Circadian clock regulates metabolism and cardiac myocyte survival during cardiovascular stress

Abstract

Disruption of the normal circadian clock has been associated with greater incidence of cardiovascular disease in shift workers. While the underlying mechanisms for this phenomenon remains poorly understood, recent evidence from our laboratory has identified a novel signaling axis that functionally connects the mechanistic target of rapamycin (mTOR), a critical regulator of cardiac hypertrophy and autophagy to the circadian gene Circadian locomotor output cycles kaput (Clock). Herein, I show that in contrast to normal cardiac myocytes, post-natal cardiac myocytes genetically deficient for Clock gene (Clock -/-) exhibited impaired mTOR signaling and autophagy gene expression in response to different cellular stresses, including hypoxia, hypertrophy, and amino acid starvation. This is highlighted by significant reduction in mTOR activity and its upstream regulators, Ras homolog enriched in brain (Rheb) and AMP activated protein kinase (AMPK), as well as its downstream targets, Unc-51-like kinase (ULK1), 4E-BP1 (eIF4E binding protein 1), and S6 kinase 1 (S6). Additionally, impaired mTOR signalling in Clock -/- cardiac myocytes was accompanied by deregulated autophagy gene activation and decreased cell viability. Interestingly, cardiac myocytes subjected to nutrient stress through absence of amino acids also displayed similar autophagy regulation deficiency. Gain of function of Clock normalized autophagy gene expression through AMPK decrease and stabilization of the mTOR pathway which increased cell survival. Western blot analysis verified that circadian clock overexpressed resulted in activation of mTOR activity that coincided with a reduction in AMPK while ULK1 levels were normalized in a nutrient stressed model. Further mutations of Clock failed to activate mTOR and stabilize autophagy and cell survival in cardiac myocytes. To our knowledge my data provides the first evidence that mechanistically links Clock to amino acid starvation response in cardiac myocytes via the mTOR pathway. This data suggests interventions that mitigate circadian disruption may prove beneficial in improving amino acid metabolism and cardiovascular disease burden in shift workers or individuals with sleep disorders.