Markers of inflammation have been associated with cardiovascular diseases and proposed as other cardiovascular risk factors (Bullón et al., 2017 ). Recently, the role of the NLR family pyrin domain containing 3 protein (NLRP3) inflammasome has been studied in cardiovascular diseases. NLRP3 inflammasome is upregulated after myocardial infarction, atherosclerosis, ischemic heart disease, diabetic cardiomyopathy, chronic heart failure, and hypertension, and recently, NLRP3 and IL‐1β have also been proposed as new cardiovascular risk biomarkers (Bullón et al., 2017 ; Liu, Zeng, Li, Mehta, & Wang, 2017 ). Previous studies have suggested a role for NLRP3 inflammasome in several events associated with aging. Genetic deletion of NLRP3 in mice has been shown to improve healthspan by attenuation of multiple age‐related degenerative changes, such as glycemic control, bone loss, cognitive function, and motor performance (Youm et al., 2013 ). Furthermore, the deletion of NLRP3 in old mice increased muscle strength and endurance and prevented from age‐related increase in the number of myopathic fibers (McBride et al., 2017 ). However, the role of the NLRP3 inflammasome in lifespan and cardiac aging has not been studied. Hence, we sought to determine whether or not genetic deletion of NLRP3 may have effect on lifespan and potentially prevent cardiac aging.

Several of the metabolic changes presented in this study corroborated a special protection for cardiac changes by NLRP3−/− deletion. The metabolic hallmarks related to aging such as glucose tolerance and lipid metabolism are potentially corrected in NLRP3−/− mice, probably related to the reduced IGF‐1 signaling and the PI3K/AKT/mTOR pathway. Notably, adiponectin was also increased in these mice during aging. Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2 (Lindgren et al., 2013 ). AdipoR2−/− mice were resistant to obesity induced by a high‐fat diet and exhibited improved glucose tolerance and decreased plasma cholesterol levels (Fontana, Vinciguerra, & Longo, 2012 ). Increased levels of AdipoR2 were observed in old WT mice when compared with NLRP3−/−, which could be associated with AdipoR2 deficiency‐dependent protection of atherosclerosis (Lindgren et al., 2013 ). Furthermore, the reduced levels of adiponectin associated with increased levels of AdipoR2 in WT mice in aging could be interpreted as an attempt to contribute to the optimization of their metabolic environment. This has a negative effect, increasing the leptin/adiponectin ratio.

Accordingly, these genes were upregulated in old WT mice compared with NLRP3−/− mice, supporting the enhancing role of NLRP3 inhibition in the cardiovascular aging process. Furthermore, the expression of transglutaminase 2 (TGM2), an arterial calcification‐related protein that is positively associated with hypertension and atherosclerosis (Mattison et al., 2014 ), and Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) linked to the pathogenesis of vascular lesions were increased in WT but not in NLRP3−/− mice (Jeanne, Jorgensen, & Gould, 2015 ). Collectively, these data support significant protection imparted by NLRP3 inhibition on cardiac aging and age‐induced stress and vascular changes that occur during aging. Furthermore, this microarray study showed upregulation of genes associated with the mTOR pathway (Arntl, Akt1 and Ddit4) from old WT mice and changes associated with the negative regulation of autophagy processes (Nampt, Stat3, Fez2 and Akt1), which are associated with our findings of an inhibited mTOR pathway and increased autophagy in NLRP3 −/− mice during aging (Table S2 and S3 ).

Rad (Ras associated with diabetes) GTPase has been established as an endogenous regulator of cardiac excitation‐contraction (Wang et al., 2010 ). Rrad gene expression was increased in WT, but not in old NLRP3−/− mice, which could explain the increased cardiac pathology. Moreover, upregulation of Thbs1 was also observed in old WT mice when compared with NLRP3−/− mice, which is associated with a disturbed flow from arterial stiffening (Kim et al., 2017 ). Tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) and tripartite motif containing 72 (TRIM72) overexpression have been associated with atherosclerosis and diabetic cardiomyopathy, respectively (Liu et al., 2015 ; Lyu et al., 2018 ).

The homocysteine inducible ER protein with ubiquitin‐like domain 1 (Herpud1), which has been proposed as necessary for adequate insulin‐induced glucose uptake (Navarro‐Marquez et al., 2018 ), was also shown to be downregulated in old WT when compared to old NLRP3 −/− mice (Table S2 ). Herpud1 inhibition has recently been associated with induced pathological cardiac hypertrophy, which could explain the reduced hypertrophy observed in NLRP3 −/− mice (Torrealba et al., 2017 ). Similarly, our transcriptomic study showed upregulated gene expression of the cardiac hypertrophy‐related gene. Expression of established biomarkers atrial natriuretic peptide A (Nppa) and B (Nppb), which are associated with cardiac hypertrophy and strongly upregulated in the ventricular myocardium during cardiac stress (Man, Barnett, & Christoffels, 2018 ; Newman, Nguyen, Watson, Hull, & Yu, 2017 ), significantly increased in old WT mice when compared with old NLRP3 mice. Acta1 was the second most upregulated gene in old WT mice when compared with old NLRP3−/− mice. Recently, Acta1 has been associated with cardiac hypertrophy through increased levels of IGF‐1, so the reduced levels of Acta1 in NLRP3−/− mice could be associated with the reduced serum levels of IGF‐1 shown in this study (Bisping et al., 2012 ). The insulin receptor substrate protein 1 (IRS‐1) was also upregulated in old WT mice when compared to old NLRP3−/− mice. IRS‐1 may contribute to longevity (Selman, Partridge, & Withers, 2011 ).

To better define the molecular basis of improved cardiac health in the absence of NLRP3, a microarray expression profiling was performed on cardiac tissues obtained from 22‐month‐old animals. In old WT mice, 202 transcripts (from the 65,956 transcripts examined) changed significantly when compared with old NLRP3−/− mice: 142 transcripts were upregulated and 60 transcripts were downregulated, as those with a fold change equal to or higher than 2, and a p‐value equal to or lower than 0.05 (Figure S7 a). The most significant changes common to aging in WT and NLRP3−/− mice are available at http://www.ncbi.nlm.nih.gov/geo/ with code GSE124483 . All the gene expression data were loaded into DAVID for gene ontology (GO) enrichment analysis. The enrichment analysis in WT young‐to‐old mice showed that for the biological process, most of the genes were enriched in the response to stress and organic substances. However, these and other important biological and molecular enriched processes were not significantly different from those in NLRP3−/− young and old mice. NLRP3 deletion conferred protection related to aging, and significant differences were found between WT and NLRP3−/− (Figure 5 a–c). To examine the differences in gene expression profiling, gene coding pathways were represented in a heatmap (Figure 5 d). Our analysis indicated that 43 pathways were significantly altered between old WT and old NLRP3−/− mice. For a deeper analysis, the downregulated changes in protein coding are presented in Table S3 and the upregulated changes are summarized in Table S4 . A subset of expression changes was verified by polymerase chain reaction with reverse transcription (RT‐qPCR) (Figure S8 ). Notably, the most significant downregulated gene expression in old WT mice compared with old NLRP3 was nicotinamide phosphoribosyltransferase (Nampt), the rate‐limiting enzyme in mammalian NAD + biosynthesis (Table S2 ). NAD + deficiency is suggested to be a common central pathological factor in a number of diseases, including cardiovascular diseases and aging (North & Sinclair, 2012 ; Zhang & Ying, 2018 ). Interestingly, Nampt‐mediated NAD + deficiency is severely associated with glucose intolerance and insulin resistance in metabolic dysfunction by a high‐fat diet (HFD) and aging (Yoshino, Mills, Yoon, & Imai, 2011 ). Accordingly, we determined NAD + levels from the heart during HFD and a high sucrose diet (HSD) (exposed 15 weeks) and aging in WT and NLRP3−/− mice. In this respect, NLRP3−/− mice showed increased NAD + levels in both cases (HFD, HSD, and aging) and increased SIRT‐1 protein expression (Figure S9 A,B). These findings could explain the improved metabolic status and autophagic flux observed in NLRP3−/− mice during aging (Fang et al., 2017 ).

Changes in the Pi3K/mTOR pathways and autophagy observed in cardiac tissues from young and old mice. (a) Western blot analysis showing reduced levels in the Pi3K/mTOR pathway in the heart of NLRP3 −/− mice compared with WT. Densitometric analysis are presented as means ± SEM , n = 10 mice; *** p < .001 young vs. old mice. aaa p < .001, WT vs. NLRP3 −/− mice. (b) Western blot analysis with representative blot including ATG12, Beclin 1, LC3, Parkin, and p62 level in the heart of Young and old mice. Densitometric analysis are presented as means ± SEM , n = 10 mice; * p < .05, ** p < .005, and *** p < .001 young vs. old mice. (c) Cardiac tissues showing typical ultrastructure with several lamellar bodies and autophagosome (black arrows) present in cardiac tissues from old mice and (white arrows). Scale bar 2 µm (low magnification) and 1 µm (high magnification)

To gain insight into metabolic “longevity regulatory” pathways, we investigated IGF‐1, PI3K, mTOR in the heart. Since NLRP3−/− mice showed low levels of IGF‐1 in young and old mice, we examined signaling changes through these pathways in the heart. Despite no significant differences in phosphorylation of PI3K (p110α), mTOR (Ser2448) was decreased in the heart of aged NLRP3−/− mice (Figure 4 a). These data are consistent with the previous observations that cardiac aging is retarded and that healthspan is increased by mTOR inhibition (Inuzuka et al., 2009 ; Wu et al., 2013 ). mTOR inhibition is associated with the important physiological process of lysosomal‐dependent recycling, known as autophagy, which is involved in cellular homeostasis through protein degradation and removal of damaged intracellular organelles (Pyo et al., 2013 ). Autophagic disfunction has also been linked to aging with blocked autophagic flux and accumulation of nondegradated substrates in the form of autophagosome (Pyo et al., 2013 ). Interestingly, NLRP3−/− mice showed increased levels of ATG12, beclin 1 expression, and LC3II protein expression in NLRP3−/− old mice, with a reduction of p62/SQSTM1 (Figure 4 b). From electron microscopic analysis, we corroborated that the numbers of accumulated autophagosomes were reduced in hearts from NLRP3−/− old mice (Figure 4 c). This could be explained by where NLRP3 inhibition induced improved autophagy quality in the heart during aging.

In order to evaluate the role of NLRP3 during aging of the heart, several markers and pathways associated with aging were studied in hearts from young and aged WT and NLRP3−/− mice. Telomeres in young animals were similar in WT and NLRP3 −/− mice, whereas in two‐year‐old animals, NLRP3 −/− mice showed slightly longer telomeres (Figure 3 a,b). This is due to an increased telomere length reduction rate in WT mice compared with NLRP3 −/− mice (Figure S5 a). Furthermore, less lipofuscin accumulation in the heart of NLRP3−/− mice was shown after a qualitative observation (Figure S5 b). Additionally, we explored classical senescence biomarkers such as IL‐6, p21, and p53. Similar to serum levels of IL‐6, cardiac tissues showed increased IL‐6 protein levels in old mice, accompanied by increased p21 and phospho‐p53 protein levels compared with young mice in WT and KO mice; however, there was a higher increase in old KO mice than WT mice (Figure S6 ).

Upon electrocardiographic examination, the mean QRS complex was not significantly wider in old WT mice; however, we observed a significant prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging (Figure 2 e) (Magnani et al., 2013 ). Due to the improvement in the cardiac function of old KO mice and the low incidence of cancer in these mice (WT showed an increased rate of hepatocarcinoma and adenocarcinoma at death), the cause of death was unknown and will require further study.

Heart weight normalized to body weight was increased in old mice in comparison with young mice, and heart weight was higher in WT in comparison with NLRP3−/− ( p < .05) (Figure 2 a and Table S2 ). Cardiac hypertrophy measured by the left ventricular wall thickness was significantly increased in elderly WT when compared to NLRP3−/− mice which was corroborated by LV mass measured by echocardiography (Figure 2 b). To assess the impact of aging and cardiac hypertrophy on myocardial histology, the cardiomyocyte cross‐sectional area and fibrosis were quantified. In the hematoxylin‐and‐eosin–stained sections, aged WT mice showed an increased cardiomyocyte transverse cross‐sectional area unlike NLRP3−/− mice (Figure 2 c,d), which was corroborated by wheat germ agglutinin staining (Figure S2 ). Further examination with Masson trichrome and Sirius red staining revealed overt interstitial and perivascular fibrosis in the aged WT group, with no significant changes in the aged NLRP3−/− group (Figure 2 c,d). Ultrastructural analysis of the left ventricle in WT and NLRP3−/− showed mitochondrial abnormalities. We compared electron microscopic images of cardiac tissues from young and old WT mice and NLRP3−/− mice. TEM studies revealed evidence of mitochondrial damage in aged WT myocardium, including mitochondrial disarray, degeneration, fragmentation, reduction of mitochondrial area, and cristae disorganization, that is,. pointing in varying oblong and oblique directions in the matrix (Figures S3 a,b and S4a,b).

NLRP3 signaling suppression in mice extend lifespan and improve metabolic homeostasis. (a) Kaplan–Meier graph showing a significant increment of the máximum lifespan in WT mice (blue) compared with NLRP3 −/− mice (red). (b, c) Body weights and average daily oral food intake normalized to body weight and to mouse of the groups over time. Images of representative mice to illustrate phenotypic body mass of the groups at 20 months of age. (d) Representative photographs of 24 months of age mice. (e, f) Oral glucose tolerance test with area under the curve (inset). (g–i) Levels of leptin, adiponectin, and ratio in plasma. Blood samples were collected after overnight fasting. All data are presented as means ± SEM , n = 10 mice; * p < .05, ** p < .005, *** p < .001 young vs. old mice. aa p < .005, WT vs. NLRP3 −/− mice

To evaluate the impact of NLRP3 deletion on survival and metabolic changes during aging, we followed NLRP3 deficient (NLRP3 −/−) and NLRP3 +/+ littermate control (WT) mice throughout the entire lifespan. The survival of NLRP3 −/− mice compared to littermate controls using a Kaplan–Meier survival curve was augmented with an increase in mean lifespan of 34% and in maximum lifespan of 29% (Figure 1 a), while body weights and food intake did not differ between the two groups during the entire observation period (Figure 1 b,c). Twenty‐four‐month‐old WT animals displayed increased age‐related alopecia than their coveal NLRP3 knockout mice (Figure 1 d). Old NLRP3−/− mice exhibited a significant decrease in glucose at the OGTT peak (>15 min), compared with old WT mice (Figure 1 e,f), indicating a higher glucose tolerance as measured as a trend toward lower values of the area under the curve (AUC) of the glucose tolerance test (insert of Figure 1 f). Fasting blood glucose and circulating IGF‐1 levels were reduced in young and old NLRP3−/− mice, indicating that the insulin sensitivity of these animals was considerably higher than sham controls during aging (Table S1 ). Reduced levels of glucose and IGF‐1 have been associated with stress resistance and an antiaging effect (Brandhorst et al., 2015 ). Furthermore, leptin is an established regulator of body weight, and leptin/adiponectin dysregulation has been associated with cardiovascular disease, metabolic syndrome, and nonalcoholic fatty liver disease (DiNicolantonio, Lucan, & O'Keefe, 2016 ). Young and old NLRP3−/− mice showed similar serum levels of leptin compared to young and old WT, but a reduced leptin/adiponectin ratio with increased levels of adiponectin was observed in old NLRP3−/− mice (Figure 1 g–i). Plasma lipid levels were reduced in NLRP3−/− old mice, accompanied by a significant reduction in hepatic transaminases, creatine phosphokinase, and lactate dehydrogenase (Table S1 ). However, plasma IL‐1β levels were not detected in old mice, but increased protein levels of active caspase 1 and IL‐1β were observed in old WT, when compared to NLRP3−/− mice (Figure S1 ), and increased levels of TNF‐α, IL‐6, and IL‐8 were observed in WT similar old mice and NLRP3−/− mice. This shows that the loss of NLRP3 did not affect the age‐related increase of other inflammatory pathways and confers an important role on inflammasome in cardiac aging (Table S1 ).

3 DISCUSSION

Aging is the principal pathological process of cardiovascular diseases in healthy people. The principal age‐dependent changes in cardiac structure and function in the heart during normal aging are not well understood and, if defined, could provide new clues for protection from aging‐specific cardiac functional decline. This study showed that NLRP3 is associated with aging by an improved lifespan and healthspan via the modification of several hallmarks of aging. Little has been studied about the role of NLRP3 inhibition during aging, and nothing has been studied about longevity; our data, such as glucose tolerance, are consistent with previous studies on the effect of NLRP3 ablation on aging (Youm et al., 2013). Inflammation is highly associated with aging and age‐related diseases and many rejuvenation strategies adopt anti‐inflammatory diets (Finkel, 2015; Fontana et al., 2012). Increased systemic inflammation is commonly concomitant with metabolic alterations and the deterioration of metabolic health, including the appearance of increased adiposity, insulin resistance, and dyslipidemia, which could prove to be a key determinant of a shortening lifespan and healthspan (Finkel, 2015). According to this, one should anticipate that an experimental manipulation of a specific inflammatory pathway would entail systemic and metabolic effects with an improvement in life expectancy and health. Our results provide convincing evidence that the NLRP3 ablation causes an increase in longevity that could be due to several of the metabolic changes induced by this manipulation. In this study, we have observed an increase in glucose tolerance, a reduction and an increase, respectively, in lectin and adiponectin levels, and a regulation of dyslipemia. All these changes are associated with common pathways, such as IGF‐1, PI3K/AKT/mTOR, autophagy, and intracellular NAD+ levels (Finkel, 2015). According to our data, the ablation of NLRP3 showed low serum levels of IGF‐1 in old mice. The role of the protective pathological effects of IGF‐1 is contradictory, but our data suggest that low serum levels of IGF‐1 are the end product of decreased insulin/IGF‐1 signaling, which is known to prolong life, both in invertebrates and in vertebrates (Finkel, 2015; Fontana et al., 2012). Thus, low levels of insulin and/or IGF‐1 signaling, along with a high sensitivity to insulin and IGF‐1, are physiological characteristics that support the prolonged lifespan of Ames dwarf mice (Finkel, 2015) in which the levels of IRS‐1 associated with longevity were reduced (Papaconstantinou & Hsieh, 2015). Interestingly, our transcriptomic analysis showed reduced IRS‐1 expression in old NLRP3 mice when compared to old WT mice.

NLRP3 ablation also showed inhibition of PI3K/AKT/mTOR. mTOR is a serine‐threonine kinase that functions as an intracellular energy sensor whose genetic and pharmacological inhibition has been shown to extend life in a wide range of organisms (Cordero, Williams, & Ryffel, 2018; Wu et al., 2013). Since it is known that mTOR induces autophagy, the ablation of NLRP3 also showed, consistent with previous data showing the effect of inhibition of NLRP3 with other stressors, such as a hypercaloric diet, an increase in autophagy during aging (Pavillard et al., 2017). Cardiac aging is characterized by the presence of hypertrophy, fibrosis, and the accumulation of misfolded proteins and dysfunctional mitochondria. Therefore, autophagy and autophagic fluxes generally were reduced in cardiac tissues during aging, and models of loss of murine autophagy function models show an increase in cardiac dysfunction associated with the accumulation of misfolded proteins and dysfunctional organelles. Accordingly, it has been shown that the stimulation of autophagy improves cardiac function by eliminating accumulated cellular content, thus relieving different aging‐associated pathologies in the heart (Shirakabe, Ikeda, Sciarretta, Zablocki, & Sadoshima, 2016). This mechanism could be key to the improvement of longevity and health induced by the inhibition of NLRP3 and the support of many of the strategies to improve the extension of lifespan and healthspan through the use of rapamycin, caloric restriction with metformin or resveratrol, which have two common mechanisms: an improvement in autophagy and NLRP3 inhibition of inflammasome (Cordero et al., 2018). Furthermore, we found a reduced telomere shortening rate in WT mice when compared to NLRP3 −/− mice. Interestingly, reductions in telomere shortening rate, rather than the initial telomere length, have been suggested as a critical variable that determines a species’ lifespan in a wide variety of species, including mice and humans (Canela, Vera, Klatt, & Blasco, 2007; Vera, Bernardes de Jesus, Foronda, Flores, & Blasco, 2012; Whittemore, Vera, Martínez‐Nevado, Sanpera, & Blasco, 2019). We also found that protein levels of senescence/DNA damage markers, such as p21 or p53, measured by Western blot increase with age in WT hearts, but their expression does not vary with age in NLRP3 −/− hearts, which could be related to a reduced DNA damage response activated by dysfunctional telomeres in these animals. Interestingly, DNA damage in dysfunctional telomeres is a key hallmark of cardiomyocytes with a senescent‐like phenotype (Anderson et al., 2019), and clearance of senescence cells in mice alleviates cardiac deterioration with aging (Anderson et al., 2019; Lewis‐McDougall et al., 2019). Similarly, inhibition or deletion of NLRP3 would improve the detrimental effect of senescence cells in the heart, opening the door to a new line of research. In this sense, the NLRP3 inflammasome activation has been shown to promote the aging of the thymus and lead to immunosenescence (Spadaro et al., 2016). Moreover, the term of inflammaging has been nurtured and associated with a low‐grade proinflammatory phenotype that accompanies aging (Latz & Duewell, 2018). The different implications of NLRP3 in metabolism during aging and the protective role of the inhibition of NLRP3 show a relevant role of this in inflammaging. In this respect, NLRP3 −/− mice showed increased inflammatory levels during aging and, despite this, cardiac aging was prevented by NLRP3 deletion. Senescence influences the cellular environment through the secretion of proinflammatory cytokines, proteases, and chemokines called senescence‐associated secretory phenotype (SASP). Since activation of the NLRP3 system is a probable driver of SASP (Latz & Duewell, 2018), our findings could suggest a role of NLRP3 in the senescence phenotype during inflammaging.

Another age‐related mechanism linked to autophagy impairment is the intracellular reduction of NAD+. NAD+ is an electron acceptor in the mitochondrial electron transport chain that is also an essential substrate for NAD+‐dependent enzymes, such as sirtuins and poly ADP ribose polymerase (Rajman, Chwalek, & Sinclair, 2018). NAD+ levels decrease with age due to Nampt downregulation, oxidative stress, inflammation, defective circadian rhythm, and accumulation of DNA damage. Nampt, a key enzyme in the salvage pathway of NAD+ biosynthesis, is downregulated in the heart in response to ischemia, which induces a decrease in NAD+ levels in the heart, inhibition of autophagic flux, and cell death (Shirakabe et al., 2016). Therefore, restoring NAD+ content by overexpressing Nampt or adding NAD+ supplements restores the level of autophagy during ischemia and reduces the extent of myocardial infarction (Rajman et al., 2018). We observed an increased level of Nampt in NLRP3−/− old mice by transcriptomic analysis, which was corroborated by real‐time PCR. This is probably connected to the high levels of NAD+ observed in hearts of elderly NLRP3−/− mice fed with hypercaloric diets.

We acknowledge the limitations of using male mice only. On the other hand, we have studied the effect of the NLRP3 ablation in the expression of several markers of senescence by Western blots; however, our results must be extended to detect the specific cell type using, among others, gH2AX‐telomere immuno FISH or gH2AX‐PML colocalization in Immunofluorescence and IHC. Thus, future investigations should account for the implication of other inflammasomes in aging and their modulation.

In conclusion, our findings suggest that NLRP3 inhibition attenuates the harmful effects of cardiac aging and extends the lifespan in male mice. NLRP3 ablation improves metabolic characteristics related to aging, such as glucose tolerance, lipid metabolism, and leptin/adiponectin. These results could be associated with reduced IGF‐1 signaling and the PI3K/AKT/mTOR pathway and with autophagy activation. Our data associate the inhibition of NLRP3 with previous interventions against aging, such as caloric restriction, metformin, resveratrol or protein restriction, and involve levels of Nampt‐dependent NAD+ and SIRT1 (Cordero et al., 2018). In addition, our transcriptomic results show a profile related to metabolic improvement and an anti‐hypertrophic effect of cardiac protection. Finally, NLRP3 inhibition could be associated with a specific inflammasome‐dependent inflammaging. Therefore, prevention of the aging process through multiple mechanisms by NLRP3 inhibition is likely to attenuate the associated decrease in cardiac function. Thus, it offers a promising goal for the prevention of cardiac aging.