Reduced expression of the PER2 protein contributes to β1-AA-induced cardiac autophagy rhythm disorders

Abstract

Heart failure may be linked to fluctuations in the rhythm of autophagy in cardiomyocytes throughout the day. Circadian rhythms depend on the regulation of core biological clock proteins, with PER2 playing a crucial role. Our previous research confirmed that the presence of β1-adrenergic receptor autoantibodies (β1-AAs) could inhibit myocardial autophagy, leading to cell death and heart failure. However, it remains unclear whether β1-AA induces cardiac autophagy rhythm disorders by affecting PER2 expression. In this study, we find that β1-AA disrupts the autophagy rhythm in cardiomyocytes, which is primarily indicated by decreased expression of the autophagy marker protein LC3. β1-AA disrupts the rhythmic expression of the PER2 protein in myocardial cells, which is manifested mainly by a decrease in PER2 protein expression. Metoprolol is used to verify that the β1-adrenergic receptor contributes to the reduction in the Per2 protein caused by β1-AA. Knockdown of Per2 with lentivirus reduces the inhibition of LC3 expression caused by β1-AA, whereas overexpression of Per2 in cardiomyocytes using lentivirus significantly restores the β1-AA-induced decrease in LC3 expression. Moreover, mTORC1 activation is found to participate in β1-AA-induced autophagy inhibition in cardiomyocytes after pretreatment with the mTORC1 inhibitor rapamycin. Furthermore, the decreased expression of the PER2 protein caused by β1-AA disrupts the myocardial autophagy rhythm by promoting mTORC1 activation through lentiviruses that knock down or overexpress the Per2 gene. This study provides an experimental basis for the precise treatment of cardiovascular diseases from the perspective of biological rhythm.

1. Introduction

Heart failure is the final stage of various cardiovascular diseases [1]. With the increasing aging of society, the prevalence and mortality of heart failure worldwide are increasing, with China accounting for 1/3 of all heart failure cases [2,3]. Lifestyles related to circadian rhythm disturbances, such as “daily staying up late”, “shift work” and “sleep disorders”, can disrupt the repair cycle of the heart and cause damage to or even death of cardiomyocytes, resulting in an increased risk of heart failure or aggravation of pre-existing heart failure [4]. Previous studies have shown that various biological activities that maintain the normal operation of cells, including autophagy, undergo circadian changes [5]. Adverse cardiac events occur frequently in the early morning, when autophagy activity is low [6], suggesting that the occurrence of heart failure may be related to the circadian rhythm of autophagy in cardiomyocytes throughout the day. Therefore, it is particularly important to identify the factors that disturb the circadian rhythm of autophagy during heart failure.

Clinical data show that autoantibodies are present in the serum of 40%–60% of patients with heart failure and continuously activate the β1-adrenergic receptor (β1-AR), namely, β1-adrenergic receptor autoantibodies (β1-AAs) [7]. The removal of β1-AA from the serum of patients with heart failure by the immunosorbent technique could significantly improve the cardiac function of patients [8]. Our previous studies revealed that β1-AA can reduce the level of myocardial autophagy and promote cardiac cell death, leading to heart failure [9]. However, it is not clear whether β1-AA is a factor causing the disturbance of the rhythm of cardiomyocyte autophagy. A previous study showed that the autophagy rhythm is regulated by the circadian rhythm [10]. The circadian rhythm is a regulated rhythmic oscillation with a 24-h cycle that exists universally in the body. Its molecular mechanism involves a transcriptional translation feedback loop composed of a series of clock genes [11], in which the negative feedback regulation of the PER2 protein plays a key role [12]. Per2 is closely related to autophagy [13]. After the Per2 gene is silenced in fibroblasts, the levels of the autophagy-related proteins LC3II and ULK1 are significantly decreased, and their rhythms are also significantly abnormal [14]. Therefore, we speculated that β1-AA could disturb the autophagy rhythm of cardiomyocytes by altering the expression of PER2 in cardiomyocytes and further explored the specific mechanism from the perspective of mTORC1 to provide a new experimental basis for the precise treatment of cardiovascular diseases from the perspective of biological rhythm.