J, Hartmann M, Rehling P. The role of mitochondrial cardiolipin in heart function and its implication in cardiac disease.Biochim Biophys Acta Mol Basis Dis. 2019; 1865:810–821. doi: 10.1016/j.bbadis.2018.08.025CrossrefMedlineGoogle Scholar2. Zhang J, Guan Z, Murphy AN, Wiley SE, Perkins GA, Worby CA, Engel JL, Heacock P, Nguyen OK, Wang JH, et al.. Mitochondrial phosphatase PTPMT1 is essential for cardiolipin biosynthesis.Cell Metab. 2011; 13:690–700. doi: 10.1016/j.cmet.2011.04.007CrossrefMedlineGoogle Scholar3. Dorn GW, Vega RB, Kelly DP. Mitochondrial biogenesis and dynamics in the developing and diseased heart.Genes Dev. 2015; 29:1981–1991. doi: 10.1101/gad.269894.115CrossrefMedlineGoogle Scholar4. Cogliati S, Frezza C, Soriano ME, Varanita T, Quintana-Cabrera R, Corrado M, Cipolat S, Costa V, Casarin A, Gomes LC, et al.. Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency.Cell. 2013; 155:160–171. doi: 10.1016/j.cell.2013.08.032CrossrefMedlineGoogle Scholar5. Pfanner N, Warscheid B, Wiedemann N. Mitochondrial proteins: from biogenesis to functional networks.Nat Rev Mol Cell Biol. 2019; 20:267–284. doi: 10.1038/s41580-018-0092-0CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails August 3, 2021Vol 144, Issue 5Article InformationMetrics Download: 1,002 © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.121.054768PMID: 34339306 Originally publishedAugust 2, 2021 Keywordscardiolipinsmitochondriamitochondrial membranesmyocytes, cardiacprotein tyrosine phosphatasesPDF download

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948-1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645delknockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn's function in tubular system organization and normal cardiac function.

HomeCirculationVol. 141, No. 10Loss of Filamin C Is Catastrophic for Heart Function Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBLoss of Filamin C Is Catastrophic for Heart Function Yangzhao Zhou, MD, Ze'e Chen, BS, Lunfeng Zhang, PhD, Mason Zhu, BS, Changming Tan, MD, PhD, Xinmin Zhou, MD, PhD, Sylvia M. Evans, PhD, Xi Fang, PhD, Wei Feng, MD, PhD and Ju Chen, PhD Yangzhao ZhouYangzhao Zhou Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (Y.Z., C.T., X.Z.). , Ze'e ChenZe'e Chen Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. , Lunfeng ZhangLunfeng Zhang Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. , Mason ZhuMason Zhu Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. , Changming TanChangming Tan Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (Y.Z., C.T., X.Z.). , Xinmin ZhouXinmin Zhou Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (Y.Z., C.T., X.Z.). , Sylvia M. EvansSylvia M. Evans Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. Pharmacology (S.M.E.), University of California San Diego, La Jolla. Skaggs School of Pharmacy and Pharmaceutical Sciences (S.M.E.), University of California San Diego, La Jolla. , Xi FangXi Fang Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. , Wei FengWei Feng Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. and Ju ChenJu Chen Ju Chen, PhD, Department of Medicine–Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, CA 92093. Email E-mail Address: [email protected] Departments of Medicine–Cardiology (Y.Z., Z.C., L.Z., M.Z., C.T., S.M.E., X.F., W.F., J.C.), University of California San Diego, La Jolla. Originally published9 Mar 2020https://doi.org/10.1161/CIRCULATIONAHA.119.044061Circulation. 2020;141:869–871Mutations in filamin C (FLNC) are associated with different forms of cardiomyopathies.1,2 The role of FLNC in mammalian cardiomyocytes remains largely unexplored. FLNC is expressed predominantly in striated muscle, localizing to Z-discs, intercalated discs (ICDs), and costameres.3 Mice expressing a C-terminal truncated FLNC mutant protein display perinatal lethality with severe defects in skeletal myogenesis but no cardiac defects.4 The latter is puzzling, given that multiple distinct FLNC mutations lead to cardiomyopathy. Because this mutant Flnc allele might be hypomorphic, a true null Flnc and an Flnc cardiomyocyte-specific knockout model are essential to investigate requirements for Flnc in cardiomyocytes fully.We generated Flnc global knockout and cardiac-specific knockout mice by crossing Sox2-Cre or cardiac troponin T-Cre to FLNC-floxed mice (Flnctm1a[EUCOMM]Hmgu),in which exons 9 through 13 of Flnc are flanked by 2 LoxP sites. Cre-mediated deletion results in loss of the Flnc region between exons 9 and 13, frameshift of Flnc, and subsequent loss of the protein. All mouse protocols were approved by the Institutional Animal Care and Use Committee. Both global knockout and cardiac-specific knockout mice were embryonic lethal, demonstrating that Flnc is critical in developing cardiomyocytes (data not shown).To study Flnc in adult cardiomyocytes, we generated icKO mice (inducible cardiomyocyte-specific knockout) by crossing Flnc-floxed mice with αMHC-MerCreMer mice and subjected them to tamoxifen treatment (TAM) at 2 months of age. FLNC protein was efficiently ablated in icKO mouse hearts 2 weeks after TAM (Figure [A and B]). Flnc-icKO mice began to die 1 week after TAM, with 73% dying by 25 weeks (Figure [C]). No icKO mice survived past 60 weeks of age (Figure [C]). icKO hearts at 2 weeks and 6 weeks after TAM displayed marked cardiac dilation and extensive fibrosis 6 weeks after TAM (Figure [D]). Analysis of heart weight to body weight ratios revealed significantly increased heart mass in icKO mice at 2 weeks after TAM compared with control mice, whereas no differences were observed in body weight. Cardiac stress markers Anf, Bnp, and Myh7 were significantly increased in icKO hearts at 2 weeks after TAM, as were profibrotic genes Col1a1 and Col3a1 (Figure [F]).Download figureDownload PowerPointFigure. Loss of filamin C is catastrophic for heart function.A and B, Representative Western blot (A) and quantitation (B) showing efficient loss of Flnc in icKO mouse (inducible cardiomyocyte-specific knockout) hearts 2 weeks after tamoxifen treatment (TAM) (40 mg/kg/d for 3 days). Flncf/f;Cre− littermates after TAM served as controls (Ctrl). Three mice per group were included. C, Kaplan-Meier survival curves of Flncf/f and Flncf/f;Cre+ mice injected with TAM or vehicle control (n=16, 8, 26, and 9, respectively). D, Microscopic cross-sectional views of hematoxylin & eosin (left; scale bar, 1 mm) and Masson trichrome (right; scale bar: 50 μm) stained hearts isolated from Flnc icKO or Ctrl mice, 2 weeks or 6 weeks after TAM (n=3). E, Heart weight to body weight (HW/BW) ratio for icKO (n=14) and Ctrl (n=12) mice 2 weeks after TAM. F, Quantitative real-time polymerase chain reaction analysis in Ctrl and icKO mouse hearts at 2 weeks after TAM. G, Representative echocardiographic images of icKO and Ctrl mice at 2 and 4 weeks after TAM. H through J, Echocardiographic measurements for Flncf/f and Flncf/f;Cre+ mice at baseline or 2, 4, or 8 weeks after TAM or vehicle control (n=8, 4, 15, and 5, respectively). K, Diagram of intercalated disc (ICD), Z-disc, and costamere components. Proteins noted in red were upregulated, whereas those in black were unchanged, in Flnc icKO hearts. L through P, Representative Western blots and quantification of integrin complex, DGC (dystrophin-associated glycoprotein complex) proteins, Z-disk protein, and desmin and ICD components in icKO and Ctrl hearts at 2 weeks after TAM (n=3). Other than FAK (focal adhesion kinase), δ-SAG (1-stearoyl-2-arachidonoyl-sn-glycerol), β-DSG (disuccinimidyl glutarate), and dystrophin, which were not detected in our mass spectrometry studies, all other proteins tested in the Western blot analysis have similar changes in mass spectrometry studies. Q, Quantitative real-time polymerase chain reaction analysis for mRNAs of proteins examined in Ctrl and icKO mouse hearts at 2 weeks after TAM injection. R, Functional enrichment analysis of proteins upregulated in icKO. For Western blot analysis, GAPDH served as a loading control. The pixel density of each protein was normalized to the level of GAPDH. In quantitative real-time polymerase chain reaction assays, mRNA levels of each gene were normalized to corresponding 18S levels, with icKO values being expressed as fold change versus control. Data are mean±SEM. CAR indicates Coxsackievirus–adenovirus receptor; DSP, dithiobis(succinimidyl propionate); ECM, extracellular matrix; ENH, enigma homolog; FLNC, Filamin C; FS, fractional shortening; ILK, integrin-linked kinase; JUP, junction plakoglobin; LIMP-2, lysosomal integral membrane protein; LVIDd, end-diastolic left ventricle internal diameter; LVIDs, end-systolic left ventricle internal diameter; MLP, muscle LIM protein; PINCH, particularly interesting new cysteine-histidine-rich protein; PKP2, plakophilin-2; T-cap, telethonin; and ZO-1, zonula occludens–1. *P<0.05 by 2-tailed Student t test.Echocardiographic studies revealed a rapid and progressive decrease in left ventricular systolic function (fractional shortening) in icKO mice relative to control mice (Figure [G and H]). Left ventricular chamber dilation was shown by a significant increase in end-diastolic left ventricle internal diameter and end-systolic left ventricle internal diameter (Figure [I and J]).FLNC interacts with multiple cytoskeletal proteins, including integrins and the dystrophin-associated glycoprotein complex at the costamere, myotilin and FATZ-1 at the Z-disk, and Xin proteins at the ICD.3 We investigated whether loss of Flnc in cardiomyocytes affected levels of major cytoskeletal proteins comprising these complexes (Figure [K through P]).5 Loss of Flnc resulted in significant increases in β1D-integrin, as well as Talin1, Kindlin2, FAK (focal adhesion kinase), vinculin, and ILK (integrin-linked kinase), whereas paxillin was not changed (Figure [L]). δ- and γ-SAG (1-stearoyl-2-arachidonoyl-sn-glycerol), dystrophin, and β-DSG (disuccinimidyl glutarate), but not α-DSG, were increased in icKO hearts (Figure [M]). Desmin, an intermediate filament protein, was also increased. Z-disc proteins myotilin, FATZ-1, α-actinin, and Cypher were not changed (Figure [N]). ICD proteins Xirp1 and Xirp2, JUP (junction plakoglobin), and DSP (dithiobis[succinimidyl propionate]) were significantly increased, whereas PKP2 (plakophilin-2) and DSG2 were not changed (Figure [O]). These results demonstrate that loss of Flnc resulted in increased levels of a subset of costameric and ICD proteins (Figure [P]). Quantitative real-time polymerase chain reaction data indicated that mRNA levels of increased proteins were not changed, with the exception of Xirp1 and Xirp2 mRNA levels, which were significantly increased (Figure [Q]). Immunostaining for upregulated proteins showed no protein aggregation or mislocalization in icKO hearts (data not shown).Mass spectrometry analysis identified 380 proteins that were upregulated more than 2-fold and 27 proteins that were downregulated more than 2-fold in icKO hearts relative to control hearts. These data confirm increased levels of specific proteins found by initial Western blot analyses. Functional clustering of upregulated proteins revealed the most significant pathway enrichment in cytoskeletal protein binding, actin binding, and extracellular matrix structural constituent (Figure [R]).Previous mutation of Flnc in mice resulted in defects in skeletal muscle development, with no evident cardiac phenotype,4 despite the observation that recessive mutations in FLNC lead to human congenital dilated cardiomyopathy.1,2 Our results have addressed this apparent contradiction by demonstrating a key and critical role for FLNC in both developing and adult cardiomyocytes. In embryonic cardiomyocytes, loss of FLNC resulted in fetal death. In adult cardiomyocytes, upon loss of FLNC, mice developed rapid and fulminant dilated cardiomyopathy within 2 weeks. Loss of FLNC was accompanied by upregulation of multiple proteins, including those that directly interact with FLNC, representing components of the costamere and ICD, and the intermediate filament protein Desmin (Figure [K]). The dire effects of loss of FLNC are likely to reflect its significant effect on all 3 of these major constituents of the cardiomyocyte cytoskeleton, which are essential for normal contraction.Sources of FundingDrs J. Chen, Fang, and Evans are funded by grants from the National Heart, Lung, and Blood Institute of the US National Institutes of Health. Dr J. Chen holds an American Heart Association Endowed Chair in Cardiovascular Research. M. Zhu was supported by an American Heart Association Summer Undergraduate Fellowship.DisclosuresNone.Footnotes*Drs Fang, Feng, and J. Chen contributed equally.https://www.ahajournals.org/journal/circThe data, analytical methods, and study materials that support the findings of this study will be available to other researchers from the corresponding authors on reasonable request.Ju Chen, PhD, Department of Medicine–Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, CA 92093. Email [email protected]eduReferences1. Ader F, De Groote P, Réant P, Rooryck-Thambo C, Dupin-Deguine D, Rambaud C, Khraiche D, Perret C, Pruny JF, Mathieu-Dramard M, et al. FLNC pathogenic variants in patients with cardiomyopathies: prevalence and genotype-phenotype correlations.Clin Genet. 2019; 96:317–329. doi: 10.1111/cge.13594CrossrefMedlineGoogle Scholar2. Reinstein E, Gutierrez-Fernandez A, Tzur S, Bormans C, Marcu S, Tayeb-Fligelman E, Vinkler C, Raas-Rothschild A, Irge D, Landau M, et al. Congenital dilated cardiomyopathy caused by biallelic mutations in Filamin C.Eur J Hum Genet. 2016; 24:1792–1796. doi: 10.1038/ejhg.2016.110CrossrefMedlineGoogle Scholar3. van der Ven PF, Ehler E, Vakeel P, Eulitz S, Schenk JA, Milting H, Micheel B, Fürst DO. Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin C and Mena/VASP.Exp Cell Res. 2006; 312:2154–2167. doi: 10.1016/j.yexcr.2006.03.015CrossrefMedlineGoogle Scholar4. Dalkilic I, Schienda J, Thompson TG, Kunkel LM. Loss of FilaminC (FLNc) results in severe defects in myogenesis and myotube structure.Mol Cell Biol. 2006; 26:6522–6534. doi: 10.1128/MCB.00243-06CrossrefMedlineGoogle Scholar5. Zhang Z, Mu Y, Zhang J, Zhou Y, Cattaneo P, Veevers J, Peter AK, Manso AM, Knowlton KU, Zhou X, et al. Kindlin-2 is essential for preserving integrity of the developing heart and preventing ventricular rupture.Circulation. 2019; 139:1554–1556. doi: 10.1161/CIRCULATIONAHA.118.038383LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Song S, Shi A, Lian H, Hu S and Nie Y (2021) Filamin C in cardiomyopathy: from physiological roles to DNA variants, Heart Failure Reviews, 10.1007/s10741-021-10172-z, 27:4, (1373-1385), Online publication date: 1-Jul-2022. Brodehl A and Gerull B (2022) Genetic Insights into Primary Restrictive Cardiomyopathy, Journal of Clinical Medicine, 10.3390/jcm11082094, 11:8, (2094) Chen S, Lam C, Wan Y, Gao S, Malak O, Zhao S, Lombardi R, Ambardekar A, Bristow M, Cleveland J, Gigli M, Sinagra G, Graw S, Taylor M, Wu J and Mestroni L (2022) Activation of PDGFRA signaling contributes to filamin C–related arrhythmogenic cardiomyopathy, Science Advances, 10.1126/sciadv.abk0052, 8:8, Online publication date: 25-Feb-2022. Bang M, Bogomolovas J and Chen J (2022) Understanding the molecular basis of cardiomyopathy, American Journal of Physiology-Heart and Circulatory Physiology, 10.1152/ajpheart.00562.2021, 322:2, (H181-H233), Online publication date: 1-Feb-2022. Celeghin R, Cipriani A, Bariani R, Bueno Marinas M, Cason M, Bevilacqua M, De Gaspari M, Rizzo S, Rigato I, Da Pozzo S, Zorzi A, Perazzolo Marra M, Thiene G, Iliceto S, Basso C, Corrado D, Pilichou K and Bauce B (2022) Filamin-C variant-associated cardiomyopathy: A pooled analysis of individual patient data to evaluate the clinical profile and risk of sudden cardiac death, Heart Rhythm, 10.1016/j.hrthm.2021.09.029, 19:2, (235-243), Online publication date: 1-Feb-2022. Powers J and McCulloch A (2022) Biomechanical signals regulating the structure of the heart, Current Opinion in Physiology, 10.1016/j.cophys.2021.100482, 25, (100482), Online publication date: 1-Feb-2022. Powers J, Kirkland N, Liu C, Razu S, Fang X, Engler A, Chen J and McCulloch A (2022) Subcellular Remodeling in Filamin C Deficient Mouse Hearts Impairs Myocyte Tension Development during Progression of Dilated Cardiomyopathy, International Journal of Molecular Sciences, 10.3390/ijms23020871, 23:2, (871) Qin Z, Sun L, Sun X, Su H and Gao X (2021) A CRISPR/Cas9 strategy for the generation of a FLNC knockout hESC line (WAe009-A-70) to model dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy, Stem Cell Research, 10.1016/j.scr.2021.102562, 56, (102562), Online publication date: 1-Oct-2021. Agarwal R, Paulo J, Toepfer C, Ewoldt J, Sundaram S, Chopra A, Zhang Q, Gorham J, DePalma S, Chen C, Gygi S, Seidman C and Seidman J (2021) Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation, Circulation Research, 129:7, (751-766), Online publication date: 17-Sep-2021. Tan C, Zhu S, Chen Z, Liu C, Li Y, Zhu M, Zhang Z, Zhang Z, Zhang L, Gu Y, Liang Z, Boyer T, Ouyang K, Evans S, Fang X and Firulli A (2021) Mediator complex proximal Tail subunit MED30 is critical for Mediator core stability and cardiomyocyte transcriptional network, PLOS Genetics, 10.1371/journal.pgen.1009785, 17:9, (e1009785) Schänzer A, Schumann E, Zengeler D, Gulatz L, Maroli G, Ahting U, Sprengel A, Gräf S, Hahn A, Jux C, Acker T, Fürst D, Rupp S, Schuld J and van der Ven P (2021) The p.Ala2430Val mutation in filamin C causes a "hypertrophic myofibrillar cardiomyopathy", Journal of Muscle Research and Cell Motility, 10.1007/s10974-021-09601-1, 42:2, (381-397), Online publication date: 1-Jun-2021. Arif M, Klevstig M, Benfeitas R, Doran S, Turkez H, Uhlén M, Clausen M, Wikström J, Etal D, Zhang C, Levin M, Mardinoglu A and Boren J (2021) Integrative transcriptomic analysis of tissue-specific metabolic crosstalk after myocardial infarction, eLife, 10.7554/eLife.66921, 10 Wadmore K, Azad A and Gehmlich K (2021) The Role of Z-disc Proteins in Myopathy and Cardiomyopathy, International Journal of Molecular Sciences, 10.3390/ijms22063058, 22:6, (3058) Eden M and Frey N (2021) Cardiac Filaminopathies: Illuminating the Divergent Role of Filamin C Mutations in Human Cardiomyopathy, Journal of Clinical Medicine, 10.3390/jcm10040577, 10:4, (577) Schuld J, Orfanos Z, Chevessier F, Eggers B, Heil L, Uszkoreit J, Unger A, Kirfel G, van der Ven P, Marcus K, Linke W, Clemen C, Schröder R and Fürst D (2020) Homozygous expression of the myofibrillar myopathy-associated p.W2710X filamin C variant reveals major pathomechanisms of sarcomeric lesion formation, Acta Neuropathologica Communications, 10.1186/s40478-020-01001-9, 8:1, Online publication date: 1-Dec-2020. Guo H, Lu Y, Lin Z, Huang Z, Liu J, Wang Y, Seok H, Hu X, Ma Q, Li K, Kyselovic J, Wang Q, Lin J, Lin J, Cowan D, Naya F, Chen Y, Pu W and Wang D (2020) Intercalated disc protein Xinβ is required for Hippo-YAP signaling in the heart, Nature Communications, 10.1038/s41467-020-18379-8, 11:1, Online publication date: 1-Dec-2020. Lamsoul I, Dupré L and Lutz P (2020) Molecular Tuning of Filamin A Activities in the Context of Adhesion and Migration, Frontiers in Cell and Developmental Biology, 10.3389/fcell.2020.591323, 8 Knyazeva A, Khudiakov A, Vaz R, Muravyev A, Sukhareva K, Sejersen T and Kostareva A (2020) FLNC Expression Level Influences the Activity of TEAD-YAP/TAZ Signaling, Genes, 10.3390/genes11111343, 11:11, (1343) Pecorari I, Mestroni L and Sbaizero O (2020) Current Understanding of the Role of Cytoskeletal Cross-Linkers in the Onset and Development of Cardiomyopathies, International Journal of Molecular Sciences, 10.3390/ijms21165865, 21:16, (5865) Pruna M and Ehler E (2020) The intercalated disc: a mechanosensing signalling node in cardiomyopathy, Biophysical Reviews, 10.1007/s12551-020-00737-x, 12:4, (931-946), Online publication date: 1-Aug-2020. Gerull B and Brodehl A (2020) Genetic Animal Models for Arrhythmogenic Cardiomyopathy, Frontiers in Physiology, 10.3389/fphys.2020.00624, 11 Bogomolovas J, Feng W, Yu M, Huang S, Zhang L, Trexler C, Gu Y, Spinozzi S and Chen J (2020) Atypical ALPK2 kinase is not essential for cardiac development and function, American Journal of Physiology-Heart and Circulatory Physiology, 10.1152/ajpheart.00249.2020, 318:6, (H1509-H1515), Online publication date: 1-Jun-2020. March 10, 2020Vol 141, Issue 10 Advertisement Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.119.044061PMID: 32150467 Originally publishedMarch 9, 2020 Keywordsmutationfilaminsmyocytes, cardiaccardiomyopathyPDF download Advertisement SubjectsCardiomyopathyHeart Failure

BACKGROUND:NEXN (nexilin) is a protein of the junctional membrane complex required for development of cardiac T-tubules. Global and cardiomyocyte-specific loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy and premature death. Therefore, little is known as to the role of NEXN in adult cardiomyocytes. Transverse-axial tubular system remodeling are well-known features in heart failure. Although NEXN is required during development for T-tubule formation, its role, if any, in mature T-tubules remains to be addressed.METHODS:Nexn inducible adult cardiomyocyte-specific KO mice were generated. Comprehensive morphological and functional analyses were performed. Heart samples (n>3) were analyzed by molecular, biochemical, and electron microscopy analyses. Isolated single adult cardiomyocytes were analyzed by confocal microscopy, and myocyte shortening/re-lengthening and Ca2+ transient studies were conducted.RESULTS:Inducible cardiomyocyte-specific loss of Nexn in adult mice resulted in a dilated cardiomyopathy with reduced cardiac function (13% reduction in percentage fractional shortening; P<0.05). In vivo and in vitro analyses of adult mouse heart samples revealed that NEXN was essential for optimal contraction and calcium handling and was required for maintenance of T-tubule network organization (transverse tubular component in Nexn inducible adult cardiomyocyte-specific KO mice reduced by 40% with respect to controls, P<0.05).CONCLUSIONS:Results here reported reveal NEXN to be a pivotal component of adult junctional membrane complexes required for maintenance of transverse-axial tubular architecture. These results demonstrate that NEXN plays an essential role in the adult cardiomyocyte and give further understanding of pathological mechanisms responsible for cardiomyopathy in patients carrying mutations in the NEXN gene.