Influence of Innervation on Diaphragm Muscle Growth

描述（由申请方提供）：在出生后发育期间，DIAm纤维显示出显著的生长，在表达不同肌球蛋白重链（MHC）亚型的纤维中不成比例。出生后DIAm纤维比力的增加在表达不同MHC亚型的纤维之间也有所不同。单侧去神经支配（DNV）显著延缓出生后DIAm纤维的生长并降低比力。这些数据表明，神经源性因子（如神经营养因子家族）在出生后肌纤维生长中起着关键作用。我们假设神经营养因子调节出生后DIAm纤维的生长和MHC蛋白的表达，从而影响特异性力。所提出的研究的主要目标是：1）探索DIAm纤维出生后生长期间调节MHC蛋白表达的机制，2）探索TrkB受体活化对MHC蛋白表达的影响，3）探索DNV（即，去除神经营养因子的影响）对MHC蛋白表达的影响，以及4）检查出生后MHC蛋白含量的变化与DIAm纤维产生的比力变化之间的关系。在所提出的研究中有四个具体目的：具体目的#1：检查DNV对DIAm纤维中肌球蛋白结构域大小的出生后变化的影响假设1a-在DNV之后，肌球蛋白结构域大小减小，并且这种影响随着出生后年龄和不同MHC同种型而变化。假设Ib-在培养的肌管/肌纤维中，TrkB受体活化增加肌钙蛋白结构域大小。具体目标#2：评价DNV对DIAm纤维中MHC亚型转录的出生后变化的影响。假设2a -DNV后，MHC转录减少，这种影响随出生后年龄和不同MHC亚型而变化。假设2b -在培养的肌管/肌纤维中，TrkB受体活化增加MHC转录。具体目标#3：评价DNV对每半肌节MHC蛋白含量和DIAm纤维中MHC蛋白周转的出生后变化的影响。假设3a -DNV后，每半个肌节的MHC蛋白质含量降低，这种影响随出生后年龄和不同MHC亚型而变化。假设3b -DNV后，MHC的合成率降低，而降解率增加，这些对MHC周转的影响随出生后年龄和不同MHC亚型而变化。假设3c -在培养的肌管/肌纤维中，TrkB受体活化增加MHC蛋白表达。具体目标#4：评价DNV对DIAm纤维比力的出生后变化的影响。假设4a -在DNV之后，DIAm纤维的比力降低，并且这种效应随着出生后年龄和表达不同MHC同种型的纤维而变化。假设4 b-DNV后，DIAm纤维比力的降低反映了每半肌节MHC含量的降低和每横桥力的降低。

DESCRIPTION (provided by applicant): During postnatal development, DIAm fibers display dramatic growth that is disproportionate across fibers expressing different myosin heavy chain (MHC) isoforms. Postnatal increase in specific force of DIAm fibers also varies across fibers expressing different MHC isoforms. Unilateral denervation (DNV) dramatically retards postnatal growth of DIAm fibers and reduces specific force. These data suggest that nerve-derived factors (such as the family of neurotrophins) play a key role in postnatal muscle fiber growth. We hypothesize that neurotrophins modulate the postnatal growth of DIAm fibers and MHC protein expression and thereby affect specific force. The major goals of the proposed studies are: 1) to explore the mechanisms regulating MHC protein expression during postnatal growth of DIAm fibers, 2) to explore the effects of TrkB receptor activation on MHC protein expression, 3) to explore the effects of DNV (i.e., removal of neurotrophin influence) on MHC protein expression, and 4) to examine the relationship between postnatal changes in MHC protein content and changes in specific force generated by DIAm fibers. There are four specific aims in the proposed studies: Specific Aim #1: To examine the impact of DNV on postnatal changes in myonuclear domain size in DIAm fibers Hypothesis la - Following DNV, myonuclear domain size decreases and this effect varies with postnatal age and across different MHC isoforms. Hypothesis lb - In cultured myotubes/myofibers, TrkB receptor activation increases myonuclear domain size. Specific Aim #2: To evaluate the impact of DNV on postnatal changes in MHC isoform transcription in DIAm fibers. Hypothesis 2a - Following DNV, MHC transcription decreases and this effect varies with postnatal age and across different MHC isoforms. Hypothesis 2b - In cultured myotubes/myofibers, TrkB receptor activation increases MHC transcription. Specific Aim #3: To evaluate the impact of DNV on postnatal changes in MHC protein content per haft sarcomere and MHC protein turnover in DIAm fibers. Hypothesis 3a - Following DNV, MHC protein content per half sarcomere decreases and this effect varies with postnatal age and across different MHC isoforms. Hypothesis 3b - Following DNV, the fractional synthesis rate of MHC decreases while degradation rate increases, and these effects on MHC turnover vary with postnatal age and across different MHC isoforms. Hypothesis 3c - In cultured myotubes/myofibers, TrkB receptor activation increases MHC protein expression. Specific Aim #4: To evaluate the impact of DNV on postnatal changes in specific force of DIAm fibers. Hypothesis 4a - Following DNV, specific force of DIAm fibers decreases and this effect varies with postnatal age and across fibers expressing different MHC isoforms. Hypothesis 4b - Following DNV, the decrease in DIAm fiber specific force reflects both a decrease in MHC content per half sarcomere and a decrease in the force per cross bridge.