Molecular insights into meat quality determination: role of calcineurin in oxidative fibre conversion and in regulation of myosin heavy chain genes

肉质测定的分子见解：钙调神经磷酸酶在氧化纤维转化和肌球蛋白重链基因调节中的作用

• 批准号: BB/F018487/1
• 负责人: Kin-Chow Chang
• 金额: $ 50.67万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF018487%2F1
• 关键词: Molecular; insights; into; meat; quality

Fibre type composition of a muscle in a live animal has a strong bearing on its subsequent meat quantity and quality at post mortem. Favourable meat quality traits, in particular pork quality, like tenderness and colour, are associated with the abundance of the most oxidative fibre types, as defined by the presence of myosin heavy chain (MyHC) slow and 2a isoforms. The most important cellular pathway that is responsible for converting fast-glycolytic fibres to oxidative fibres is calcineurin signalling. Little is known about the downstream protein factors or genes that are under the control of calcineurin, a calcium-dependent serine-threonine phosphatase. This project is about the use of an integrated genomics approach to identify novel molecular targets (protein factors and DNA regulatory elements) of calcineurin, which will greatly advance our understanding of how calcineurin differentially and co-ordinately converts fast-glycolytic to oxidative fibres. The scientific impetus behind the proposal is based on 2 recent key findings. Firstly, we discovered that calcineurin differentially regulates the post-natal MyHC genes (2a, 2x and 2b), which reveals a novel mechanistic process in which calcineurin mediates an oxidative fibre type outcome (Fig.1). We found that an early response to calcineurin is the induction of the fast-oxidative MyHC2a, and the down-regulation of the faster and more glycolytic MyHC2x and MyHC2b genes. By contrast, the up-regulation of the most oxidative MyHCslow by calcineurin is only detected after the differential regulation of the post-natal fast MyHC genes has been well established. Hence calcineurin appears to play a crucial role in the early down-regulation of the fast-glycolytic fibre phenotype (MyHC2x and 2b) to facilitate the oxidative outcome of MyHC2a and subsequent MyHCslow expression. Secondly, we demonstrated that calcineurin complex is able to bind MyHC chromatin DNA (Fig.2), which suggests that calcineurin-chromatin interaction could be an important mechanism of calcineurin signalling. We hypothesise that chromatin binding is functionally important for calcineurin to mediate the coordinated isoform-specific programme of an oxidative phenotype. The strategy proposed is to integrate the use of ChIP-on-chip analysis for the genome-wide detection of protein-factor binding (such as calcineurin) to promoters, with our recently acquired expression microarray data from calcineurin over-expression studies. This combined approach would greatly enhance the identification of novel trans-regulatory factors and DNA regulatory cis-acting elements at MyHC and other promoters in the genome that contribute to oxidative fibre conversion. This project will provide much needed underpinning knowledge of the molecular mechanisms of calcineurin signalling that are responsible for the promotion of favourable meat quality fibres. A possible project outcome that is of commercial importance is the identification of candidate target genes of calcineurin that could be developed into DNA markers for marker-assisted selection to improved meat quality. The exploitation of such markers needs to take into account the costs of target-species SNP development (e.g. in pigs) and association studies, potential commercial benefit and possible additional effects of selection. Another possible commercial outcome is the identification of candidate effector genes as pharmacological targets. The precise targeting of downstream genes has the potential to minimise unwanted side effects, and to safely improve specific muscle parameters. In farm animal production, the direct pharmacological modification of a target gene or protein activity to improve meat quality is not necessarily the only approach available. Depending on the target, the strategic use of an immunisation approach, as exemplified by the GnRH vaccine (Improvac) for sterilisation of boars, could be an effective strategy in improving animal production.

活体动物肌肉的纤维类型组成对其死后的肉的数量和质量有很大的影响。良好的肉类品质特征，特别是猪肉品质，如嫩度和颜色，与最具氧化性的纤维类型的丰富有关，如肌球蛋白重链(MyHC)慢型和2a亚型的存在。负责将快速糖酵解纤维转化为氧化纤维的最重要的细胞途径是钙调神经磷酸酶信号。人们对钙调神经磷酸酶(一种钙依赖的丝氨酸-苏氨酸磷酸酶)控制下的下游蛋白因子或基因知之甚少。这个项目是关于使用一种集成的基因组学方法来确定钙调神经磷酸酶的新的分子靶点(蛋白质因子和DNA调控元件)，这将极大地促进我们对钙调神经磷酸酶如何差异和协调地将快速糖酵解转化为氧化纤维的理解。这项提议背后的科学推动力是基于最近的两项关键发现。首先，我们发现钙调神经磷酸酶对出生后MyHC基因(2a、2x和2b)有不同的调节作用，这揭示了钙调神经磷酸酶介导氧化纤维型结果的新机制(图1)。我们发现，对钙调神经磷酸酶的早期反应是诱导快速氧化的MyHC2a，以及更快和更具糖酵解作用的MyHC2x和MyHC2b基因的下调。相比之下，只有在出生后快速MyHC基因的差异调控已经很好地建立之后，才能检测到钙调神经磷酸酶上调氧化程度最高的MyHClow。因此，钙调神经磷酸酶似乎在快速糖酵解纤维表型(MyHC2x和2b)的早期下调中发挥关键作用，以促进MyHC2a的氧化结果和随后的MyHClow表达。其次，我们证明了钙调神经磷酸酶复合体能够结合MyHC染色质DNA(图2)，这表明钙调神经磷酸酶与染色质的相互作用可能是钙调神经磷酸酶信号转导的一个重要机制。我们假设染色质结合对于钙调神经磷酸酶调节氧化表型的协调异构体特异性程序具有重要的功能。建议的策略是将芯片上分析用于全基因组检测蛋白质因子结合(如钙调神经磷酸酶)与启动子的结合，与我们最近从钙调神经磷酸酶过度表达研究中获得的表达微阵列数据相结合。这种结合的方法将极大地增强在MyHC和基因组中有助于氧化纤维转换的其他启动子上的新的反式调节因子和DNA调节顺式作用元件的识别。该项目将为促进良好肉类品质纤维的钙调神经磷酸酶信号转导的分子机制提供急需的基础知识。一个可能具有商业意义的项目成果是鉴定钙调神经磷酸酶的候选目标基因，这些基因可以开发成DNA标记，用于标记辅助选择，以改善肉类品质。利用这种标记需要考虑到目标物种SNP开发(例如在猪身上)和关联研究的成本、潜在的商业利益和可能的额外选择影响。另一个可能的商业成果是确定候选效应基因作为药理靶点。对下游基因的精确定位有可能将不良副作用降至最低，并安全地改善特定的肌肉参数。在农场动物生产中，直接对目标基因或蛋白质活性进行药理学修饰以改善肉类品质并不一定是唯一可用的方法。根据目标的不同，战略性地使用免疫方法，如用于公猪绝育的促性腺激素释放激素疫苗(Improvac)，可能是提高动物产量的有效战略。

项目成果

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/beta gene expression via acetylation of nuclear factor of activated T cells c1.

• DOI: 10.1093/nar/gkr162
• 发表时间: 2011-08
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Meissner JD;Freund R;Krone D;Umeda PK;Chang KC;Gros G;Scheibe RJ
• 通讯作者: Scheibe RJ

Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

• DOI: 10.1128/jvi.03421-14
• 发表时间: 2015-03
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Baquero-Perez B;Kuchipudi SV;Ho J;Sebastian S;Puranik A;Howard W;Brookes SM;Brown IH;Chang KC
• 通讯作者: Chang KC

Extended 2D myotube culture recapitulates postnatal fibre type plasticity.

• DOI: 10.1186/s12860-015-0069-1
• 发表时间: 2015-09-17
• 期刊: BMC cell biology
• 作者: Sebastian S;Goulding L;Kuchipudi SV;Chang KC
• 通讯作者: Chang KC

A simplified but robust method for the isolation of avian and mammalian muscle satellite cells.

• DOI: 10.1186/1471-2121-13-16
• 发表时间: 2012-06-21
• 期刊: BMC cell biology
• 作者: Baquero-Perez B;Kuchipudi SV;Nelli RK;Chang KC
• 通讯作者: Chang KC

Mitogen-activated protein kinase-activated protein kinases 2 and 3 regulate SERCA2a expression and fiber type composition to modulate skeletal muscle and cardiomyocyte function.

丝裂原激活蛋白激酶激活蛋白激酶 2 和 3 调节 SERCA2a 表达和纤维类型组成，以调节骨骼肌和心肌细胞功能。

• DOI: 10.1128/mcb.01692-12
• 发表时间: 2013
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Scharf M