Cardiomyocyte-specific long noncoding RNA Trdn-as induces mitochondrial calcium overload by promoting the m6A modification of calsequestrin 2 in diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM), a major complication of diabetes mellitus, involves cardiac remodeling and dysfunction, often progressing to heart failure. Key pathological features include sarcoplasmic reticulum (SR) and mitochondrial calcium overload in cardiomyocytes, though the underlying mechanisms remain unclear. This study investigates the role of a cardiomyocyte-specific long noncoding RNA, Trdn-as, in DCM pathogenesis. Findings reveal that Trdn-as is significantly upregulated in cardiac tissues of diabetic mice and high glucose-treated cardiomyocytes. Experimental manipulation of Trdn-as in vivo and in vitro demonstrates its critical role in driving cardiac dysfunction and mitochondrial damage. Overexpression of Trdn-as in healthy mice induces DCM-like cardiac abnormalities, while silencing it in diabetic models alleviates structural and functional deficits, highlighting its potential as a therapeutic target.

Mechanistically, Trdn-as promotes SR and mitochondrial calcium overload by enhancing the stability and expression of calsequestrin 2 (Casq2), a key calcium-binding protein in the junctional SR. RNA sequencing and bioinformatics analyses identify Casq2 as a downstream target of Trdn-as. Further experiments show that Trdn-as recruits the methyltransferase METTL14 to Casq2 mRNA, increasing N6-methyladenosine (m6A) modification. This epigenetic alteration stabilizes Casq2 mRNA, leading to elevated protein levels. Elevated Casq2 disrupts calcium homeostasis, causing excessive SR calcium storage and subsequent mitochondrial calcium influx. The resultant mitochondrial dysfunction—evidenced by reduced membrane potential, impaired respiration, oxidative stress, and ATP depletion—exacerbates cardiomyocyte injury. Knocking down Casq2 mitigates these effects, confirming its central role in Trdn-as-mediated pathology.

In diabetic mice, Trdn-as upregulation correlates with cardiac hypertrophy, fibrosis, and impaired systolic and diastolic function. Echocardiography and histopathological analyses reveal that Trdn-as overexpression mimics DCM phenotypes, while its knockdown restores cardiac function. Cellular studies using neonatal mouse cardiomyocytes exposed to high glucose conditions replicate these findings, linking Trdn-as to calcium dysregulation and mitochondrial damage. Transmission electron microscopy and JC-1 staining confirm mitochondrial structural abnormalities and depolarization in Trdn-as-overexpressing cells. Additionally, Trdn-as exacerbates oxidative stress by increasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels while reducing superoxide dismutase (SOD) activity, further compromising cellular integrity.

The study also explores transcriptional regulation of Trdn-as, identifying C/EBPα as a potential inducer under high glucose conditions. This transcription factor binds to the Trdn-as promoter, suggesting a feedback loop in diabetic cardiac pathology. While Trdn-as does not directly affect SERCA2a, a key regulator of SR calcium reuptake, its interaction with METTL14 underscores the importance of RNA methylation in post-transcriptional regulation. Methylated RNA immunoprecipitation (MeRIP) assays pinpoint specific m6A sites on Casq2 mRNA, with Trdn-as enhancing methylation at the 748 locus. These modifications increase mRNA stability, perpetuating calcium mishandling and mitochondrial dysfunction.

Clinical relevance is emphasized by the conserved role of Trdn-as across species and its specific expression in cardiomyocytes. Previous studies link Trdn-as to arrhythmias and heart failure, but this work expands its role to metabolic cardiomyopathy. The findings propose a novel pathway where Trdn-as-METTL14-Casq2 axis drives calcium overload and mitochondrial injury, offering insights into DCM progression. Targeting this axis could disrupt pathological calcium signaling, presenting opportunities for therapeutic intervention. Future research may explore broader RNA methylation networks and validate Casq2’s role in animal models to refine therapeutic strategies.

In summary, the study delineates a mechanism by which Trdn-as exacerbates DCM through m6A-dependent regulation of Casq2, linking RNA epigenetics to calcium homeostasis and mitochondrial function. These discoveries advance understanding of DCM pathogenesis and highlight Trdn-as as a potential biomarker or therapeutic target for diabetic heart disease.