IMPACT OF CARDIOMYOCYTE-SPECIFIC BMAL1 DELETION ON MYOCARDIAL GENE EXPRESSION, METABOLISM, AND CONTRACTILE FUNCTION

Circadian clocks are cell autonomous, transcriptionally-based, molecular mechanisms that confer the selective advantage of anticipation, enabling cells/organs to respond to environmental factors in a temporally appropriate manner. Critical to circadian clock function are two transcription factors, CLOCK and BMAL1. Previous studies in our laboratory have highlighted roles for CLOCK in cardiac physiology/pathophysiology. Here, we describe transcriptional, metabolic, and functional consequences of cardiomyocyte-specific Bmal1 knockout (CBK). Microarray analysis revealed 2037 differentially expressed genes in CBK hearts, many of which were previously identified in cardiomyocyte-specific Clock mutant (CCM) hearts. Subsequent analysis showed that Beta-hydroxybutyrate dehydrogenase 1 mRNA, protein, and enzymatic activity are markedly depressed in both CBK and CCM hearts, as is myocardial Beta-hydroxybutyrate oxidation, revealing a novel role for the circadian clock in ketone body utilization. A number of genes encoding for collagen isoforms were identified as oscillating in a time-of-day-dependent manner in wild-type, but not CBK, hearts, including col3a1, col4a1, and col4a2. Chronic induction of collagen isoform genes in CBK hearts was associated with severe age-dependent depression of cardiac function. Development of cardiomyopathy in CBK mice was associated with early mortality; all CBK mice die by one year of age. These studies highlight novel critical functions for BMAL1 in the heart, including regulation of ketone body metabolism and the extracellular matrix. RNA from whole hearts collected every 3 hours for 24 hours from wildtype and CBK mice was isolated and analyzed using MouseRef-8_V2 BeadChips (Illumina, Inc.). The 24-hour data were examined for rhythmicity using cosinor analysis and differences in rhythmicity between genotype groups were further examined for differences in the model fitting parameters.昼夜节律钟（circadian clock）是一类细胞自主性的、基于转录调控的分子机制，其赋予机体预期性选择优势，使细胞与器官能够以时间适配的方式响应环境因素。昼夜节律钟发挥功能的核心调控因子为两类转录因子：CLOCK与BMAL1。本实验室既往研究已阐明CLOCK在心脏生理与病理生理过程中的重要作用。本研究针对心肌细胞特异性BMAL1敲除（cardiomyocyte-specific Bmal1 knockout, CBK）模型，系统阐述其转录、代谢及功能层面的异常改变。基因芯片分析显示，CBK小鼠心脏中存在2037个差异表达基因，其中多数已在心肌细胞特异性CLOCK突变（cardiomyocyte-specific Clock mutant, CCM）小鼠心脏中被报道。后续分析表明，CBK与CCM小鼠心脏中，β-羟丁酸脱氢酶1（Beta-hydroxybutyrate dehydrogenase 1）的mRNA水平、蛋白表达量及酶活性均显著下调，心肌β-羟丁酸氧化过程亦受到抑制，这一发现揭示了昼夜节律钟在酮体利用中的全新功能。多项编码胶原蛋白亚型的基因在野生型小鼠心脏中呈现出昼夜节律性表达波动，但在CBK小鼠心脏中无此节律特征，其中包括col3a1、col4a1及col4a2。CBK小鼠心脏中胶原蛋白亚型基因的长期诱导表达，与严重的年龄依赖性心脏功能减退密切相关。CBK小鼠的心肌病发生与早期死亡率升高相关，所有CBK小鼠均在1岁龄前死亡。本研究揭示了BMAL1在心脏中的全新关键功能，包括调控酮体代谢与细胞外基质稳态。研究人员从野生型与CBK小鼠心脏中每隔3小时采集一次样本，持续24小时，随后提取全心脏RNA并使用MouseRef-8_V2 BeadChips（Illumina公司）进行分析。研究采用余弦分析法对24小时的检测数据进行节律性鉴定，并进一步比较不同基因型组间的模型拟合参数差异。