TgfB-dependent regulation of extracellular matrix-cardiomyocyte crosstalk

细胞外基质-心肌细胞串扰的 TgfB 依赖性调节

• 批准号: 10231433
• 负责人: Rachel Minerath
• 金额: $ 6.77万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231433
• 关键词: Acute; Address; Adult; Age; Attenuated; Automobile Driving; Back; Calcium; Cardiac; Cardiac Myocytes; Cardiac development; Cardiomyopathies; Cell physiology; Cells; Chronic; Complement Factor B; Complex; Cytoskeleton; Data; Defect; Deposition; Development; Embryo; Extracellular Matrix; Feedback; Fibroblasts; Fibrosis; Gene Deletion; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Growth Factor; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Histology; Homeostasis; Image; Impairment; Individual; Infarction; Injury; Lead; Ligands; Maintenance; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Microarray Analysis; Modeling; Molecular; Mus; Muscle Cells; Myocardial dysfunction; Myofibroblast; Pathologic; Production; Property; Protein Isoforms; RNA analysis; Regulation; Role; Series; Signal Transduction; Source; Stress; Structural Protein; Structure; TGFBR2 gene; Tamoxifen; Tensile Strength; Testing; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Transforming Growth Factors; cell growth; constriction; coronary fibrosis; heart cell; heart function; in vitro Model; loss of function; mechanical properties; mouse model; programs; receptor; response

Project Summary The extracellular matrix (ECM) is essential for maintaining cardiac structure and function. Aside from providing structural support, the ECM relays molecular signals to cells via cellular attachment complexes and ECM- sequestered growth factors to maintain cellular homeostasis. In response to acute injury or pathological stress, excess ECM deposition occurs that can contribute to cardiac dysfunction. Transforming growth factor β (TGFβ) is an ECM-sequestered growth factor that is released and activated by cardiac stress. Once released from the ECM, TGFβ induces differentiation of quiescent fibroblasts into myofibroblasts which secrete ECM components. However, the requirement for baseline TGFβ signaling in the heart remains unknown. Three TGFβ isoforms exist encoded by three separate genes (Tgfb1, Tgfb2, and Tgfb3) although the specific roles of each TGFβ isoform are not understood. To investigate the role of TGFβ ligands in regulating cardiac homeostasis, we generated mice with cardiomyocyte (CM)-specific deletion of TGFβ ligands (Tgfb1/2/3fl/fl-αMHC-Cre). These mice developed heart failure by 10 weeks of age accompanied by cardiac dilation without signs of fibrosis suggesting defective ECM deposition. In contrast to previous studies demonstrating that TGFβ receptor deletion had minimal effect on cardiac development, microarray analysis of Tgfb1/2/3fl/fl-αMHC-Cre hearts demonstrated a defect in CM differentiation indicating a key role for the ECM in driving CM maturation. To circumvent a developmental role of TGFβ, we employed a Tamoxifen-inducible CM-specific mouse model (Tgfb1/2/3fl/fl-αMHC-MCM) to delete TGFβ ligands from the adult heart. These mice developed heart failure 12-16 weeks post-tamoxifen treatment suggesting that TGFβ is required to maintain cardiac function in the adult heart. Surprisingly, fibroblast-specific deletion of Tgfb1/2/3 did not develop cardiac dysfunction indicating that TGFβ is primarily generated by CMs. Altogether, we hypothesize that TGFβ acts as a critical regulator of ECM-cell cross talk in the heart by signaling to fibroblasts to promote ECM production, which ultimately provides signals back to CMs to maintain their maturation. To address this hypothesis, we will use Tgfb1/2/3fl/fl-αMHC-MCM mice to determine the role of TGFβ in maintaining CM differentiation. Specifically, we will assess these mice for cardiac function, CM maturation and ECM composition and organization. Furthermore, we will perform RNA analysis at multiple timepoints both before and after the onset of cardiac dysfunction to determine the transcriptional role of TGFβ in maintaining CM maturation. Additionally, we aim to decipher the differential roles of each specific ligand (TGFβ 1, 2, or 3) in regulating cardiac function. The proposed studies will further assess the role of TGFβ in mediating CM-cardiac fibroblast crosstalk by studying fibroblast activity using in vitro models. Lastly, we will examine the composition, organization, and mechanical properties of the ECM. Using mass spectrometry, we will identify potential ECM structural proteins and growth factors that may have a role in maintaining CM maturation. Altogether, these studies aim to understand the ECM-cellular crosstalk that occurs in the heart to regulate cardiac homeostasis.

项目摘要 细胞外基质(ECM)是维持心脏结构和功能所必需的。除了提供 结构支持，ECM通过细胞附着复合体和ECM将分子信号传递给细胞- 隔离生长因子以维持细胞动态平衡。作为对急性损伤或病理性压力的反应， 过多的细胞外基质沉积会导致心功能不全。转化生长因子β(转化生长因子β) 是一种ECM隔离的生长因子，由心脏应激释放和激活。一旦从 细胞外基质、转化生长因子β诱导静止的成纤维细胞分化为分泌细胞外基质成分的肌成纤维细胞。 然而，对心脏中基线转化生长因子β信号的需求仍然未知。三种转化生长因子β亚型 存在由三个独立的基因(Tgfb1、Tgfb2和TgFB3)编码的基因，尽管每个转化生长因子β的特定作用 异构体是不被理解的。为了研究转化生长因子β配体在调节心脏内稳态中的作用，我们 心肌细胞(CM)特异性缺失转化生长因子β配体的小鼠(Tgfb1/2/3fl/fl-αMHC-Cre)。这些老鼠 在10周龄时出现心力衰竭，并伴有心脏扩张，没有纤维化的迹象 有缺陷的ECM沉积。与以前的研究表明转化生长因子β受体缺失有 对心脏发育的最小影响，Tgfb1/2/3fl/fl-αMHC-Cre心脏的微阵列分析显示存在缺陷 在CM分化中，表明ECM在推动CM成熟中起着关键作用。绕过发展中的 我们采用三苯氧胺诱导的CM特异性小鼠模型(Tgfb1/2/3fl/fl-βMHC-α)删除 转化生长因子β配体来自成人心脏。这些小鼠在他莫昔芬治疗12-16周后出现心力衰竭。 提示转化生长因子β是维持成人心脏功能所必需的。令人惊讶的是，成纤维细胞特异性 Tgfb1/2/3缺失未发生心功能障碍，提示转化生长因子β主要由CMS产生。 综上所述，我们假设转化生长因子β在心脏中作为细胞外基质-细胞串扰的关键调节器通过信号传递起作用。 到成纤维细胞以促进ECM的产生，而ECM最终向CMS提供信号以维持其 成熟。为了解决这一假设，我们将使用Tgfb1/2/3fl/fl-αMHC-MCM小鼠来确定转化生长因子β在 保持CM的差异化。具体来说，我们将评估这些小鼠的心脏功能、CM成熟度和 ECM的组成和组织。此外，我们将在多个时间点进行RNA分析 心功能不全前后转化生长因子β转录在维持心功能障碍中的作用 成熟。此外，我们的目标是破译每个特定配体(转化生长因子β1、2或3)在 调节心脏功能。拟议的研究将进一步评估转化生长因子β在介导心肌梗死中的作用。 利用体外模型研究成纤维细胞的活性。最后，我们来检查一下这个构图， ECM的组织和机械性能。利用质谱学，我们将识别潜在的ECM 可能对维持CM成熟有作用的结构蛋白和生长因子。总而言之，这些 研究的目的是了解细胞外基质-细胞串扰发生在心脏，以调节心脏的动态平衡。