Functional Consequences of FHC-linked RLC Mutations.

FHC 相关 RLC 突变的功能后果。

• 批准号: 8242787
• 负责人: Danuta Szczesna-Cordary
• 金额: $ 38.39万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242787
• 关键词: ATP phosphohydrolase; Actins; Address; Affect; Affinity; Animal Model; Atrial Myosins; Atrial Natriuretic Factor; Attenuated; Binding; Binding Proteins; Binding Sites; Biological Assay; Buffers; Calcium; Calmodulin; Cardiac; Cardiac Death; Cardiac Muscle Contraction; Cardiovascular Diseases; Cells; Contractile Proteins; Development; Disease; Dissociation; Dyspnea; EF Hand Motifs; Echocardiography; Electrocardiogram; Energy Metabolism; Event; Evolution; Familial Hypertrophic Cardiomyopathy; Family; Fatigue; Filament; Functional disorder; Generations; Genes; Health; Heart; Heart Hypertrophy; Heart failure; Hematoxylin and Eosin Staining Method; Histopathology; Human; Hypertrophy; Immunofluorescence Immunologic; In Vitro; Induced Mutation; Intervention; Kinetics; Knowledge; Laboratories; Lead; Light; Link; Measures; Mechanics; Mediating; Medical; Metals; Microfilaments; Molecular; Monitor; Morphology; Mus; Muscle; Muscle Contraction; Muscle Fibers; Muscle relaxation phase; Mutant Strains Mice; Mutate; Mutation; Myocardium; Myofibrils; Myosin ATPase; Myosin Alkali Light Chains; Myosin Heavy Chains; Myosin Light Chain Kinase; Myosin Regulatory Light Chains; Organ; Pathology; Performance; Performance at work; Phenotype; Phosphorylation; Physiological; Play; Preparation; Process; Production; Property; Proteins; Pump; Regulation; Relaxation; Research; Role; Sarcomeres; Sarcoplasmic Reticulum; Severities; Site; Skin; Solutions; Staining method; Stains; Techniques; Testing; Therapeutic; Tissues; Transcript; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Troponin C; Troponin I; Ventricular; Work; age related; base; blood pump; cell motility; heart function; hemodynamics; improved; in vivo; innovation; insight; interdisciplinary approach; intermolecular interaction; mRNA Expression; mortality; mouse model; mutant; myosin-binding protein C; novel; optical traps; papillary muscle; phospholamban; premature; prevent; protein expression; research study; single molecule; skeletal; sudden cardiac death

DESCRIPTION (provided by applicant): Familial hypertrophic cardiomyopathy (FHC) is one of the pathological compensatory manifestations found in the heart resulting from its inability to adequately pump blood, thus leading to hypertrophy and often to premature cardiac death. Over the past 4 years our laboratory has been studying the functional consequences of several FHC mutations in the regulatory light chain (RLC) of myosin expressed in transgenic mice. We hypothesize that by changing the properties of the RLC Ca2+-Mg2+ binding site, the FHC mutations interfere with the intracellular function of RLC as a temporary delayed Ca2+- buffer and lead to increased or decreased kinetics of muscle relaxation. Another hypothesis pertains to the mutation controlled metal occupancy of the Ca2+-Mg2+ binding site of RLC and the mechanism by which Ca2+ or Mg2+ binding to RLC may influence the interaction of myosin with actin and tension generation. We further hypothesize that an FHC induced pathological cardiac phenotype can be rescued by Ca2+-calmodulin activated MLCK phosphorylation of the RLC-mutated myocardium. This application will continue the use of integrated multidisciplinary approaches from single molecule, cell to organ levels and novel transgenic mouse models to address the following questions: SPECIFIC AIM 1: Do FHC induced changes in the properties of the RLC Ca2+-Mg2+ binding site inhibit or facilitate the function of RLC as a temporary intracellular calcium buffer? Do FHC mutations shift the metal occupancy of the RLC Ca2+-Mg2+ binding site during muscle contraction? SPECIFIC AIM 2: Is RLC phosphorylation by Ca2+-calmodulin (CaM) activated myosin light chain kinase (MLCK) affected by FHC-linked RLC mutations? Can MLCK phosphorylation rescue a mutation induced pathological cardiac phenotype? SPECIFIC AIM 3: Do FHC-associated mutations in RLC alter intermolecular interactions between RLC and myosin heavy chain (HC) and ultimately myosin and actin? Do these changes lead to myofilament disarray, cardiac hypertrophy and dysfunction of the mutated myocardium? Successful execution of this proposal will result in new mechanical, physiological and histological information regarding the role of the RLC in cardiac muscle contraction in health and disease. Relevance: Cardiovascular diseases are the number one cause of mortality worldwide with heart failure being highly prevalent in most affluent parts of the world. There is an urgent need for a better understanding of the mechanisms underlying Familial Hypertrophic Cardiomyopathy (FHC) that often leads to premature sudden cardiac death (SCD). This proposal addresses the mechanisms by which the mutations in myosin regulatory light chain (RLC) cause FHC and lead to SCD. Determining the mechanisms of the RLC- mediated regulation of contraction in the healthy and hypertrophic heart will provide insight and be instrumental in developing specific therapeutic strategies that can be employed to reverse or prevent FHC RLC pathology.

描述（由申请人提供）：家族性肥厚性心肌病（FHC）是心脏中发现的病理性代偿表现之一，其原因是心脏无法充分泵血，从而导致肥大，并经常导致心源性过早死亡。在过去的4年里，我们的实验室一直在研究转基因小鼠中表达的肌球蛋白的调节轻链（RLC）中的几个FHC突变的功能后果。我们推测，通过改变RLC的Ca 2 +-Mg 2+结合位点的性质，FHC突变干扰RLC作为暂时延迟的Ca 2+缓冲液的细胞内功能，并导致肌肉松弛动力学的增加或减少。另一种假说涉及RLC的Ca ~（2+）-Mg ~（2+）结合位点的突变控制的金属占据以及Ca ~（2+）或Mg ~（2+）与RLC的结合可能影响肌球蛋白与肌动蛋白的相互作用和张力产生的机制。我们进一步假设FHC诱导的病理性心脏表型可以通过RLC突变心肌的Ca 2 +-钙调蛋白激活的MLCK磷酸化来挽救。本申请将继续使用从单分子、细胞到器官水平的综合多学科方法和新型转基因小鼠模型来解决以下问题：具体目的1：FHC诱导的RLC Ca 2 +-Mg 2+结合位点性质的变化是否抑制或促进RLC作为临时细胞内钙缓冲剂的功能？FHC突变是否改变了肌肉收缩过程中RLC Ca ~（2+）-Mg ~（2+）结合位点的金属占有率？特异性目的2：钙调蛋白（CaM）激活的肌球蛋白轻链激酶（MLCK）引起的RLC磷酸化是否受FHC相关RLC突变的影响？MLCK磷酸化能否挽救突变诱导的病理性心脏表型？具体目标3：RLC中FHC相关突变是否改变RLC与肌球蛋白重链（HC）以及最终肌球蛋白与肌动蛋白之间的分子间相互作用？这些变化是否会导致突变心肌的肌丝紊乱、心肌肥大和功能障碍？成功地执行这一建议将导致新的机械，生理和组织学信息的RLC在心肌收缩中的作用在健康和疾病。 相关性：心血管疾病是世界范围内的头号死亡原因，心力衰竭在世界上最富裕的地区非常普遍。家族性肥厚型心肌病（FHC）常导致心源性猝死（SCD），目前迫切需要更好地了解其发病机制。该建议阐述了肌球蛋白调节轻链（RLC）突变引起FHC并导致SCD的机制。确定健康和肥大心脏中RLC介导的收缩调节的机制将提供洞察力，并有助于开发可用于逆转或预防FHC RLC病理的特定治疗策略。

项目成果

Deletion of 1-43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca(2+) sensitivity and cross-bridge detachment kinetics.

心肌肌球蛋白必需轻链中 1-43 个氨基酸的缺失减弱了 Ca(2) 敏感性和跨桥脱离动力学的长度依赖性。

• DOI: 10.1152/ajpheart.00572.2012
• 发表时间: 2013
• 期刊: American journal of physiology. Heart and circulatory physiology
• 作者: Michael,JohnJeshurun;Gollapudi,SampathK;Ford,StevenJ;Kazmierczak,Katarzyna;Szczesna-Cordary,Danuta;Chandra,Murali
• 通讯作者: Chandra,Murali

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice.

心肌肌球蛋白调节轻链的组成型磷酸化可预防小鼠肥厚型心肌病的发生。

• DOI: 10.1073/pnas.1505819112
• 发表时间: 2015
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Yuan,Chen-Ching;Muthu,Priya;Kazmierczak,Katarzyna;Liang,Jingsheng;Huang,Wenrui;Irving,ThomasC;Kanashiro-Takeuchi,RosemeireM;Hare,JoshuaM;Szczesna-Cordary,Danuta
• 通讯作者: Szczesna-Cordary,Danuta

The E22K mutation of myosin RLC that causes familial hypertrophic cardiomyopathy increases calcium sensitivity of force and ATPase in transgenic mice.

导致家族性肥厚型心肌病的肌球蛋白 RLC E22K 突变会增加转基因小鼠中力和 ATP 酶的钙敏感性。

• DOI: 10.1242/jcs.02492
• 发表时间: 2005
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Szczesna-Cordary,Danuta;Guzman,Georgianna;Zhao,Jiaju;Hernandez,Olga;Wei,Jianqin;Diaz-Perez,Zoraida
• 通讯作者: Diaz-Perez,Zoraida

The effect of myosin RLC phosphorylation in normal and cardiomyopathic mouse hearts.

• DOI: 10.1111/j.1582-4934.2011.01371.x
• 发表时间: 2012-04
• 期刊: Journal of cellular and molecular medicine
• 影响因子: 5.3
• 作者: Muthu P;Kazmierczak K;Jones M;Szczesna-Cordary D
• 通讯作者: Szczesna-Cordary D

The spatial distribution of actin and mechanical cycle of myosin are different in right and left ventricles of healthy mouse hearts.

肌动蛋白的空间分布和肌球蛋白的机械循环在健康小鼠心脏的左右心室不同。

• DOI: 10.1021/bi501175s
• 发表时间: 2014-12-09
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Nagwekar, J.;Duggal, D.;Rich, R.;Raut, S.;Fudala, R.;Gryczynski, I.;Gryczynski, Z.;Borejdo, J.
• 通讯作者: Borejdo, J.