Abstract：
UNASSIGNED: Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear.
UNASSIGNED: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF.
UNASSIGNED: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury.
UNASSIGNED: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.
UNASSIGNED: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF.
UNASSIGNED: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury.
UNASSIGNED: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.
摘要：
■心肌线粒体功能障碍是心力衰竭（HF）的发病机理，然而，恢复心肌线粒体功能的治疗选择很少。线粒体DNA（mtDNA）的表观遗传修饰，比如甲基化，在调节线粒体稳态中起关键作用。然而,他们是否参与HF仍不清楚.
■通过连续血管紧张素II和去氧肾上腺素（AngII/PE）输注或延长的心肌缺血/再灌注损伤建立实验性HF模型。使用高分辨率质谱和甲基化DNA免疫沉淀测序来表征衰竭心肌细胞mtDNA中N6-甲基腺嘌呤（6mA）甲基化的景观。使用他莫昔芬诱导的心肌细胞特异性Mettl4敲除小鼠模型和设计用于心肌细胞靶向操作METTL4（甲基转移酶样蛋白4）表达的腺相关病毒载体来确定mtDNA6mA及其甲基转移酶METTL4在HF中的作用。
■METTL4主要位于成年心肌细胞线粒体内。6mA修饰在mtDNA中比核DNA中明显更丰富。出生后心肌细胞成熟表现为mtDNA内6mA水平降低，与METTL4表达减少相吻合。然而,在衰竭的成年心肌细胞中观察到mtDNA6mA水平和METTL4表达的增加，表明向新生儿样状态的转变。METTL4优先靶向mtDNA启动子区，导致转录起始复合物组装受到干扰，mtDNA转录停滞，最终线粒体功能障碍。通过METTL4过表达扩增心肌细胞mtDNA6mA导致自发性线粒体功能障碍和HF表型。转录因子p53被确定为响应HF引起的应激的METTL4转录的直接调节因子，从而揭示了控制METTL4表达和mtDNA6mA的应激反应机制。Mettl4基因的心肌细胞特异性缺失消除了mtDNA6mA过量，保留的线粒体功能，并在连续输注AngII/PE后减轻HF的发展。此外,心肌细胞中METTL4的特异性沉默可恢复线粒体功能，并在先前存在HF的小鼠中提供治疗性缓解，无论该病症是由AngII/PE输注还是心肌缺血/再灌注损伤引起。
■我们的发现确定了心肌细胞mtDNA6mA和相应的甲基转移酶的关键作用，METTL4，在线粒体功能障碍和HF的发病机理中。有针对性地抑制METTL4以纠正mtDNA6mA过量，是开发以线粒体为重点的HF干预措施的有希望的策略。
■通过连续血管紧张素II和去氧肾上腺素（AngII/PE）输注或延长的心肌缺血/再灌注损伤建立实验性HF模型。使用高分辨率质谱和甲基化DNA免疫沉淀测序来表征衰竭心肌细胞mtDNA中N6-甲基腺嘌呤（6mA）甲基化的景观。使用他莫昔芬诱导的心肌细胞特异性Mettl4敲除小鼠模型和设计用于心肌细胞靶向操作METTL4（甲基转移酶样蛋白4）表达的腺相关病毒载体来确定mtDNA6mA及其甲基转移酶METTL4在HF中的作用。
■METTL4主要位于成年心肌细胞线粒体内。6mA修饰在mtDNA中比核DNA中明显更丰富。出生后心肌细胞成熟表现为mtDNA内6mA水平降低，与METTL4表达减少相吻合。然而,在衰竭的成年心肌细胞中观察到mtDNA6mA水平和METTL4表达的增加，表明向新生儿样状态的转变。METTL4优先靶向mtDNA启动子区，导致转录起始复合物组装受到干扰，mtDNA转录停滞，最终线粒体功能障碍。通过METTL4过表达扩增心肌细胞mtDNA6mA导致自发性线粒体功能障碍和HF表型。转录因子p53被确定为响应HF引起的应激的METTL4转录的直接调节因子，从而揭示了控制METTL4表达和mtDNA6mA的应激反应机制。Mettl4基因的心肌细胞特异性缺失消除了mtDNA6mA过量，保留的线粒体功能，并在连续输注AngII/PE后减轻HF的发展。此外,心肌细胞中METTL4的特异性沉默可恢复线粒体功能，并在先前存在HF的小鼠中提供治疗性缓解，无论该病症是由AngII/PE输注还是心肌缺血/再灌注损伤引起。
■我们的发现确定了心肌细胞mtDNA6mA和相应的甲基转移酶的关键作用，METTL4，在线粒体功能障碍和HF的发病机理中。有针对性地抑制METTL4以纠正mtDNA6mA过量，是开发以线粒体为重点的HF干预措施的有希望的策略。
