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Severe Attenuation of Circadian Clock Output in the Heart following Sustained Augmentation of Cardiomyocyte Protein O-GlcNAcylation

ABSTRACT: Background. Changes in circadian-related behaviors (e.g., the timing of food intake, sleep cycles) and the environment (e.g., light-dark cycles) increase the risk of numerous cardiometabolic diseases, including diabetes mellitus and cardiac disease. Recent studies indicate a close interrelationship between circadian clocks and the posttranslational modification, protein O-GlcNAcylation. The current study was designed to investigate whether a modest elevation of protein O-GlcNAcylation in the adult mouse heart, similar to levels observed during pathologic states, influenced circadian governance of the heart. Methods and Results. Cardiomyocyte-specific expression of a dominant negative O-GlcNAcase (dnOGAh) for a 2-week period resulted in an approximate 1.5-fold increase in cardiac protein O-GlcNAcylation, impacting 70% of core circadian clock components in the heart at the mRNA level. Further interrogation of cardiac mRNA species in dnOGAh hearts at candidate (RT-PCR) and unbiased (RNAseq) levels revealed a 95% loss of circadian governance of the cardiac transcriptome. This was despite persistent/augmented 24hr oscillations of the core circadian clock proteins BMAL1, REVERB, and PER2 in dnOGAh hearts. Direct comparison of dnOGAh hearts with cardiomyocyte-specific BMAL1 knockout (CBK) hearts underscored an apparent uncoupling of the core clock mechanism from clock control of downstream target genes in dnOGAh hearts, and highlighted that loss of circadian governance results in interstitial fibrosis. Conclusions. Sustained protein O-GlcNAcylation in the heart causes loss of circadian governance, likely downstream of the core circadian clock mechanism. Moreover, interstitial fibrosis appears to be a universal adverse outcome following impaired circadian governance.

ORGANISM(S): Mus musculus

PROVIDER: GSE301657 | GEO | 2025/07/07

REPOSITORIES: GEO

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Project description:Circadian clocks are cell autonomous timekeeping mechanisms that govern critical biological processes. Regarding the heart, the cardiomyocyte circadian clock regulates a diverse array of processes, ranging from transcription and translation, to signaling, metabolism, electrophysiology, and contractility. The importance of this mechanism is underscored by observations that genetic disruption of the cardiomyocyte circadian clock in murine models leads to adverse cardiac remodeling, heart failure, and reduced lifespan. However, the precise molecular links between the cardiomyocyte circadian clock and cardiac physiology/pathology have not been characterized fully. Given that recent studies have highlighted that small RNA species (such as miRNAs) influence both cardiac physiology and pathology, we sought to determine the extent to which cardiomyocyte circadian clock disruption impacts cardiac small RNA species. Accordingly, hearts were collected from cardiomyocyte-specific Bmal1 knockout (CBK) and littermate control (CON) mice at distinct times of the day. Small RNA-seq revealed 47 differentially expressed miRNA species in CBK hearts (in the absence of significant time-of-day-dependent effects). Subsequent bioinformatic analyses predicted that differentially expressed miRNA species in CBK hearts potentially influence processes such as circadian rhythmicity, cellular signaling, and metabolism. Of the induced miRNAs in CBK hearts, 7 were predicted to be targeted by the transcriptional repressors REV-ERB/ (integral circadian clock components that are directly regulated by BMAL1). Similar to CBK hearts, cardiomyocyte-specific Rev-erb/ double knockout (CM-RevDKO) mouse hearts exhibited increased let-7c-1-3p, miR-23b-5p, miR-139-3p, miR-5123, and miR-7068-3p levels. Importantly, 19 putative targets of these 5 miRNAs were commonly repressed in both CBK and CM-RevDKO heart (of which 16 are targeted by let-7c-1-3p). These observations suggest that disruption of the BMAL1–REV-ERB/ axis in the heart leads to induction of a subset of miRNAs, whose predicted mRNA targets have established functions in biological processes such as metabolism and cellular signaling.

Project description:Over the past decade, genome-wide assays have underscored the broad sweep of circadian gene expression. A substantial fraction of the transcriptome undergoes oscillations in many organisms and tissues, which governs the many biochemical, physiological and behavioral functions under circadian control. Based predominantly on the transcription feedback loops important for core circadian timekeeping, it is commonly assumed that this widespread mRNA cycling reflects circadian transcriptional cycling. To address this issue, we directly measured dynamic changes in mouse liver transcription using Nascent-Seq. Many genes are rhythmically transcribed over the 24h day, which include precursors of several non-coding RNAs as well as the expected set of core clock genes. Surprisingly however, nascent RNA rhythms overlap poorly with mRNA abundance rhythms assayed by RNA-seq. This is because most mouse liver genes with rhythmic mRNA expression manifest poor transcriptional rhythms, indicating a prominent role of post-transcriptional regulation in setting mRNA cycling amplitude. To gain further insight into circadian transcriptional regulation, we also characterized the rhythmic transcription of liver genes targeted by the transcription factors CLOCK and BMAL1; they directly target other core clock genes and sit at the top of the molecular circadian clock hierarchy in mammals. CLK:BMAL1 rhythmically bind at the same discrete phase of the circadian cycle to all target genes, which not surprisingly have a much higher percentage of rhythmic transcription than the genome as a whole. However, there is a surprisingly heterogeneous set of cycling transcription phases of direct target genes, which even include core clock genes. This indicates a disconnect between rhythmic DNA binding and the peak of transcription, which is likely due to other transcription factors that collaborate with CLK:BMAL1. In summary, the application of Nascent-Seq to a mammalian tissue provides surprising insights into the rhythmic control of gene expression and should have broad applications beyond the analysis of circadian rhythms. CLK and BMAL1 DNA binding profile in the mouse liver at ZT8, sequenced along an Input sample using GAII (ChIP-Seq) Supplementary file ChIPSeq_Mouse_Liver_Processed_data_Table1.txt represents annotated CLK and BMAL1 peaks.

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Severe Attenuation of Circadian Clock Output in the Heart following Sustained Augmentation of Cardiomyocyte Protein O-GlcNAcylation

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