Chronodisruption: melatonin deficiency and innate immunity. Connection with mitochondrial dysfunction during aging

Abstract

Cardiovascular diseases are the leading cause of death in the world, with aging being the main risk factor associated with these pathologies (Global Health and Aging, 2019). The world’s population is aging at an unprecedented rate, and study of the mechanisms underlying this process is vitally important from a health, economic and social point of view. Aging is characterized by a deregulation of the immune system resulting in a subclinical, sterile, asymptomatic and chronic pro-inflammatory state known as inflammaging (Franceschi and Campisi, 2014). This inflammatory condition, coupled with oxidative stress, leads to mitochondrial dysfunction and subsequent apoptosis, facilitating the release of reactive species, ATP, and mtDNA. These hazard signals are recognized by the NLRP3 inflammasome, a multiprotein complex responsible for the maturation of pro-inflammatory cytokines dependent on NF-κB, including IL-1β. In this way, this process perpetuates a vicious cycle that results in systemic inflammation that is accompanied by symptoms of immunosenescence and activation of the innate immune pathway. Alterations in the regulation of mitochondrial homeostasis, including mechanisms of mitochondrial dynamics, autophagy, apoptosis, as well as decreased antioxidant defense that occurs with aging, such as the Nrf2-dependent pathway, may be necessary for activation of NLRP3 inflammasome. In addition, the effect this inflammasome may have on mitochondrial function or on the antioxidant pathway of Nrf2 during cardiac aging remains unknown. Interestingly, numerous scientific studies relate inflammaging to the disruption of circadian rhythms, which allow the organism to adapt and anticipate environmental changes to ensure optimal physiological performance (Acuña-Castroviejo et al., 2017; Acuña-Fernández et al., 2020; Volt et al., 2016). In mammals, circadian rhythms are regulated by a central clock, located in the suprachiasmatic nucleus, and by peripheral clocks, located in virtually all tissues, including the heart. While there seems to be a connection between aging, clock genes and innate immune response, the molecular mechanisms that link these processes remain a mystery. To date, the influence of NF-κB on the disruption of circadian rhythms during aging has been demonstrated. However, little is known about NLRP3's involvement in aging-associated chronodisruption. It should be noted that aging manifests the progressive loss of strength and muscle mass. This process is defined as sarcopenia and is considered one of the main causes of reduced physical performance and impaired cardiorespiratory function in patients with heart failure (Curcio et al., 2020). Numerous clinical and experimental studies have shown that aging is associated with histological, structural, and functional changes in cardiac tissue (Lakatta, 2002). Our group demonstrated that the absence of the NLRP3 inflammasome reduced sarcopenia in skeletal muscle (Sayed et al., 2019). Given these results, we consider it of interest to analyze the involvement of NLRP3 inflammasome activation in structural alterations in the aging heart. Melatonin is a hormone synthesized by the pineal gland, as well as by most organs and tissues, including the heart. Pineal melatonin has chronobiotic actions and its production decreases with age. This decline has been linked to changes in circadian rhythms, increased inflammation, and development of cardiac pathologies (Hardeland, 2012). Extrapineal melatonin has antioxidant and anti-inflammatory properties (Hawthorn et al., 2012). In experimental models that include chronic and acute inflammation as well as aging in the mouse heart, melatonin decreased innate immune response, counteracted oxidative stress, and improved the activity of cardiac mitochondria (García et al., 2015; Rodríguez et al., 2007). In addition, melatonin administration has been shown to improve muscle function and reduce inflammation in athletes (Leonardo-Mendonca et al., 2017). Considering this theoretical framework, our work focused on the study of the causal relationship between chronodisruption, melatonin deficiency, and innate immunity, as well as the involvement of the NLRP3 inflammasome-mediated immune response during cardiac aging. To accomplish this, the aims were to evaluate cardiac tissue of wild type C57/Bl6 mice and mice with a C57/Bl6 background knocked-out for NLRP3 inflammasome (NLRP3-/-) of 3, 12 and 24 months of age in the following parameters: 1.- Mitochondrial pathway: mitochondrial dynamics, autophagy, apoptosis and mitochondria ultrastructure. 2.- Antioxidant pathway dependent on Nrf2. 3.- Biological clocking: expression of clock genes, rhythmicity, acrophase, amplitude and mesor. 4.- Histological study and MRI of heart muscle. 5.- Effects of melatonin treatment on the parameters mentioned above. The results of this doctoral thesis show the deleterious effect NLRP3 inflammasome has on mitochondrial function during aging, as its absence prevented damage to mitochondrial dynamics and structure. Melatonin treatment also reestablished mitochondrial dynamics, had an anti-apoptotic action, restored the Nrf2 dependent antioxidant pathway, and preserved mitochondrial structure during aging. With reference to the biological clock pathway, it could be found that aging, melatonin, and presence of the NLRP3 inflammasome had significant effects on expression observed in the clock genes, except for the Rev-erbα gene, which was not affected by the mouse genotype. Small phase changes were observed in the Clock gene, loss of rhythmicity in Per2 and Rorα and a tendency for mesor to decrease with aging. The NLRP3 inflammasome influenced the acrophase of Clock, Per2 and Rorα, suggesting some negative impact on the function of the myocardium. Melatonin restored rhythms and acrophases in cardiac tissue, highlighting its clinical potential in the prevention and treatment of chronodisruption. Besides these changes, the results indicate that the local chronobiotic system of the heart is highly protected against aging. Finally, it was concluded that NLRP3 is involved in cardiac sarcopenia, as 24-month-old mutant mice had less thickening of the ventricular wall, less fibrosis, lower expression of inflammatory cytokines, and lower mitochondrial damage compared to wild type mice. Again, we observed a prophylactic effect of melatonin in preserving the structure and number of cardiomyocytes and reducing pro-inflammatory and hypertrophic markers as well as apoptosis. It is therefore inferred that suppression of the NLRP3 inflammasome and implementation of melatonin therapy may be beneficial therapeutic approaches to ameliorate cardiac aging and sarcopenia.