N6-methyladenosine (6 mA or m6dA) represents one of the direct DNA epigenetic modifications, distinct from RNA N6-methyladenosine (m6A). 6 mA destabilizes the DNA double helix, resulting in DNA deconvolution. METTL4 is a 6 mA methyltransferase that can modify mitochondrial DNA (mtDNA) through 6 mA and plays an important role in the regulation of mitochondrial homeostasis [1]. However, the relationship between 6 mA and heart diseases remains largely unclear, and more evidence is needed to reveal the role of METTL4 in the regulation of mitochondrial function in cardiomyocytes.

It has been previously known that DNA 6 mA modifications are widely present in the genomes of prokaryotes. A recent study has demonstrated that in mammals, DNA 6 mA modifications are mainly enriched in mitochondrial DNA [2]. The mitochondrial DNA 6 mA content, regulated by the methylation enzyme METTL4, is significantly increased under hypoxic conditions. This suggests a crucial function of mitochondrial DNA 6 mA modification in the regulation of mitochondrial activity and stress response in mammals. Cardiomyocytes, which contain a large number of mitochondria that produce ample ATP for the heart, have shown an inextricable link between mitochondrial dysfunction and cardiovascular diseases. The 6 mA modifications in mtDNA are much higher than in genomic DNA, emphasizing the importance of studying the function of 6 mA in mtDNA. 6 mA methylation plays a key role in regulating DNA replication, repair, and transcription [3]. It has been suggested that DNA 6 mA modification is extensively present in human cells and the decrease of genomic DNA 6 mA promotes human tumorigenesis [4]. Nonetheless, few studies have explored the link between 6 mA modifications in mitochondria and hypoxia- or stress-induced heart diseases.

In a recent study by Zhang et al., the authors reported that the expression level of METTL4 was elevated in the mitochondria of failing adult cardiomyocytes, along with a significant increase of 6 mA abundance in mtDNA [5]. Based on adeno-associated virus 9 (AAV9)-mediated overexpression of METTL4 in mice, increasing METTL4 induced spontaneous cardiac dysfunction and mitochondrial structural and functional abnormalities, which was accompanied with obvious reduced expressions of electron transport chain (ETC) genes encoded by mtDNA. Conversely, using cardiomyocyte-specific METTL4 knockout adult mice or mice with AAV9-mediated METTL4 knockdown, the authors demonstrated that specific downregulation of METTL4 in cardiomyocytes can restore mitochondrial function and protect against pathological hypertrophy- or ischemia/reperfusion-induced cardiac injury by reducing 6 mA excess in mtDNA. These findings reveal the critical role of mtDNA 6 mA and its methyltransferase METTL4 in the regulation of mitochondrial function in cardiomyocytes, providing new insights and potential strategies for the treatment of stress-induced cardiac diseases from a mitochondrial-epigenetic perspective.

To elucidate the consequence of METTL4-mediated 6 mA excess in mtDNA, the authors combined 6 mA-specific DNA methylation immunoprecipitation sequencing (MeDIP-Seq) and chromatin immunoprecipitation (ChIP) assay showing that METTL4 can inhibit the assembly of mitochondrial transcription initiation complexes, such as TFAM and POLRMT, in the mtDNA promoter region by catalyzing 6 mA modification of mtDNA promoter sequences, leading to mtDNA transcriptional inhibition and mitochondrial dysfunction in cardiomyocytes.

To further explore the upstream regulator of METTL4 in cardiomyocyte mitochondria, the authors conducted ChIP experiments using HEK293T cells, revealing an interaction between the transcription factor p53 and the promoter of the Mettl4 gene. METTL4 expressions in adult mouse cardiomyocytes was induced after angiotensin II and phenylephrine treatment, which can be further modified by altering p53 levels in several ways: 1) using pifithrin-α which inhibits p53 transcription activity; 2) utilizing p53 knockout (KO) mice to isolate adult cardiomyocytes; 3) using Nutlin-3a which is a p53 agonist. These results emphasized the transcriptional regulation of p53 on METTL4 in cardiomyocytes, and this interaction was further enhanced under hypertrophic stress.

In conclusion, this paper is highly innovative in exploring the role and mechanism of methyltransferase METTL4 in cardiomyocyte mitochondria from the perspective of mitochondrial 6 mA modification. Under pathological stimuli that leads to heart failure, p53 activates the transcription of METTL4 which further inhibits the assembly of the mitochondrial transcription initiation complexes by catalyzing the 6 mA modifications within the mtDNA promoter, leading to mtDNA transcriptional inhibition and mitochondrial dysfunction. It is expected to further explore the functions of METTL4 in regulating human cardiomyocyte mtDNA 6 mA, which will support and pave the way to the development of new drug therapies targeting cardiomyocyte mitochondria.