Loss of the core circadian clock transcription factor BMAL1 (ARNTL) selectively in cardiomyocytes causes dilated cardiomyopathy preceded by a coordinated decrease in the expression of genes for fatty-acid oxidation (FAO), the tricarboxylic-acid (TCA) cycle, and the electron-transport-chain/oxidative-phosphorylation (ETC/OXPHOS) system, together with a parallel fall in both NAD+ and NADH that indicates uncoupling of the TCA cycle from the respiratory chain. Which molecular pathway mediates this coordinated transcriptional shutdown — direct CLOCK:BMAL1 activation of these metabolic genes, or indirect action through clock-controlled regulators (the REV-ERB nuclear receptors and the transcriptional repressor E4BP4, the transcription factor KLF15, the fatty-acid-oxidation nuclear receptor PPARα and its coactivator PGC-1α) — and what causes the TCA–ETC uncoupling, remains unresolved, as does the extent to which the fall in the NAD+/NADH (NAD(H)) pool feeds back onto the cardiac oscillator.

Background

In mammals, nearly every tissue, including the heart, contains a molecular circadian clock — a transcription–translation feedback circuit that generates ~24-hour rhythms of gene expression. The core activator of this circuit is the heterodimer of the transcription factors CLOCK and BMAL1 (also named ARNTL), which binds short promoter DNA motifs called E-boxes (consensus CACGTG) to drive target genes. Among its targets are the clock's own repressors (the PER/CRY proteins and the nuclear receptors REV-ERBα/β) as well as clock-controlled output regulators such as KLF15 (a cardiac transcription factor regulating metabolic genes) and E4BP4 (a repressor binding D-box promoter motifs, a second clock-responsive element), so BMAL1 sits at the top of a timed transcriptional cascade that paces organ physiology. The adult heart depends almost continuously on mitochondrial oxidative metabolism — fatty-acid oxidation (FAO) feeding the tricarboxylic-acid (TCA) cycle, which reduces NAD+ to NADH for the electron-transport chain (ETC) driving oxidative phosphorylation (OXPHOS) — and much of the cardiac metabolic gene program is rhythmically expressed.

Kohsaka et al. (PLOS ONE, 2014) generated heart-specific Bmal1 knockout mice (H-Bmal1−/−) and showed that cardiomyocyte BMAL1 loss causes progressive dilated cardiomyopathy preceded by a coordinated decrease in expression of genes for FAO, the TCA cycle, and the ETC/OXPHOS system, reduced respiratory-chain complex I activity, and a parallel fall in both NAD+ and NADH. Because the TCA cycle normally supplies NADH to the ETC, which regenerates NAD+ in return, the authors interpreted this co-reduction as uncoupling of the TCA cycle from the respiratory chain. The mechanisms causing the coordinated transcriptional shutdown were left open, with the discussion pointing both to possible direct CLOCK:BMAL1 action and to indirect routes via metabolic regulators that physically interact with clock proteins — PPARα (the nuclear receptor that drives FAO genes) and its transcriptional coactivator PGC-1α — and to the CLOCK:BMAL1–NAMPT (nicotinamide phosphoribosyltransferase, the rate-limiting enzyme of the NAD+ salvage pathway) redox axis. The study used only male mice, one light–dark-reversal protocol, and phenylephrine (PE, a hypertrophic stimulus delivered by osmotic pump) as the cardiomyopathy model.

Subsequent work has resolved parts of the broader clock–cardiac-metabolism question. Dierickx et al. (Nat Cardiovasc Res, 2022) showed that cardiomyocyte REV-ERBα/β double deletion causes dilated cardiomyopathy in which FAO-gene downregulation is mediated by derepression of E4BP4, and that E4BP4 directly represses Nampt, so that the clock's repressor limb controls cardiac NAD+ biosynthesis; a 2024 review (Lal et al., Circ Res) summarizes that cardiac Nampt is regulated by direct CLOCK:BMAL1 E-box binding, KLF15, and E4BP4 D-box repression. However, the same review states that direct evidence for cardiomyocyte clock control of several metabolic processes 'does not currently exist', no cardiomyocyte BMAL1 cistrome (genome-wide in-vivo binding map) has been published, and no study has explained the co-reduction of NAD+ and NADH or tested the NAD+/NADH feedback onto the cardiac oscillator in the Bmal1-deficient heart. Sex-specific cardiac transcriptomes (Zhang et al., Function, 2025) and sex-dependent mitochondrial rhythms (Kane et al., Sci Rep, 2023) have only begun to address the sex-difference aspect.

Problem Statement

Determine the molecular pathway by which loss of cardiomyocyte BMAL1 produces the coordinated transcriptional suppression of the fatty-acid-oxidation, TCA-cycle, and ETC/OXPHOS gene programs and the associated mitochondrial defects observed in heart-specific Bmal1 knockout mice (H-Bmal1−/−). A second, independently generated cardiomyocyte-specific Bmal1 knockout (CBK) shares the age-onset dilated cardiomyopathy and reduced lifespan, represses individual metabolic genes (Nampt, Bdh1, Acsm5, Coq10b, Aldob, Dgat2), and partially overlaps at the transcriptome level (121 of 300 genes downregulated in CM-RevDKO hearts are also downregulated in CBK), but its reported oxidative-metabolism phenotype differs in direction (increased ex vivo oleate oxidation with decreased glucose and ketone utilization, unchanged citrate synthase activity and unchanged myocardial oxygen consumption; unchanged complex I with increased complex IV activity), so the coordinated suppression of the FAO/TCA/ETC gene programs and the NAD+/NADH co-reduction remain documented in the H-Bmal1−/− model. Specifically: (1) Are these metabolic genes direct CLOCK:BMAL1 targets in cardiomyocytes (i.e., E-box-mediated transcriptional activation), or is the suppression indirect — and through which intermediates (REV-ERB loss with E4BP4 derepression, KLF15, PPARα and PGC-1α perturbation, microRNA species, or others)? (2) What causes the observed TCA-cycle/ETC uncoupling, i.e., the simultaneous decrease of both NAD+ and NADH in Bmal1-deficient hearts, and the associated mitochondrial defects (in particular the reduced respiratory-chain complex I activity)? (3) To what extent does the fall in the NAD+/NADH (NAD(H)) pool feed back onto the circadian oscillator within cardiomyocytes in vivo? The three sub-questions are jointly posed.

Later literature raises separate comparative questions of contextual interest — whether circadian regulation of cardiovascular metabolism is sex-dependent, and whether these cardiac responses generalize beyond the specific light–dark-reversal and phenylephrine-infusion protocols used, including to human shift-work-associated heart failure (Lal et al. 2024; Kane et al. 2023; Zhang et al. 2025). Both source models used male mice only, so these comparative extensions are context for, rather than required components of, the mechanistic question posed here.

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