Abstract Body: Introduction: Prior studies indicate that the cardiomyocyte circadian clock contributes towards 24-hour rhythms in ischemia/reperfusion (I/R) tolerance (which peaks at the beginning of the sleep period). However, the mechanisms by which this occurs are unknown. E4 promoter binding protein 4 (E4BP4) is a novel transcriptional repressor regulated by the circadian clock. Our published studies revealed that E4BP4 regulates cardiac gene expression, metabolism, and electrophysiology. However, the role of E4BP4 in I/R tolerance remains unknown and was, therefore, the subject of this study.
Methods: 12-week-old male cardiomyocyte-specific E4BP4 knockout (CEK) and littermate flox control (CON) mice were used in this study. At the beginning of the sleep phase, mice were subjected to I/R (45-min ischemia, 24-hour reperfusion) in vivo. Left ventricular ejection fraction (LVEF) was assessed by ECHO both before and 24 hours after I/R. Hearts were collected 24 hours after I/R. Infarct size was evaluated by TTC staining. Neonatal rat ventricular myocyte (NRVM) cell death following simulated I/R was detected by an MTT assay in vitro. Knockdown of E4BP4 was achieved in NRVMs through the use of siRNA. Overexpressing E4BP4 in NRVM was achieved via adenovirus hosting a troponin-promoter-driven E4BP4. Adult mouse ventricular myocytes (AMVM) isolated from CEK and CON were also subjected to simulated I/R.
Results: Infarct size following I/R was larger in CEK versus CON mice by 35% (p=0.0257). LVEF in the two groups was similar at baseline, but after I/R surgery, the LVEF of the CEK was reduced by 25% compared with the CON (p=0.003). Knockdown of E4BP4 in NRVMs increased cell death by 34% (p=0.0233) after simulated I/R in vitro. In contrast, overexpressing E4BP4 increased cell viability after simulated I/R in vitro. Loss of E4BP4 attenuated autophagy in both NRVMs and AMVMs, as evidenced by altered P62 and LC3-II levels.
Conclusions: Loss of E4BP4 in cardiomyocytes reduces cardiac I/R tolerance both in vivo and in vitro (potentially through attenuated autophagic flux). Augmenting E4BP4 function may, therefore, be a therapeutic target to reduce infarct size in patients with acute myocardial infarction.