Cell biology
Abstract
Cardiac hypertrophy, as a response to hemodynamic stress, is associated with cardiac dysfunction and death, but whether hypertrophy itself represents a pathological process remains unclear. Hypertrophy is driven by changes in myocardial gene expression that require the MEF2 family of DNA-binding transcription factors, as well as the nuclear lysine acetyltransferase p300. Here we used genetic and small-molecule probes to determine the effects of preventing MEF2 acetylation on cardiac adaptation to stress. Both nonacetylatable MEF2 mutants and 8MI, a molecule designed to interfere with MEF2-coregulator binding, prevented hypertrophy in cultured cardiac myocytes. 8MI prevented cardiac hypertrophy in 3 distinct stress models, and reversed established hypertrophy in vivo, associated with normalization of myocardial structure and function. The effects of 8MI were reversible, and did not prevent training effects of swimming. Mechanistically, 8MI blocked stress-induced MEF2 acetylation, nuclear export of class II histone deacetylases HDAC4 and -5, and p300 induction, without impeding HDAC4 phosphorylation. Correspondingly, 8MI transformed the transcriptional response to pressure overload, normalizing almost all 232 genes dysregulated by hemodynamic stress. We conclude that MEF2 acetylation is required for development and maintenance of pathological cardiac hypertrophy, and that blocking MEF2 acetylation can permit recovery from hypertrophy without impairing physiologic adaptation.
Authors
Jianqin Wei, Shaurya Joshi, Svetlana Speransky, Christopher Crowley, Nimanthi Jayathilaka, Xiao Lei, Yongqing Wu, David Gai, Sumit Jain, Michael Hoosien, Yan Gao, Lin Chen, Nanette H. Bishopric
Abstract
Heart disease remains the leading cause of death worldwide, highlighting a pressing need to identify novel regulators of cardiomyocyte (CM) function that could be therapeutically targeted. The mammalian Hippo/Tead pathway is critical in embryonic cardiac development and perinatal CM proliferation. However, the requirement of Tead1, the transcriptional effector of this pathway, in the adult heart is unknown. Here, we show that tamoxifen-inducible adult CM–specific Tead1 ablation led to lethal acute-onset dilated cardiomyopathy, associated with impairment in excitation-contraction coupling. Mechanistically, we demonstrate Tead1 is a cell-autonomous, direct transcriptional activator of SERCA2a and SR-associated protein phosphatase 1 regulatory subunit, Inhibitor-1 (I-1). Thus, Tead1 deletion led to a decrease in SERCA2a and I-1 transcripts and protein, with a consequent increase in PP1-activity, resulting in accumulation of dephosphorylated phospholamban (Pln) and decreased SERCA2a activity. Global transcriptomal analysis in Tead1-deleted hearts revealed significant changes in mitochondrial and sarcomere-related pathways. Additional studies demonstrated there was a trend for correlation between protein levels of TEAD1 and I-1, and phosphorylation of PLN, in human nonfailing and failing hearts. Furthermore, TEAD1 activity was required to maintain PLN phosphorylation and expression of SERCA2a and I-1 in human induced pluripotent stem cell–derived (iPS-derived) CMs. To our knowledge, taken together, this demonstrates a nonredundant, novel role of Tead1 in maintaining normal adult heart function.
Authors
Ruya Liu, Jeongkyung Lee, Byung S. Kim, Qiongling Wang, Samuel K. Buxton, Nikhil Balasubramanyam, Jean J. Kim, Jianrong Dong, Aijun Zhang, Shumin Li, Anisha A. Gupte, Dale J. Hamilton, James F. Martin, George G. Rodney, Cristian Coarfa, Xander H.T. Wehrens, Vijay K. Yechoor, Mousumi Moulik
Abstract
Adrenergic signaling is known to promote tumor growth and metastasis, but the effects on tumor stroma are not well understood. An unbiased bioinformatics approach analyzing tumor samples from patients with known biobehavioral profiles identified a prominent stromal signature associated with cancer-associated fibroblasts (CAFs) in those with a high biobehavioral risk profile (high Center for Epidemiologic Studies Depression Scale [CES-D] score and low social support). In several models of epithelial ovarian cancer, daily restraint stress resulted in significantly increased CAF activation and was abrogated by a nonspecific β-blocker. Adrenergic signaling–induced CAFs had significantly higher levels of collagen and extracellular matrix components than control tumors. Using a systems-based approach, we found INHBA production by cancer cells to induce CAFs. Ablating inhibin β A decreased CAF phenotype both in vitro and in vivo. In preclinical models of breast and colon cancers, there were increased CAFs and collagens following daily restraint stress. In an independent data set of renal cell carcinoma patients, there was an association between high depression (CES-D) scores and elevated expression of ACTA2, collagens, and inhibin β A. Collectively, our findings implicate adrenergic influences on tumor stroma as important drivers of CAFs and establish inhibin β A as an important regulator of the CAF phenotype in ovarian cancer.
Authors
Archana S. Nagaraja, Robert L. Dood, Guillermo Armaiz-Pena, Yu Kang, Sherry Y. Wu, Julie K. Allen, Nicholas B. Jennings, Lingegowda S. Mangala, Sunila Pradeep, Yasmin Lyons, Monika Haemmerle, Kshipra M. Gharpure, Nouara C. Sadaoui, Cristian Rodriguez-Aguayo, Cristina Ivan, Ying Wang, Keith Baggerly, Prahlad Ram, Gabriel Lopez-Berestein, Jinsong Liu, Samuel C. Mok, Lorenzo Cohen, Susan K. Lutgendorf, Steve W. Cole, Anil K. Sood
Abstract
The architectural integrity of tissues requires complex interactions, both between cells and between cells and the extracellular matrix. Fundamental to cell and tissue homeostasis are the specific mechanical forces conveyed by the actomyosin cytoskeleton. Here we used super-resolution imaging methods to visualize the actin cytoskeleton in the kidney glomerulus, an organized collection of capillaries that filters the blood to make the primary urine. Our analysis of both mouse and human glomeruli reveals a network of myosin IIA–containing contractile actin cables within podocyte cell bodies and major processes at the outer aspects of the glomerular tuft. These likely exert force on an underlying network of myosin IIA–negative, noncontractile actin fibers present within podocyte foot processes that function to both anchor the cells to the glomerular basement membrane and stabilize the slit diaphragm against the pressure of fluid flow. After injuries that disrupt the kidney filtration barrier and cause foot process effacement, the podocyte’s contractile actomyosin network relocates to the basolateral surface of the cell, manifesting as sarcomere-like structures juxtaposed to the basement membrane. Our findings suggest a new model of the podocyte actin cytoskeleton in health and disease and suggest the existence of novel mechanisms that regulate podocyte architecture.
Authors
Hani Y. Suleiman, Robyn Roth, Sanjay Jain, John E. Heuser, Andrey S. Shaw, Jeffrey H. Miner
Abstract
Myocardial atrophy is a wasting of cardiac muscle due to hemodynamic unloading. Doxorubicin is a highly effective anticancer agent but also induces myocardial atrophy through a largely unknown mechanism. Here, we demonstrate that inhibiting transient receptor potential canonical 3 (TRPC3) channels abolishes doxorubicin-induced myocardial atrophy in mice. Doxorubicin increased production of ROS in rodent cardiomyocytes through hypoxic stress–mediated upregulation of NADPH oxidase 2 (Nox2), which formed a stable complex with TRPC3. Cardiomyocyte-specific expression of TRPC3 C-terminal minipeptide inhibited TRPC3-Nox2 coupling and suppressed doxorubicin-induced reduction of myocardial cell size and left ventricular (LV) dysfunction, along with its upregulation of Nox2 and oxidative stress, without reducing hypoxic stress. Voluntary exercise, an effective treatment to prevent doxorubicin-induced cardiotoxicity, also downregulated the TRPC3-Nox2 complex and promoted volume load–induced LV compliance, as demonstrated in TRPC3-deficient hearts. These results illustrate the impact of TRPC3 on LV compliance and flexibility and, focusing on the TRPC3-Nox2 complex, provide a strategy for prevention of doxorubicin-induced cardiomyopathy.
Authors
Tsukasa Shimauchi, Takuro Numaga-Tomita, Tomoya Ito, Akiyuki Nishimura, Ryosuke Matsukane, Sayaka Oda, Sumio Hoka, Tomomi Ide, Norimichi Koitabashi, Koji Uchida, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida
Abstract
The Mediator complex regulates gene transcription by linking basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is mainly controlled by a kinase submodule that is composed of 4 proteins, including MED12. Although ubiquitously expressed, Mediator subunits can differentially regulate gene expression in a tissue-specific manner. Here, we report that MED12 is required for normal cardiac function, such that mice with conditional cardiac-specific deletion of MED12 display progressive dilated cardiomyopathy. Loss of MED12 perturbs expression of calcium-handling genes in the heart, consequently altering calcium cycling in cardiomyocytes and disrupting cardiac electrical activity. We identified transcription factors that regulate expression of calcium-handling genes that are downregulated in the heart in the absence of MED12, and we found that MED12 localizes to transcription factor consensus sequences within calcium-handling genes. We showed that MED12 interacts with one such transcription factor, MEF2, in cardiomyocytes and that MED12 and MEF2 co-occupy promoters of calcium-handling genes. Furthermore, we demonstrated that MED12 enhances MEF2 transcriptional activity and that overexpression of both increases expression of calcium-handling genes in cardiomyocytes. Our data support a role for MED12 as a coordinator of transcription through MEF2 and other transcription factors. We conclude that MED12 is a regulator of a network of calcium-handling genes, consequently mediating contractility in the mammalian heart.
Authors
Kedryn K. Baskin, Catherine A. Makarewich, Susan M. DeLeon, Wenduo Ye, Beibei Chen, Nadine Beetz, Heinrich Schrewe, Rhonda Bassel-Duby, Eric N. Olson
Abstract
Our previous work showed myocellular differences in pediatric and adult dilated cardiomyopathy (DCM). However, a thorough characterization of the molecular pathways involved in pediatric DCM does not exist, limiting the development of age-specific therapies. To characterize this patient population, we investigated the transcriptome profile of pediatric patients. RNA-Seq from 7 DCM and 7 nonfailing (NF) explanted age-matched pediatric left ventricles (LV) was performed. Changes in gene expression were confirmed by real-time PCR (RT-PCR) in 36 DCM and 21 NF pediatric hearts and in 20 DCM and 10 NF adult hearts. The degree of myocyte hypertrophy was investigated in 4 DCM and 7 NF pediatric hearts and in 4 DCM and 9 NF adult hearts. Changes in gene expression in response to pluripotency-inducing factors were investigated in neonatal rat ventricular myocytes (NRVMs). Transcriptome analysis identified a gene expression profile in children compared with adults with DCM. Additionally, myocyte hypertrophy was not observed in pediatric hearts but was present in adult hearts. Furthermore, treatment of NRVMs with pluripotency-inducing factors recapitulated changes in gene expression observed in the pediatric DCM heart. Pediatric DCM is characterized by unique changes in gene expression that suggest maintenance of an undifferentiated state.
Authors
Philip D. Tatman, Kathleen C. Woulfe, Anis Karimpour-Fard, Danielle A. Jeffrey, James Jaggers, Joseph C. Cleveland, Karin Nunley, Matthew R.G. Taylor, Shelley D. Miyamoto, Brian L. Stauffer, Carmen C. Sucharov
Abstract
Familial hemophagocytic lymphohistiocytosis 5 (FHL5) is an autosomal recessive disease caused by mutations in STXBP2, coding for Munc18-2, which is required for SNARE-mediated membrane fusion. FHL5 causes hematologic and gastrointestinal symptoms characterized by chronic enteropathy that is reminiscent of microvillus inclusion disease (MVID). However, the molecular pathophysiology of FHL5-associated diarrhea is poorly understood. Five FHL5 patients, including four previously unreported patients, were studied. Morphology of duodenal sections was analyzed by electron and fluorescence microscopy. Small intestinal enterocytes and organoid-derived monolayers displayed the subcellular characteristics of MVID. For the analyses of Munc18-2–dependent SNARE-protein interactions, a Munc18-2 CaCo2–KO model cell line was generated by applying CRISPR/Cas9 technology. Munc18-2 is required for Slp4a/Stx3 interaction in fusion of cargo vesicles with the apical plasma membrane. Cargo trafficking was investigated in patient biopsies, patient-derived organoids, and the genome-edited model cell line. Loss of Munc18-2 selectively disrupts trafficking of certain apical brush-border proteins (NHE3 and GLUT5), while transport of DPPIV remained unaffected. Here, we describe the molecular mechanism how the loss of function of Munc18-2 leads to cargo-selective mislocalization of brush-border components and a subapical accumulation of cargo vesicles, as it is known from the loss of polarity phenotype in MVID.
Authors
Georg F. Vogel, Jorik M. van Rijn, Iris M. Krainer, Andreas R. Janecke, Carsten Posovzsky, Marta Cohen, Claire Searle, Prevost Jantchou, Johanna C. Escher, Natalie Patey, Ernest Cutz, Thomas Müller, Sabine Middendorp, Michael W. Hess, Lukas A. Huber
Abstract
Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.
Authors
Luke M. Judge, Juan A. Perez-Bermejo, Annie Truong, Alexandre J.S. Ribeiro, Jennie C. Yoo, Christina L. Jensen, Mohammad A. Mandegar, Nathaniel Huebsch, Robyn M. Kaake, Po-Lin So, Deepak Srivastava, Beth L. Pruitt, Nevan J. Krogan, Bruce R. Conklin
Abstract
Magnesium (Mg2+) plays pleiotropic roles in cellular biology, and it is essentially required for all living organisms. Although previous studies demonstrated intracellular Mg2+ levels were regulated by the complex of phosphatase of regenerating liver 2 (PRL2) and Mg2+ transporter of cyclin M (CNNMs), physiological functions of PRL2 in whole animals remain unclear. Interestingly, Mg2+ was recently identified as a regulator of circadian rhythm–dependent metabolism; however, no mechanism was found to explain the clock-dependent Mg2+ oscillation. Herein, we report PRL2 as a missing link between sex and metabolism, as well as clock genes and daily cycles of Mg2+ fluxes. Our results unveil that PRL2-null animals displayed sex-dependent alterations in body composition, and expression of PRLs and CNNMs were sex- and circadian time–dependently regulated in brown adipose tissues. Consistently, PRL2-KO mice showed sex-dependent alterations in thermogenesis and in circadian energy metabolism. These physiological changes were associated with an increased rate of uncoupled respiration with lower intracellular Mg2+ in PRL2-KO cells. Moreover, PRL2 deficiency causes inhibition of the ATP citrate lyase axis, which is involved in fatty acid synthesis. Overall, our findings support that sex- and circadian-dependent PRL2 expression alter intracellular Mg2+ levels, which accordingly controls energy metabolism status.
Authors
Noriko Uetani, Serge Hardy, Simon-Pierre Gravel, Silke Kiessling, Adam Pietrobon, Nau Nau Wong, Valérie Chénard, Nicolas Cermakian, Julie St-Pierre, Michel L. Tremblay
No posts were found with this tag.
