Czornobil Roman, Abou-Assali Obada, Remily-Wood Elizabeth, Lynch David R, Noujaim Sami F, Chidipi Bojjibabu
bioRxiv. 2023 Nov 13:2023.11.09.566141. doi: 10.1101/2023.11.09.566141.
Friedreich's ataxia (FA) is an inherited neurodegenerative disorder that causes progressive nervous system damage resulting in impaired muscle coordination. FA is the most common autosomal recessive form of ataxia and is caused by an expansion of the DNA triplet guanine-adenine-adenine (GAA) in the first intron of the Frataxin gene (FXN), located on chromosome 9q13. In the unaffected population, the number of GAA repeats ranges from 6 to 27 repetitions. In FA patients, GAA repeat expansions range from 44 to 1,700 repeats which decreases frataxin protein expression. Frataxin is a mitochondrial protein essential for various cellular functions, including iron metabolism. Reduced frataxin expression is thought to negatively affect mitochondrial iron metabolism, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, FA patients display heart disease that includes hypertrophy, heart failure, arrhythmias, conduction abnormalities, and cardiac fibrosis.
In this work, we investigated whether abnormal Ca handling machinery is the molecular mechanism that perpetuates cardiac dysfunction in FA.
We used the frataxin knock-out (FXN-KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. ECG and echocardiography were used to assess cardiac function in the mice. Expression of calcium handling machinery proteins was assessed with proteomics and western blot. In left ventricular myocytes from FXN-KO and FXN-WT mice, the IonOptix system was used for calcium imaging, the seahorse assay was utilized to measure oxygen consumption rate (OCR), and confocal imaging was used to quantify the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS).
We found that major contractile proteins, including SERCA2a and Ryr2, were downregulated in human left ventricular samples from deceased donors with FA compared to unaffected donors, similar to the downregulation of these proteins in the left ventricular tissue from FXN-KO compared to FXN-WT. On the ECG, the RR, PR, QRS, and QTc were significantly longer in the FXN-KO mice compared to FXN-WT. The ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the FXN-KO mice versus FXN-WT. The mitochondrial membrane potential Δψm was depolarized, ROS levels were elevated, and OCR was decreased in ventricular myocytes from FXN-KO versus FXN-WT.
The development of left ventricular contractile dysfunction in FA is associated with reduced expression of calcium handling proteins and mitochondrial dysfunction.
弗里德赖希共济失调（FA）是一种遗传性神经退行性疾病，会导致进行性神经系统损伤，进而造成肌肉协调受损。FA是共济失调最常见的常染色体隐性遗传形式，由位于9号染色体q13上的铁调素基因（FXN）第一个内含子中的DNA三联体鸟嘌呤 - 腺嘌呤 - 腺嘌呤（GAA）扩增引起。在未受影响的人群中，GAA重复序列的数量为6至27次重复。在FA患者中，GAA重复扩增范围为44至1700次重复，这会降低铁调素蛋白的表达。铁调素是一种对包括铁代谢在内的各种细胞功能至关重要的线粒体蛋白。铁调素表达降低被认为会对线粒体铁代谢产生负面影响，导致氧化损伤增加。尽管FA被认为是一种神经退行性疾病，但FA患者会出现包括肥厚、心力衰竭、心律失常、传导异常和心脏纤维化在内的心脏病。
在本研究中，我们调查了异常的钙处理机制是否是导致FA心脏功能障碍持续存在的分子机制。
我们使用了FA的铁调素基因敲除（FXN - KO）小鼠模型以及来自FA供体和未受影响供体的人类心脏样本。通过心电图和超声心动图评估小鼠的心脏功能。用蛋白质组学和蛋白质印迹法评估钙处理机制蛋白的表达。在FXN - KO和FXN - WT小鼠的左心室心肌细胞中，使用IonOptix系统进行钙成像，利用海马体分析法测量氧消耗率（OCR），并使用共聚焦成像来量化线粒体膜电位（Δψm）和活性氧（ROS）。
我们发现，与未受影响的供体相比，来自已故FA供体的人类左心室样本中，包括肌浆网钙ATP酶2a（SERCA2a）和兰尼碱受体2（Ryr2）在内的主要收缩蛋白下调，这与FXN - KO小鼠左心室组织中这些蛋白的下调情况类似。在心电图上，与FXN - WT相比，FXN - KO小鼠的RR、PR、QRS和QTc明显更长。与FXN - WT相比，FXN - KO小鼠的射血分数和缩短分数显著降低，左心室壁厚度和直径显著增加。与FXN - WT相比，FXN - KO小鼠心室肌细胞中的线粒体膜电位Δψm去极化，ROS水平升高，OCR降低。
FA中左心室收缩功能障碍的发展与钙处理蛋白表达降低和线粒体功能障碍有关。
