Melatonin/Nrf2/NLRP3 Connection in Mouse Heart Mitochondria during Aging

antioxidants Article Melatonin/Nrf2/NLRP3 Connection in Mouse Heart Mitochondria during Aging Marisol Fernández-Ortiz 1, Ramy K. A. Sayed 1,2 , JoséFernández-Martínez 1, Antonia Cionfrini 1, Paula Aranda-Martínez 1, Germaine Escames 1,3 , Tomás de Haro 4 and Darío Acuña-Castroviejo 1,3,4,* 1Centro de Investigación Biomédica, Departamento de Fisiología, Facultad de Medicina, Instituto de Biotecnología, Parque Tecnológico de Ciencias de la Salud, Universidad de Granada, 18016 Granada, Spain; [email protected].es (M.F.-O.); ramy[email protected] (R.K.A.S.); [email protected] (J.F.-M.); [email protected].es (A.C.); [email protected].es (P.A.-M.); [email protected] (G.E.) 2Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Sohag University, Sohag 82524, Egypt 3CIBERfes, Ibs. Granada, 18016 Granada, Spain 4UGC de Laboratorios Clínicos, Hospital Universitario San Cecilio, 18016 Granada, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-958-241-000 (ext. 20169) Received: 29 October 2020; Accepted: 23 November 2020; Published: 27 November 2020   Abstract: Aging is a major risk for cardiovascular diseases (CVD). Age-related disorders include oxidative stress, mitochondria dysfunction, and exacerbation of the NF- κ B/NLRP3 innate immune response pathways. Some of the molecular mechanisms underlying these processes, however, remain unclear. This study tested the hypothesis that NLRP3 inflammasome plays a role in cardiac aging and melatonin is able to counteract its effects. With the aim of investigating the impact of NLRP3 inflammasome and the actions and target of melatonin in aged myocardium, we analyzed the expression of proteins implied in mitochondria dynamics, autophagy, apoptosis, Nrf2-dependent antioxidant response and mitochondria ultrastructure in heart of wild-type and NLRP3-knockout mice of 3, 12, and 24 months-old, with and without melatonin treatment. Our results showed that the absence of NLRP3 prevented age-related mitochondrial dynamic alterations in cardiac muscle with minimal effects in cardiac autophagy during aging. The deficiency of the inflammasome affected Bax/Bcl2 ratio, but not p53 or caspase 9. The Nrf2-antioxidant pathway was also unaffected by the absence of NLRP3. Furthermore, NLRP3-deficiency prevented the drop in autophagy and mice showed less mitochondrial damage than wild-type animals. Interestingly, melatonin treatment recovered mitochondrial dynamics altered by aging and had few effects on cardiac autophagy. Melatonin supplementation also had an anti-apoptotic action in addition to restoring Nrf2-antioxidant capacity and improving mitochondria ultrastructure altered by aging. Keywords: melatonin; mitochondria; NLRP3 inflammasome; Nrf2; heart ultrastructure; apoptosis; mitochondrial dynamics 1. Introduction Cardiovascular diseases (CVD) constitute the leading cause of death in the world, especially in industrialized countries [ 1 ]. Genetics, hypertension, diabetes, obesity, smoking, and physical inactivity have been identified as risk factors for these diseases [ 2 ]. However, aging is by far the major risk factor for cardiac dysfunction, since its prevalence increases dramatically in aged people. Antioxidants 2020,9, 1187; doi:10.3390/antiox9121187 www.mdpi.com/journal/antioxidants Antioxidants 2020,9, 1187 2 of 22 The connection between aging and these cardiac pathologies have been widely reported [ 3 – 5 ]. Cardiac aging correlates with hemodynamic and metabolic alterations together, with changes in the structure and function of cardiovascular tissues. Furthermore, the increase in reactive oxygen species (ROS) and the activation of inflammation-related pathways have also been documented [ 6 – 8 ]. Aging is characterized by an increase in oxidative damage and persistent activation of innate immunity resulting in immunosenescence. This immune dysregulation results in a state of age-associated chronic inflammation termed ‘inflammaging’, which plays an important role in the onset and progression of cardiovascular diseases, in addition to other age-related disorders [9–12]. The main components of the innate immunity include NF-kB and NLRP3 inflammasome. Focusing on the NLRP3 inflammasome, it consists of the scaffold protein NLRP3, the adaptor protein ASC and caspase-1, forming a multiprotein complex [ 13 ]. The NLRP3 inflammasome is induced upon different signs of cellular ‘danger’ and is responsible for the maturation of the NF-kB-dependent pro-inflammatory cytokines including interleukin-1 β (IL-1 β ), potentiating the inflammatory response [ 14 ]. Some of these danger signals, such as ROS and mitochondrial DNA (mtDNA), come from impaired mitochondria during inflammation [ 15 ]. Additionally, age-related alterations in processes that maintain mitochondrial homeostasis, including fusion, fission, autophagy (mitophagy), and mitochondrial biogenesis, have been described. The resulting accumulation of dysfunctional mitochondria enhances ROS production and mtDNA release [ 16 , 17 ]. Another fact that could contribute to NLRP3 inflammasome activation is the reduced endogenous antioxidant defense capacity, which occurs during aging, in particular, the decline of transcription factor Nrf2 [ 18 , 19 ]. Thus, there seems to be a close relationship between aging, NF-kB/NLRP3 inflammasome response, cardiac and mitochondrial dysfunction, ROS formation, and decrease in Nrf2. Melatonin (N-acetyl-5-methoxytryptamine, aMT) is an ubiquitous molecule that, aside from the pineal gland [ 20 ], is synthesized by most body organs and tissues, including the heart [ 21 , 22 ]. In addition to its chronobiotic effects, this indoleamine presents important anti-oxidative and anti-inflammatory properties that depend on the high levels of extrapineal melatonin [ 23 – 25 ]. Within the cell, melatonin acts on its main target, the mitochondria, boosting their bioenergetic properties, enhancing the ATP levels and reducing the formation of free radicals [ 26 – 30 ]. In multiple experimental conditions including acute and chronic inflammation, and aging in mouse heart, melatonin consistently prevented oxidative stress, reduced the innate immunity activation, and boosted cardiac mitochondria function [ 12 , 23 , 25 , 31 ]. The mechanisms by which NLRP3 contributes to cardiovascular disorders are still unclear [ 32 ]. We hypothesized that NLPR3 inflammasome has a role in aged cardiac muscle and we considered it worthwhile to evaluate its association with molecular mechanisms underlying the development of cardiovascular diseases with age. Moreover, we also hypothesized that melatonin is able to counteract the age-related changes in the myocardium and we investigated where it exerts its action. For this purpose, we assessed age-associated disturbances regarding mitochondrial dynamics (fusion/fission), autophagy (mitophagy), apoptosis, Nrf2-dependent antioxidant response, and mitochondrial ultrastructure in the heart of the wild-type and NLRP3-knockout mice at 3, 12, and 24 months of age, with and without melatonin treatment. 2. Materials and Methods 2.1. Animals and Treatment Wild-type C57BL/6J and NLRP3-knockout mice NLRP3 −/− (B6.129S6-NLRP3tm1Bhk/J) on the wild-type C57BL/6J background (>10 backcrosses) aged 3 weeks were purchased from Charles River (Barcelona, Spain) and The Jackson Laboratory (Bar Harbor, ME, USA), respectively. Mice were housed in the animal facility of the University of Granada under a specific pathogen-free barrier and were kept under controlled temperature (22 ◦ C ± 1 ◦ C). Room illumination was on automated 12 h light/dark cycle (lights on at 08:00 h). Animals had ad libitum access to tap water and pelleted rodent chow. Antioxidants 2020,9, 1187 3 of 22 This study was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy of Sciences, Bethesda, MD, USA), the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (CETS #123), and the Spanish law for animal experimentation (R.D. 53/2013). The protocol was approved by the Andalusian’s Ethical Committee (05/07/2016/130). Wild-type (WT) and NLRP3 −/− mice were divided into five experimental groups (n=7 animals per group) (Figure 1): (I) young (Y, 3-months old), (II) early-aged (EA, 12-months old), (III) early-aged plus melatonin (EA +aMT), (IV) old-aged (OA, 24-months old), and (V) old-aged plus melatonin (OA +aMT) mice. Melatonin (aMT) was orally administered at 10 mg/kg/day in the chow during the last two months before early and old-aged treated mice were sacrificed (EA +aMT at the age of 10 months and OA +aMT at the age of 22 months). The other groups of animals (Y, EA and OA) were fed with normal chow without melatonin. The melatonin pelleted chow was prepared by the Diet Production Unit facility of the University of Granada. The amount of melatonin in the pellets was calculated according to the average daily food intake, number, weight and age of mice [ 33 ]. The use of 10 mg/kg/day was selected on the basis of previous studies that demonstrated the effectiveness of this dose on the aging process [ 11 , 34 ] and mitochondrial function [ 35 , 36 ]. C57/BL6A was reported to be a strain of mice that responds well to melatonin therapy [ 37 , 38 ]. Therefore, we deemed them suitable for the purpose of this study. Antioxidants 2020, 9, x FOR PEER REVIEW 3 of 23 Wild-type (WT) and NLRP3 −/− mice were divided into five experimental groups (n = 7 animals per group) (Figure 1): (I) young (Y, 3-months old), (II) early-aged (EA, 12-months old), (III) earlyaged plus melatonin (EA + aMT), (IV) old-aged (OA, 24-months old), and (V) old-aged plus melatonin (OA + aMT) mice. Melatonin (aMT) was orally administered at 10 mg/kg/day in the chow during the last two months before early and old-aged treated mice were sacrificed (EA + aMT at the age of 10 months and OA + aMT at the age of 22 months). The other groups of animals (Y, EA and OA) were fed with normal chow without melatonin. The melatonin pelleted chow was prepared by the Diet Production Unit facility of the University of Granada. The amount of melatonin in the pellets was calculated according to the average daily food intake, number, weight and age of mice [33]. The use of 10 mg/kg/day was selected on the basis of previous studies that demonstrated the effectiveness of this dose on the aging process [11,34] and mitochondrial function [35,36]. C57/BL6A was reported to be a strain of mice that responds well to melatonin therapy [37,38]. Therefore, we deemed them suitable for the purpose of this study. Figure 1. Study design summary: experimental groups and melatonin treatment. Animals were killed by cervical dislocation after ketamine plus xylazine anesthesia, and hearts were collected. The left ventricle was dissected and divided into two parts. One part was washed in saline, and rapidly fixed in 2.5% glutaraldehyde for transmission electron microscopy analysis, while the other part was stored at −80 °C for further western blot analysis. 2.2. Western Blot Analysis Pure cytosolic subcellular fraction was isolated from heart tissue according to Dimauro et al. [39] with some adjustments described in Rahim et al. [30]. Briefly, heart tissue was homogenized on ice at 800 rpm in 500 μL of STM buffer containing 250 mM sucrose, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 0.5 mM DTT, 5% phosphatase inhibitor buffer (125 mM NaF, 250 mM β-glycerophosphate, 250 mM p-nitrophenyl phosphate, and 25 mM NaVO3), and a protease inhibitor cocktail (Cat. 78429, Thermo Fisher Scientific, Waltham, MA, USA) with a Teflon pestle. The homogenate was maintained on ice for 30 min, then centrifuged at 800 g for 15 min at 4 °C. The supernatant was labeled as S0 and used for subsequent isolation of cytosolic fractions. S0 was centrifuged at 800× g for 10 min at 4 °C and the supernatant S1 was centrifuged at 11,000× g for 10 min. The resulting supernatant S2, containing cytosol and microsomal fraction, was precipitated in cold 100% acetone at −20 °C for 1 h followed by centrifugation at 12,000× g for 5 min. The pellet was then resuspended in 300 μL STM buffer and labeled as cytosolic fraction. Western blot analysis was performed on cytosolic fractions of mice hearts. Denatured protein samples (40 μg/fraction) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12% or 15% acrylamide/bis-acrylamide gels. Proteins were then wet transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Life Science S.L.U., Madrid, Spain). The membrane was blocked in 5% bovine serum albumin (BSA) in PBST (PBS with 0.1% Tween-20) at room temperature and then incubated overnight at 4ºC with the primary antibodies Figure 1. Study design summary: experimental groups and melatonin treatment. Animals were killed by cervical dislocation after ketamine plus xylazine anesthesia, and hearts were collected. The left ventricle was dissected and divided into two parts. One part was washed in saline, and rapidly fixed in 2.5% glutaraldehyde for transmission electron microscopy analysis, while the other part was stored at −80 ◦C for further western blot analysis. 2.2. Western Blot Analysis Pure cytosolic subcellular fraction was isolated from heart tissue according to Dimauro et al. [ 39 ] with some adjustments described in Rahim et al. [ 30 ]. Briefly, heart tissue was homogenized on ice at 800 rpm in 500 µ L of STM buffer containing 250 mM sucrose, 50 mM Tris-HCl pH 7.4, 5 mM MgCl 2 , 0.5 mM DTT, 5% phosphatase inhibitor buffer (125 mM NaF, 250 mM β -glycerophosphate, 250 mM p-nitrophenyl phosphate, and 25 mM NaVO3), and a protease inhibitor cocktail (Cat. 78429, Thermo Fisher Scientific, Waltham, MA, USA) with a Teflon pestle. The homogenate was maintained on ice for 30 min, then centrifuged at 800 g for 15 min at 4 ◦ C. The supernatant was labeled as S0 and used for subsequent isolation of cytosolic fractions. S0 was centrifuged at 800 × gfor 10 min at 4 ◦ C and the supernatant S1 was centrifuged at 11,000 × gfor 10 min. The resulting supernatant S2, containing cytosol and microsomal fraction, was precipitated in cold 100% acetone at − 20 ◦ C for 1 h followed by centrifugation at 12,000 × gfor 5 min. The pellet was then resuspended in 300 µ L STM buffer and labeled as cytosolic fraction. Antioxidants 2020,9, 1187 4 of 22 Western blot analysis was performed on cytosolic fractions of mice hearts. Denatured protein samples (40 µ g/fraction) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12% or 15% acrylamide/bis-acrylamide gels. Proteins were then wet transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Life Science S.L.U., Madrid, Spain). The membrane was blocked in 5% bovine serum albumin (BSA) in PBST (PBS with 0.1% Tween-20) at room temperature and then incubated overnight at 4 º C with the primary antibodies (Table S1) diluted in blocking buffer as per manufacturer’s specification. Membranes were washed with PBST 3 × 10 min and incubated for 1 h at room temperature with anti-mouse (BD Biosciences Pharmigen, San Jose, CA, USA) or anti-rabbit (Thermo Scientific, Madrid, Spain) IgG-horseradish peroxidase conjugated secondary antibodies diluted according to the manufacturer’s instructions. After washing with PBST, immunoreaction was detected using ClarityTM Western ECL Substrate (Bio-Rad, Madrid, Spain) and revealed in Kodak Image Station 4000MM PRO (Carestream Health, Rochester, NY, USA). Bands were analyzed and quantified using Kodak Molecular Imaging Software v. 4.5.1 (Carestream Health, Rochester, NY, USA). GAPDH protein content was used to normalize the cytosolic subcellular fraction. Data obtained from early and old-aged mice were always compared to young mice of the same group. The value of WT Y mice group was defined as 100%. Full images of western blots are available in Figure S1. 2.3. Transmission Electron Microscopy (TEM) Small pieces from the left ventricle of the heart were rapidly immersed in a 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for fixation, then post-fixed in 0.1 M cacodylate buffer-with 1% osmium tetroxide and 1% potassium ferrocyanide for 1 h. The specimens were then immersed in 0.15% tannic acid for 50 s, incubated in 1% uranyl acetate for 1.5 h, dehydrated in ethanol, and embedded in resin. Ultrathin sections of 65 nm thickness were cut using a Reichert-Jung Ultracut E ultramicrotome. These sections were double stained with uranyl acetate and lead citrate [ 40 ], and examined by a Carl Zeiss Leo 906E electron microscope and digital electron micrographs were acquired. 2.4. Morphometric Analyses Using electron micrographs, mitochondrial number and percentage of the mitochondrial damage (as number of damaged mitochondria/total mitochondrial number 100) were analyzed in areas with a width and height of 5.24 µ m and 3.99 µ m, respectively. Moreover, some morphometric analyses, including cross-sectional area (CSA) and Feret’s diameter of the intermyofibrillar mitochondria of cardiac muscle fibers were performed on images of electron microscopy using Image J processing software. 2.5. Statistical Analyses Data are expressed as mean ± standard error of the mean (SEM) of n=7 animals per group. All statistical analyses were carried out using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA). One-way ANOVA with a Tukey’s post hoc test was used to compare the differences between experimental groups. The values were found to be significantly different when p<0.05. 3. Results 3.1. NLRP3 Deficiency Prevents, and Melatonin Treatment Restores Cardiac Muscle Mitochondrial Dynamics Altered by Aging Anomalies in mitochondrial dynamics (fusion/fission) are typical of aged cardiac muscle [ 16 ]. Here, we showed that aging induced a decrease in the levels of proteins involved in mitochondrial dynamics, including Mfn2, Opa1, and Drp1, in WT mice, an effect absent in NLRP3 −/− mice (Figure 2A–C). Melatonin supplementation counteracted the decline of Mfn2, Opa1, and Drp1 caused by aging in WT mice. Interestingly, no significant effect of melatonin was observed in fusion proteins Mfn2 and Opa1 in NLRP3 −/− mice at the age of 12 and 24 months (Figure 2A,B). A slight, but not significant enhancement Antioxidants 2020,9, 1187 5 of 22 in fission protein Drp1 was noted in EA and OA NLRP3 −/− mice with melatonin supplementation (Figure 2C). Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 23 Figure 2. Changes in mitochondrial dynamics (fusion/fission) in WT and NLRP3−/− mice during aging and melatonin treatment. (A) Protein levels of Mfn2. (B) Protein levels of Opa1. (C) Protein levels of Drp1. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA + aMT), old-aged (OA), and old-aged with melatonin (OA + aMT) wild type and NLRP3−/− mice. Data are expressed as means ± SEM (n = 7 animals/group). * p < 0.05, ** p < 0.01 vs. Y; # p < 0.05, ## p < 0.01 vs. group without melatonin treatment. 3.2. NLRP3 Deficiency and Melatonin Therapy Had Minimal Effects in Autophagy in Cardiac Muscle during Aging A drop in the autophagic capacity observed in cardiac aging is associated with the accumulation of dysfunctional mitochondria, exaggerated ROS production, and mtDNA release [16,17]. Unsurprisingly, the conversion of LC3I to LC3II, a hallmark of autophagy [41], was significantly reduced in WT mice during aging, as reflected in the decrease in the LC3II/LC3I ratio in WT EA and OA mice (Figure 3). LC3II/LC3I ratio trends to increase in NLRP3−/− EA and OA mice, which may explain the attempt to restore autophagy events. Melatonin administration had minimal effects on the LC3II/LC3I ratio in all cases. Figure 3. Changes in autophagy in WT and NLRP3−/− mice during aging and melatonin treatment. LC3II/LC3I ratio. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA + aMT), old-aged (OA), and old-aged with melatonin (OA + aMT) wild type and NLRP3−/− mice. Data are expressed as means ± SEM (n = 7 animals/group). * p < 0.05 vs. Y. Figure 2. Changes in mitochondrial dynamics (fusion/fission) in WT and NLRP3 −/− mice during aging and melatonin treatment. ( A ) Protein levels of Mfn2. ( B ) Protein levels of Opa1. ( C ) Protein levels of Drp1. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA +aMT), old-aged (OA), and old-aged with melatonin (OA +aMT) wild type and NLRP3 −/− mice. Data are expressed as means ± SEM (n=7 animals/group). * p<0.05, ** p<0.01 vs. Y; # p<0.05, ## p<0.01 vs. group without melatonin treatment. 3.2. NLRP3 Deficiency and Melatonin Therapy Had Minimal Effects in Autophagy in Cardiac Muscle during Aging A drop in the autophagic capacity observed in cardiac aging is associated with the accumulation of dysfunctional mitochondria, exaggerated ROS production, and mtDNA release [ 16 , 17 ]. Unsurprisingly, the conversion of LC3I to LC3II, a hallmark of autophagy [ 41 ], was significantly reduced in WT mice during aging, as reflected in the decrease in the LC3II/LC3I ratio in WT EA and OA mice (Figure 3). LC3II/LC3I ratio trends to increase in NLRP3 −/− EA and OA mice, which may explain the attempt to restore autophagy events. Melatonin administration had minimal effects on the LC3II/LC3I ratio in all cases. Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 23 Figure 2. Changes in mitochondrial dynamics (fusion/fission) in WT and NLRP3−/− mice during aging and melatonin treatment. (A) Protein levels of Mfn2. (B) Protein levels of Opa1. (C) Protein levels of Drp1. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA + aMT), old-aged (OA), and old-aged with melatonin (OA + aMT) wild type and NLRP3−/− mice. Data are expressed as means ± SEM (n = 7 animals/group). * p < 0.05, ** p < 0.01 vs. Y; # p < 0.05, ## p < 0.01 vs. group without melatonin treatment. 3.2. NLRP3 Deficiency and Melatonin Therapy Had Minimal Effects in Autophagy in Cardiac Muscle during Aging A drop in the autophagic capacity observed in cardiac aging is associated with the accumulation of dysfunctional mitochondria, exaggerated ROS production, and mtDNA release [16,17]. Unsurprisingly, the conversion of LC3I to LC3II, a hallmark of autophagy [41], was significantly reduced in WT mice during aging, as reflected in the decrease in the LC3II/LC3I ratio in WT EA and OA mice (Figure 3). LC3II/LC3I ratio trends to increase in NLRP3−/− EA and OA mice, which may explain the attempt to restore autophagy events. Melatonin administration had minimal effects on the LC3II/LC3I ratio in all cases. Figure 3. Changes in autophagy in WT and NLRP3−/− mice during aging and melatonin treatment. LC3II/LC3I ratio. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA + aMT), old-aged (OA), and old-aged with melatonin (OA + aMT) wild type and NLRP3−/− mice. Data are expressed as means ± SEM (n = 7 animals/group). * p < 0.05 vs. Y. Figure 3. Changes in autophagy in WT and NLRP3 −/− mice during aging and melatonin treatment. LC3II/LC3I ratio. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA +aMT), old-aged (OA), and old-aged with melatonin (OA +aMT) wild type and NLRP3−/−mice. Data are expressed as means ±SEM (n=7 animals/group). * p<0.05 vs. Y. Antioxidants 2020,9, 1187 6 of 22 3.3. Melatonin Treatment and, to a Lesser Extent NLRP3 Deficiency, Reduced Apoptosis in Cardiac Muscle during Aging Despite being intensively studied over the past three decades, many of the mechanisms of apoptotic cell death remain unknown. Although the relationship between aging and apoptosis have been a subject of controversy in scientific community, there seems to be consensus that apoptosis plays a significant role in cardiac aging [ 42 ]. Here, we showed that aging induced a rise in the levels of some proteins involved in apoptotic processes, including p53 and caspase 9 in both WT and NLRP3 −/− mice. Melatonin treatment significantly diminished the levels of p53 and caspase 9 in EA WT mice and in EA and OA mutant mice (Figure 4A,B). The pro-apoptotic protein Bax and the anti-apoptotic Bcl2 were significantly enhanced by aging in WT mice. Mutant mice only showed Bcl2 increased in OA animal’s group (Figure 4C,D). We observed a slight rise in Bax/Bcl2 ratio in EA and a significantly increase in WT OA mice (Figure 4E). The absence of NLRP3, however, prevented the apoptotic process associated with aging since Bax/Bcl2 ratio remained at similar levels as that of Y mutant mice. Melatonin supplementation significantly decreased the Bax/Bcl2 ratio in EA and OA WT mice, but had no effect in NLRP3−/−mice. Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 23 3.3. Melatonin Treatment and, to a Lesser Extent NLRP3 Deficiency, Reduced Apoptosis in Cardiac Muscle during Aging Despite being intensively studied over the past three decades, many of the mechanisms of apoptotic cell death remain unknown. Although the relationship between aging and apoptosis have been a subject of controversy in scientific community, there seems to be consensus that apoptosis plays a significant role in cardiac aging [42]. Here, we showed that aging induced a rise in the levels of some proteins involved in apoptotic processes, including p53 and caspase 9 in both WT and NLRP3−/− mice. Melatonin treatment significantly diminished the levels of p53 and caspase 9 in EA WT mice and in EA and OA mutant mice (Figure 4A,B). The pro-apoptotic protein Bax and the antiapoptotic Bcl2 were significantly enhanced by aging in WT mice. Mutant mice only showed Bcl2 increased in OA animal’s group (Figure 4C,D). We observed a slight rise in Bax/Bcl2 ratio in EA and a significantly increase in WT OA mice (Figure 4E). The absence of NLRP3, however, prevented the apoptotic process associated with aging since Bax/Bcl2 ratio remained at similar levels as that of Y mutant mice. Melatonin supplementation significantly decreased the Bax/Bcl2 ratio in EA and OA WT mice, but had no effect in NLRP3−/− mice. Figure 4. Changes in apoptosis in WT and NLRP3−/− mice during aging and melatonin treatment. (A) Protein levels of p53. (B) Protein levels of caspase 9. (C) Protein levels of Bax. (D) Protein levels of Bcl2. (E) Bax/Bcl2 ratio. Experiments were performed in hearts of young (Y), early-aged (EA), earlyaged with melatonin (EA + aMT), old-aged (OA), and old-aged with melatonin (OA + aMT) wild type and NLRP3−/− mice. Data are expressed as means ± SEM (n = 7 animals/group). * p < 0.05, ** p < 0.01 vs. Y; # p < 0.05, ## p < 0.01 vs. group without melatonin treatment. 3.4. Melatonin Treatment, but not NLRP3 Deficiency, Recovered the Nrf2-Dependent Antioxidant Capacity in Cardiac Muscle Declined by Aging In recent years, emerging evidence has indicated that aging leads to a gradual reduction of the Nrf2-dependent antioxidant response, which in turn contributes to the accumulation of oxidative stress [18,19]. Our results showed a significant decrease in the protein levels of Nrf2 and its active form pNrf2 (Ser40) in WT and NLRP3−/− mice with age, suggesting that NLRP3 deficiency was unable to ameliorate the age-related decline of Nrf2 and pNrf2 (Ser40) in these animals (Figure 5A,B). Melatonin supplementation markedly recovered the levels of Nrf2 and pNrf2 (Ser40) in both WT and mutant EA and OA mice. Aging and melatonin therapy did not significantly modify the levels of the Nrf2 inhibitor, Keap1, in either mouse strain (Figure 5C). Hmox1, Nqo1, and γGclc, three Figure 4. Changes in apoptosis in WT and NLRP3 −/− mice during aging and melatonin treatment. ( A ) Protein levels of p53. ( B ) Protein levels of caspase 9. ( C ) Protein levels of Bax. ( D ) Protein levels of Bcl2. ( E ) Bax/Bcl2 ratio. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA +aMT), old-aged (OA), and old-aged with melatonin (OA +aMT) wild type and NLRP3 −/− mice. Data are expressed as means ± SEM (n=7 animals/group). * p<0.05, ** p<0.01 vs. Y; #p<0.05, ## p<0.01 vs. group without melatonin treatment. 3.4. Melatonin Treatment, but not NLRP3 Deficiency, Recovered the Nrf2-Dependent Antioxidant Capacity in Cardiac Muscle Declined by Aging In recent years, emerging evidence has indicated that aging leads to a gradual reduction of the Nrf2-dependent antioxidant response, which in turn contributes to the accumulation of oxidative stress [ 18 , 19 ]. Our results showed a significant decrease in the protein levels of Nrf2 and its active form pNrf2 (Ser40) in WT and NLRP3 −/− mice with age, suggesting that NLRP3 deficiency was unable to ameliorate the age-related decline of Nrf2 and pNrf2 (Ser40) in these animals (Figure 5A,B). Melatonin supplementation markedly recovered the levels of Nrf2 and pNrf2 (Ser40) in both WT and mutant EA and OA mice. Aging and melatonin therapy did not significantly modify the levels of the Nrf2 inhibitor, Keap1, in either mouse strain (Figure 5C). Hmox1, Nqo1, and γ Gclc, three cytoprotective Antioxidants 2020,9, 1187 7 of 22 enzymes transcriptionally regulated by Nrf2, also remarkably decreased in WT OA mice (Figure 5D–F). The levels of Hmox1 and γ Gclc significantly dropped in NLRP3 −/− EA and OA mice (Figure 5D,F). Protein content of Nqo1 enzyme was not modified by aging in mutant animals (Figure 5E). Again, melatonin treatment greatly enhanced the levels of Hmox1, Nqo1, and γ Gclc in WT and NLRP3 −/− mice. Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 23 cytoprotective enzymes transcriptionally regulated by Nrf2, also remarkably decreased in WT OA mice (Figure 5D–F). The levels of Hmox1 and γGclc significantly dropped in NLRP3−/− EA and OA mice (Figure 5D,F). Protein content of Nqo1 enzyme was not modified by aging in mutant animals (Figure 5E). Again, melatonin treatment greatly enhanced the levels of Hmox1, Nqo1, and γGclc in WT and NLRP3−/− mice. Figure 5. Changes in the Nrf2-dependent antioxidant pathway in WT and NLRP3−/− mice during aging and melatonin treatment. (A) Protein levels of Nrf2. (B) Protein levels of pNrf2 (Ser40). (C) Protein levels of Keap1. (D) Protein levels of Hmox1. (E) Protein levels of Nqo1. (F) Protein levels of γGclc. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA + aMT), old-aged (OA), and old-aged with melatonin (OA + aMT) wild type and NLRP3−/− mice. Data are expressed as means ± SEM (n = 7 animals/group). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Y; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. group without melatonin treatment. 3.5. NLRP3 Deficiency and Melatonin Therapy Improved Mitochondria Ultrastructure Altered by Age in Cardiac Muscle Transmission electron microscopy of the cardiac muscles of Y WT mice revealed the presence of normally intact and compacted mitochondria with clearly organized cristae distributed in the intermyofibrillar spaces (Figure 6A,B). At the age of 12 months (EA), most of these mitochondria were found normally; however, a few showed cristae damage (Figure 6C,D). These changes were exacerbated and numerous mitochondria were severely damaged, hypertrophied, and vacuolated with completely destroyed cristae in WT OA mice (Figure 6G,H). Melatonin supplementation, however, preserved the normal ultrastructure of the cardiac mitochondria in EA (Figure 6E,F) and OA WT mice (Figure 6I,J), maintaining their healthy and compact appearance. Figure 5. Changes in the Nrf2-dependent antioxidant pathway in WT and NLRP3 −/− mice during aging and melatonin treatment. ( A ) Protein levels of Nrf2. ( B ) Protein levels of pNrf2 (Ser40). ( C ) Protein levels of Keap1. ( D ) Protein levels of Hmox1. ( E ) Protein levels of Nqo1. ( F ) Protein levels of γ Gclc. Experiments were performed in hearts of young (Y), early-aged (EA), early-aged with melatonin (EA +aMT) , old-aged (OA), and old-aged with melatonin (OA +aMT) wild type and NLRP3 −/− mice. Data are expressed as means ± SEM (n=7 animals/group). * p<0.05, ** p<0.01, *** p<0.001 vs. Y; #p<0.05, ## p<0.01, ### p<0.001 vs. group without melatonin treatment. 3.5. NLRP3 Deficiency and Melatonin Therapy Improved Mitochondria Ultrastructure Altered by Age in Cardiac Muscle Transmission electron microscopy of the cardiac muscles of Y WT mice revealed the presence of normally intact and compacted mitochondria with clearly organized cristae distributed in the intermyofibrillar spaces (Figure 6A,B). At the age of 12 months (EA), most of these mitochondria were found normally; however, a few showed cristae damage (Figure 6C,D). These changes were exacerbated and numerous mitochondria were severely damaged, hypertrophied, and vacuolated with completely destroyed cristae in WT OA mice (Figure 6G,H). Melatonin supplementation, however, preserved the normal ultrastructure of the cardiac mitochondria in EA (Figure 6E,F) and OA WT mice (Figure 6I,J), maintaining their healthy and compact appearance. Cardiac muscle fibers of NLRP3 −/− Y mice presented normal highly compacted mitochondria with densely packed cristae (Figure 7A,B). Mitochondrial structure did not change in EA mice, except one that showed damage in peripheral cristae (Figure 7C,D). The mitochondrial damage was less prevalent at 24 months in comparison with WT OA mice. Mitochondria were characterized by their widely-separated and organized cristae, with the presence of small-sized membranous vacuoles of possibly autophagic nature (Figure 7G,H). Melatonin treatment exhibited an obvious protective effect at the age of 12 (Figure 7E,F) and 24 months (Figure 7I,J), where it kept normal mitochondrial architecture with aging, in addition to formation of multivesicular bodies, which reflect the induction of the autophagic processes. Antioxidants 2020,9, 1187 8 of 22 Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 23 Figure 6. Age-associated ultrastructural changes of mitochondria in cardiac muscle fibers of WT mice and melatonin treatment. (A,B) Electron micrographs of cardiac muscle fibers of Y WT mice revealing the presence of normally intact and compacted mitochondria (M) distributed among myofibrils (Mf). (C,D) Electron micrographs of cardiac muscle fibers of EA WT mice demonstrating the presence of normal mitochondria (M) with few demonstrating cristae damage (asterisk). (E,F) Electron micrographs of cardiac muscle fibers of EA + aMT WT mice showing the protective effect of melatonin supplementation in preserving normal mitochondrial structure (M) with the presence of lipid droplets (L), N; nucleus. (G,H) Electron micrographs of cardiac muscle fibers of OA WT mice clarifying the presence of numerous severely damaged hypertrophied vacuolated mitochondria with completely destructed cristae (asterisk). (I,J) Electron micrographs of cardiac muscle fibers of OA + aMT WT mice exhibiting the beneficial effect of melatonin supplementation in keeping normal mitochondrial architecture (M). (A,C,E,G,I): bar = 2 μm and (B,D,F,H,J): bar = 1 μm. Cardiac muscle fibers of NLRP3−/− Y mice presented normal highly compacted mitochondria with densely packed cristae (Figure 7A,B). Mitochondrial structure did not change in EA mice, except one that showed damage in peripheral cristae (Figure 7C,D). The mitochondrial damage was less prevalent at 24 months in comparison with WT OA mice. Mitochondria were characterized by their widely-separated and organized cristae, with the presence of small-sized membranous vacuoles of possibly autophagic nature (Figure 7G,H). Melatonin treatment exhibited an obvious protective effect at the age of 12 (Figure 7E,F) and 24 months (Figure 7I,J), where it kept normal mitochondrial architecture with aging, in addition to formation of multivesicular bodies, which reflect the induction of the autophagic processes. Figure 7. Age-related ultrastructural changes of mitochondria in cardiac muscle fibers of NLRP3−/− mice and melatonin treatment. (A,B) Electron micrographs of cardiac muscle fibers of Y NLRP3−/− mice showing the presence of normally highly compacted mitochondria with densely packed cristae (M) distributed among myofibrils (Mf). (C,D) Electron micrographs of cardiac muscle fibers of EA NLRP3−/− mice demonstrating intact mitochondria (M) with individual ones depicting damaged peripherally cristae (asterisk). (E,F) Electron micrographs of cardiac muscle fibers of EA + aMT Figure 6. Age-associated ultrastructural changes of mitochondria in cardiac muscle fibers of WT mice and melatonin treatment. ( A , B ) Electron micrographs of cardiac muscle fibers of Y WT mice revealing the presence of normally intact and compacted mitochondria (M) distributed among myofibrils (Mf). ( C , D ) Electron micrographs of cardiac muscle fibers of EA WT mice demonstrating the presence of normal mitochondria (M) with few demonstrating cristae damage (asterisk). ( E , F ) Electron micrographs of cardiac muscle fibers of EA +aMT WT mice showing the protective effect of melatonin supplementation in preserving normal mitochondrial structure (M) with the presence of lipid droplets (L), N; nucleus. ( G , H ) Electron micrographs of cardiac muscle fibers of OA WT mice clarifying the presence of numerous severely damaged hypertrophied vacuolated mitochondria with completely destructed cristae (asterisk). ( I , J ) Electron micrographs of cardiac muscle fibers of OA +aMT WT mice exhibiting the beneficial effect of melatonin supplementation in keeping normal mitochondrial architecture (M). (A,C,E,G,I): bar =2µm and (B,D,F,H,J): bar =1µm. Antioxidants 2020, 9, x FOR PEER REVIEW 8 of 23 Figure 6. Age-associated ultrastructural changes of mitochondria in cardiac muscle fibers of WT mice and melatonin treatment. (A,B) Electron micrographs of cardiac muscle fibers of Y WT mice revealing the presence of normally intact and compacted mitochondria (M) distributed among myofibrils (Mf). (C,D) Electron micrographs of cardiac muscle fibers of EA WT mice demonstrating the presence of normal mitochondria (M) with few demonstrating cristae damage (asterisk). (E,F) Electron micrographs of cardiac muscle fibers of EA + aMT WT mice showing the protective effect of melatonin supplementation in preserving normal mitochondrial structure (M) with the presence of lipid droplets (L), N; nucleus. (G,H) Electron micrographs of cardiac muscle fibers of OA WT mice clarifying the presence of numerous severely damaged hypertrophied vacuolated mitochondria with completely destructed cristae (asterisk). (I,J) Electron micrographs of cardiac muscle fibers of OA + aMT WT mice exhibiting the beneficial effect of melatonin supplementation in keeping normal mitochondrial architecture (M). (A,C,E,G,I): bar = 2 μm and (B,D,F,H,J): bar = 1 μm. Cardiac muscle fibers of NLRP3−/− Y mice presented normal highly compacted mitochondria with densely packed cristae (Figure 7A,B). Mitochondrial structure did not change in EA mice, except one that showed damage in peripheral cristae (Figure 7C,D). The mitochondrial damage was less prevalent at 24 months in comparison with WT OA mice. Mitochondria were characterized by their widely-separated and organized cristae, with the presence of small-sized membranous vacuoles of possibly autophagic nature (Figure 7G,H). Melatonin treatment exhibited an obvious protective effect at the age of 12 (Figure 7E,F) and 24 months (Figure 7I,J), where it kept normal mitochondrial architecture with aging, in addition to formation of multivesicular bodies, which reflect the induction of the autophagic processes. Figure 7. Age-related ultrastructural changes of mitochondria in cardiac muscle fibers of NLRP3−/− mice and melatonin treatment. (A,B) Electron micrographs of cardiac muscle fibers of Y NLRP3−/− mice showing the presence of normally highly compacted mitochondria with densely packed cristae (M) distributed among myofibrils (Mf). (C,D) Electron micrographs of cardiac muscle fibers of EA NLRP3−/− mice demonstrating intact mitochondria (M) with individual ones depicting damaged peripherally cristae (asterisk). (E,F) Electron micrographs of cardiac muscle fibers of EA + aMT Figure 7. Age-related ultrastructural changes of mitochondria in cardiac muscle fibers of NLRP3 −/− mice and melatonin treatment. ( A , B ) Electron micrographs of cardiac muscle fibers of Y NLRP3 −/− mice showing the presence of normally highly compacted mitochondria with densely packed cristae (M) distributed among myofibrils (Mf). ( C , D ) Electron micrographs of cardiac muscle fibers of EA NLRP3 −/− mice demonstrating intact mitochondria (M) with individual ones depicting damaged peripherally cristae (asterisk). ( E , F ) Electron micrographs of cardiac muscle fibers of EA +aMT NLRP3 −/− mice revealing the clearly apparent prophylactic effect of melatonin supplementation in keeping normal mitochondrial architecture (M) with aging. ( G , H ) Electron micrographs of cardiac muscle fibers of OA NLRP3 −/− mice indicating less detectable mitochondrial damage compared with WT mice, with the presence of numerous mitochondria showing widely-separated organized cristae (asterisk) and small-sized membranous vacuoles of possibly autophagic nature (V). ( I , J ) Electron micrographs of cardiac muscle fibers of OA +aMT NLRP3 −/− mice showing the protective effect of melatonin supplementation in preserving normal mitochondrial structure (M), with formation of multivesicular bodies (MVB), which reflect the induction of the autophagic processes, N; nucleus. (A,C,E,G,I): bar =2µm and (B,D,F,H,J): bar =1µm. Antioxidants 2020,9, 1187 9 of 22 3.6. Lack of NLRP3 Reduced Mitochondria Number Loss and Mitochondrial Damage, an Effect Shared by Melatonin Morphometric analysis of cardiac mitochondria revealed that mitochondrial number exhibited initial non-significant decline in cardiac muscles of WT and NLRP3 −/− EA mice. Nevertheless, mitochondrial number was significantly decreased in OA, being more pronounced in WT mice than NLRP3 −/− one, an effect significantly counteracted after melatonin therapy (Figure 8A). Furthermore, the percentage of the mitochondrial damage was significantly increased in aged mice, especially in WT animals, and it was counteracted by melatonin supplementation (Figure 8B). Morphometrical analysis of the mitochondrial CSA illustrated a non-significant increase in cardiac muscle of WT and NLRP3 −/− EA mice, whereas the former increased in aged animals (Figure 8C). Mitochondrial diameter showed non-significant increase in WT EA mice, increasing in OA animals. NLRP3 −/− mice revealed non-significant changes in mitochondrial diameter among all experimental groups (Figure 8D). Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 23 NLRP3−/− mice revealing the clearly apparent prophylactic effect of melatonin supplementation in keeping normal mitochondrial architecture (M) with aging. (G,H) Electron micrographs of cardiac muscle fibers of OA NLRP3−/− mice indicating less detectable mitochondrial damage compared with WT mice, with the presence of numerous mitochondria showing widely-separated organized cristae (asterisk) and small-sized membranous vacuoles of possibly autophagic nature (V). (I,J) Electron micrographs of cardiac muscle fibers of OA + aMT NLRP3−/− mice showing the protective effect of melatonin supplementation in preserving normal mitochondrial structure (M), with formation of multivesicular bodies (MVB), which reflect the induction of the autophagic processes, N; nucleus. (A,C,E,G,I): bar = 2 μm and (B,D,F,H,J): bar = 1 μm. 3.6. Lack of NLRP3 Reduced Mitochondria Number Loss and Mitochondrial Damage, an Effect Shared by Melatonin Morphometric analysis of cardiac mitochondria revealed that mitochondrial number exhibited initial non-significant decline in cardiac muscles of WT and NLRP3−/− EA mice. Nevertheless, mitochondrial number was significantly decreased in OA, being more pronounced in WT mice than NLRP3−/− one, an effect significantly counteracted after melatonin therapy (Figure 8A). Furthermore, the percentage of the mitochondrial damage was significantly increased in aged mice, especially in WT animals, and it was counteracted by melatonin supplementation (Figure 8B). Morphometrical analysis of the mitochondrial CSA illustrated a non-significant increase in cardiac muscle of WT and NLRP3−/− EA mice, whereas the former increased in aged animals (Figure 8C). Mitochondrial diameter showed non-significant increase in WT EA mice, increasing in OA animals. NLRP3−/− mice revealed non-significant changes in mitochondrial diameter among all experimental groups (Figure 8D). Figure 8. Age-associated morphometrical changes of intermyofibrillar mitochondria in cardiac muscle fibers of WT and NLRP3−/− mice and melatonin treatment. (A) Analysis of mitochondrial number. (B) Analysis of mitochondrial damage percentage. (C) Analysis of cross-section area (CSA, μm2). (D) Analysis of mitochondrial Feret’s diameter (μm). Data are expressed as means ± SEM (n = 7 animals/group). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Y; # p < 0.05, ## p < 0.01 vs. group without melatonin treatment. Figure 8. Age-associated morphometrical changes of intermyofibrillar mitochondria in cardiac muscle fibers of WT and NLRP3 −/− mice and melatonin treatment. ( A ) Analysis of mitochondrial number. ( B ) Analysis of mitochondrial damage percentage. ( C ) Analysis of cross-section area (CSA, µ m 2 ). ( D ) Analysis of mitochondrial Feret’s diameter ( µ m). Data are expressed as means ± SEM (n=7 animals/group). * p<0.05, ** p<0.01, *** p<0.001 vs. Y; # p<0.05, ## p<0.01 vs. group without melatonin treatment. 4. Discussion Immunosenescence and inflammaging are caused by persistent activation of NF- κ B/NLRP3 inflammasome pathways generates chronic low-grade inflammation, which leads to, among other detriments, accumulation of cardiac mitochondrial dysfunction, characterized by dysregulation of mitochondrial dynamics, autophagy, apoptosis, Nrf2 antioxidant pathway, and maintenance of ultrastructure of mitochondria [ 43 ]. Another hallmark of aging is a decline in melatonin levels and its protective roles [ 44 ]. This brings about increased oxidative damage, chronodisruption, upregulation of pro-inflammatory cytokines, and downregulation of anti-oxidant/-inflammatory processes that contribute to inflammaging by facilitating mitochondrial disruption [ 45 ]. The role of the NLRP3 inflammasome and melatonin levels in regulation of mitochondrial dysfunction, associated with Antioxidants 2020,9, 1187 16 of 22 14. Zhou, R.; Tardivel, A.; Thorens, B.; Choi, I.; Tschopp, J. 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