Abstract Body: Background: Epigenetic regulation of cardiac transcriptional reprogramming during pathological hypertrophy is mediated, in part, by bromodomain and extra-terminal domain (BET) reader proteins that bind acetylated histone marks, eventually recruiting transcription factors to facilitate gene expression. Targeted siRNA depletion of BET family member bromodomain-containing protein 4 (BRD4) in cardiomyocytes, resulted in attenuation of the hypertrophic response. In contrast, our group has previously shown that cardiomyocyte-specific BRD4 deletion in adult heart causes contractile and mitochondrial dysfunction. In this study we resolve this paradox and examine the two major isoforms of BRD4, BRD4-L and BRD4-S, to determine if they have distinct roles in governing cardiac homeostasis and pathophysiology.

Methods: We employed isoform-specific siRNA knockdown in cardiomyocytes and inducible cardiomyocyte-specific transgenic isoform re-expression models in wild-type or BRD4-depleted murine hearts to investigate isoform-specific requirements in cardiac homeostasis and stress-induced hypertrophy. We performed bulk transcriptome and CUT&RUN sequencing to identify isoform-specific gene regulation.

Results: Whereas knockout of BRD4 in adult hearts is lethal, transgenic BRD4-L expression in endogenous BRD4 knockout hearts rescued lethality, preserved contractility, and restored mitochondrial electron transport chain gene expression. Consistent with this, BRD4-L enrichment is observed at promoters of mitochondrial regulatory genes. In contrast, transgenic BRD4-S expression induced cardiac hypertrophy and, in the absence of endogenous BRD4 expression, accelerated mortality. BRD4-S bound to promoters of calcium signaling and hypertrophic genes and was uniquely required for in vitro and in vivo hypertrophy. Transcriptomic profiling identified calcium signaling and hypertrophic cardiomyopathy pathways as BRD4-S targets and uncovered Ccn1 as a novel BRD4-S effector. Ccn1 was upregulated in murine hypertrophic and failing hearts, and its knockdown blunted the hypertrophic response.

Conclusions: BRD4 isoforms govern non-redundant functions in the heart: BRD4-L is required for mitochondrial homeostasis whereas BRD4-S drives pathological remodeling through calcium signaling and Ccn1. These findings reconcile the paradox of BET inhibition versus BRD4 deletion and highlight isoform-selective targeting of BRD4 as a potential therapeutic strategy.