The leading proponents of myocardial IRI are oxidative stress, pH and calcium paradox, inflammation, metabolic disorders, autophagy and apoptosis [14]. Numerous studies on myocardial injury have demonstrated that during IRI, ion accumulation, damage to the mitochondrial membrane, formation of ROS, disturbances in NO metabolism, and endothelial dysfunction are observed [14]. It is known that mitochondria, and enzymes like xanthine oxidase and NOX are the most important contributors to the exposure of cells to oxidants in IRI [14]. However, it is not fully understood which molecular mechanisms are fundamental in myocardial IRI yet. A combination of additional or synergistic multi-target treatments, and searching for new potential factors are required for optimal cardioprotection. In 1997 Kuro et al. reported an "aging suppressor" gene that encoded a transmembrane protein named Klotho [15]. Disrupted secretion of Klotho protein expedited aging, whereas its high expression extended lifespan in mice [16]. The analysis from the last decade showed that upregulated expression of Klotho is emerging as a promising therapeutic strategy for chronic kidney disease (CKD), acute kidney injury (AKI), and for cardiac hypertrophy, fibrosis and dysfunction [17–19]. FGF23-Klotho axis was proposed as predictive factor of fractures in type 2 diabetics with early CKD [20]. Low Klotho level was associated with mortality and cardiovascular events in haemodialysis patients [21]. For this reason, Klotho can be considered a potential protective factor in cardiovascular diseases as well. The present study confirmed that exogenous Klotho protein contributed to reduction of oxidative stress and damage in hearts subjected to IRI. This effect may be associated with the impact on the IGF1R/PI3K/AKT signalling pathway and increased antioxidant expression. The hypothesis of the influence of Klotho on the different pathway factors has been illustrated in Fig. 9.
IGF1 has pleiotropic actions in the heart. It plays a role in heart contractility, metabolism, hypertrophy, autophagy, senescence, and apoptosis. The binding of IGF1 to its receptor in the cardiomyocytes, plasma membrane IGF1R, leads to receptor autophosphorylation and activation of a complex signalling cascade (Fig. 9) [22]. It is known that IGF1R can activate two canonical pathways – PI3K/AKT pathway and the extracellular signal-regulated kinase (ERK) pathway [22]. As a result of IGF1R activation, phosphorylation of an intracellular adaptor protein insulin receptor substrate (IRS) occurs. Then, IRS recruits and phosphorylates PI3K, which is followed by AKT phosphorylation (Fig. 9) [23]. In our study, phosphorylation and activation of IGF1R and increase in PI3K level due to IRI was observed. The level of PI3K correlated with phosphorylation of IGF1R, which confirms activation of the pathway during IRI. It was shown that dysregulation of IGF signalling played a role in several kidney diseases, such as proteinuric CKD and polycystic kidneys [24]. Downregulating the IGF1R/PI3K pathway limited loss of podocytes in mice model of diabetic nephropathy [25]. Then, suppression of IGF1R signalling reduced cell death and inflammation, and protected against AKI in mice [26]. Inhibition of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway reduced oxidative stress and apoptosis, and enhanced autophagy during rat AKI [27]. Considering heart diseases, increased ROS-mediated PI3K/AKT signalling in lipopolysaccharide-induced endotoxemic in vitro and in vivo myocardial injury was shown [28]. It was reported that an underlying mechanism in induced cardiotoxicity was the PI3K/AKT/mTOR pathway. Inhibition of this axis improved cardiac dysfunction, and reduced inflammation, histopathological changes and myocardial apoptosis [29]. PI3K/AKT/mTOR signalling was also related to cardiac fibrosis in mouse ischemia-induced heart failure [30]. Similarly, increased cardiomyocyte hypertrophy through the activation of the PI3K/AKT/mTOR pathway in rat chronic intermittent hypoxia model was shown [31]. Limited activation of PI3K/AKT axis reduced cardiac hypertrophy [32].
The influence of Klotho on protective mechanisms was well-studied in several kidney diseases. It was reported that Klotho restrained IGF signalling, which protected against renal hypertrophy in diabetic mice or cystic expansion in mice with polycystic kidney disease [33, 34]. Reduced nephrotoxicity via negative regulation of the PI3K/AKT pathway by Klotho was shown as well [6]. Scientists reported that the up-regulation of Klotho expression decreased AKT phosphorylation, followed by improved aging-related memory deficits and oxidative stress in mice [35]. Similarly, Klotho was related to IGF1 signalling inhibition, thus to neuroprotection in Alzheimer’s disease mouse model [36]. In human umbilical vein endothelial cells, Klotho ameliorated oxidative stress by regulating the PI3K/AKT/endothelial nitric oxide synthase (eNOS) pathway [37]. IGF/PI3K downregulation was related to antiaging and anticancer activity of Klotho [38, 39]. The present results demonstrated that Klotho protein significantly contributed to inhibition of the IGF1R/PI3K/AKT signalling during heart IRI. A similar downregulation of the insulin/IGF1/PI3K/AKT signalling cascade by Klotho and reduction of oxidative stress was observed in HeLa cells [40]. Data showed that IGF1 deficiency and Klotho may reduce the cardiomyocytes’ sensitivity to aging-induced mechanical dysfunction [41]. Importantly, it was established that Klotho is involved in type 2 diabetes and insulin resistance. The anti-aging feature of Klotho was related to inhibition of insulin and IGF1 signalling [42]. Klotho induced insulin resistance in adipocytes by regulation of glucose transporter type 4 and intracellular insulin signalling through AKT [43]. This could explain the influence of Klotho on IGF signalling. Considering the role of IGF1 in cardiac disorders, the ability of Klotho to inhibit IGF in several heart hypertrophy models was investigated. Then, Klotho downregulated IGF/PI3K-dependent calcium channels in the mouse heart, thus protected against cardiac hypertrophy and remodelling [44]. Similarly, increased level of Klotho and inhibition of insulin/IGF1/AKT axis was related to abolished myocardial hypertrophy and fibrosis in mice [45]. It was reported that the level of circulating Klotho was increased in coronary artery disease (CAD) aerobically treated patients compared to non-treated group, but the level of IGF1 was lower [46]. Afterward, Klotho was proposed as a negative regulator for IGF1 and exercise-induced cardiac hypertrophy in women [47]. However, long-lasting chronic resistive training in young adults was related to increased level of IGF1 and heart hypertrophy, but did not influence the level of circulating Klotho [48].
There are four FOXO family members in mammals: FOXO1, FOXO3, FOXO4 and FOXO6. FOXO3 is predominantly expressed in the heart, brain, kidneys and ovaries [49]. FOXOs regulate their target genes related to metabolism, apoptosis, and cell cycle progression [50]. The association of IGFR/PI3K/AKT and FOXOs was studied in several cell lines and animal models. It was shown that activation of IGFR/PI3K/AKT pathway by IGF1 resulted in phosphorylation of FOXO1/3 in cardiac stem cells, myotubes or cancer cells [50]–[52]. As a result, translocation from the nucleus to the cytoplasm, and inactivation of FOXO are observed. The phosphorylation of FOXO1/3 by AKT impairs its transcriptional regulation function and triggers its degradation by the ubiquitin-proteasome system (Fig. 9). It was found that inhibition of PI3K/AKT axis could induce the translocation of FOXO1/3 between cytoplasm and nucleus, and its activation [8, 49, 53]. Interestingly, FOXO1/3 triggered AKT phosphorylation in cardiac myocytes. Then, FOXO-activated AKT feedback inhibition of FOXO was observed [54]. The current study confirmed the phosphorylation of FOXO3 in rat hearts during IRI, leading to its degradation. Inactivation of FOXO3 positively correlated with activation of IGF1R/PI3K signalling pathway. Likewise, Hu et al. (2022) observed low level of FOXO3 due to prolonged myocardial ischemia (MI) in rats [55]. Similar to our results, FOXO proteins were negatively regulated by the IGF1R/PI3K/AKT signalling cascade in breast cancer cells, human brain microvascular endothelial cell IRI, rat spontaneous intracerebral haemorrhage, or mouse cerebral IRI models [56]–[59]. Inhibition of FOXO3 was related to cardiac hypertrophy and heart failure in mice [60]. Then, downregulation of FOXO1 or FOXO3 expression in mouse heart reduced cardiac function, increased scar formation, enhanced stress-responsive signalling, and strengthened apoptotic cell death after MI [61]. Low expression of FOXO3 was observed also in retinal, hepatic or renal IRI in rats, and in patients with ovarian cancer [62–64]. Importantly, FOXOs were proposed as new therapeutic targets for cardiac diseases [53]. In this study, perfusion of the hearts with Klotho protein significantly inhibited inactivation of FOXO3 during IRI, showing reduced injury and cardioprotection. Interestingly, Frad et al. (2021) reported positive correlation between expression of Klotho and FOXO1 genes in patients with CAD. It was also shown that activation of FOXO1/3 resulted in upregulation of antioxidants and downregulation of proapoptotic molecules, and thus protected against myocardial or cerebral IRI [58, 59, 65, 66]. Then, increased FOXO1/3 expression in the cardiomyocytes inhibited oxidative stress and cell death caused by IRI [61]. These observations were consistent with our results, where Klotho administration inhibited FOXO3 phosphorylation and its subsequent ubiquitination and degradation. An anti-hypertrophic role of FOXO3 in the heart was also shown [67]. The inhibition of cardiomyocyte hypertrophy by FOXO3 was due to enhancing of antioxidant genes and subsequently reduction of ROS levels [68].
Heart failure is related to oxidative stress, when the production of ROS exceeds the capacity of antioxidant defence. Dependent upon the severity, depletion of endogenous antioxidants in IRI heart is observed [1]. It is known that the FOXOs target genes are related to antioxidative defence. FOXO3 activates the transcription of many antioxidant enzymes, including GPx, MnSOD, peroxiredoxin or catalase (Fig. 9) [49]. In this report, the production of antioxidants was negatively correlated with inactivation of FOXO3. Lim et al. (2019) reported decreased expression of MnSOD in tacrolimus (Tac)-induced renal injury mouse model, which was accompanied by increase in PI3K/pAKT levels and inactivation of FOXO3. Then, activated PI3K/AKT factors, FOXO3 phosphorylation, and decreased MnSOD expression due to Tac treatment in the human kidney 2 (HK-2) proximal tubule cell line were observed [69]. The expression of genes involved in oxidative stress resistance was related to FOXO3 activity in neural stem cells [70]. Moreover, FOXO1 upregulated expression of antioxidant enzymes and protected pancreatic β cells against oxidative stress [71]. This study reported that Klotho played a role in the enhancement of SOD and GPx levels in IRI hearts. Similar to the results previously shown, Klotho contributed to PI3K/AKT/FOXO3-related increase in MnSOD expression in Tac-injured HK-2 cells and mouse kidneys or rat renal IRI models [69, 72]. Importantly, Ramez et al. (2020) showed that an increase in plasma and myocardial levels of Klotho may boost antioxidant defence during heart IRI [73]. Klotho-enhanced MnSOD and catalase expression were observed in the senescence-accelerated mouse (SAMP8) brain as well. The antioxidative effect of Klotho was related to the inhibition of AKT/FOXO1 pathway [35]. Contrary, aging-related Klotho deficiency was associated with FOXO1 inactivation and downregulation of antioxidant enzymes in aged SAMP8 mice [35]. Upregulating endogenous Klotho expression resulted in the inhibition of IGF1R/AKT pathway, thus enhancement in FOXO1 and antioxidants expression in the 293T cell line [36]. Klotho was also proposed as a novel therapeutic target for cerebral ischemia, due to its antioxidative effect through AKT/FOXO1 pathway [59].
The mitochondrial electron transport chain, oxidoreductase, NOX, cytochrome P450 oxidases, and uncoupled NOS are the major enzymatic sources of cellular ROS. NOXs include a group of membrane-bound enzymes and are active in the extracellular space. It is known that during IRI, NOX can produce O2Ÿ− and other ROS that triggers oxidative stress (Fig. 9) [1]. NOX2 is expressed in the phagocytes, however its production was noted also in many other cell types, e.g., in the cardiomyocytes or endothelial cells [74]. NOX2 is mainly responsible for synthesis of O2Ÿ−, and its increased expression in human cardiomyocytes after acute myocardial infarction was shown [75]. There are numerous studies in cell lines and animal models confirming the impact of oxidative stress during IRI in many organs [59, 63, 64, 72–74]. We have previously reported higher level of NOX2 in human cardiomyocytes injured by IRI, which was accompanied by increased ROS/RNS production [74]. It was indicated that NOX4 activity is regulated via phosphorylation by several kinases, including involvement of IGF1/PI3K/AKT signalling [76–78]. In this study, the amount of NOX2 was positively correlated with p-IGFR1 and PI3K levels, and with FOXO3 inactivation, which shows the axis between IGF1R, NOX, and oxidative stress. IRI led to significantly increased production of total ROS/RNS, including H2O2 synthesis, which resulted in heart injury. The induction of oxidative stress correlated with activation of IGFR1/PI3K pathway and inactivation of FOXO3. There was also a positive correlation between ROS/RNS and NOX2 levels, which confirms the influence of NOX2 on oxidants formation. Finally, impaired IGF1R/PI3K/FOXO3 signalling and oxidative stress resulted in heart injury. Administration of Klotho protein significantly reduced NOX2 production, and then the oxidative stress and injury in IRI hearts. These results are consistent with our previous observations, where Klotho contributed to the reduction in NOX2/4 level, protected from oxidative stress, and enhanced total antioxidant capacity in the cardiomyocytes subjected to IRI [74]. Similarly, Klotho ameliorated oxidative stress caused by IRI in mouse kidneys and renal tubular epithelial cells [79]. Klotho efficiently suppressed NOX2/4 and ROS overproduction in hypertrophic neonatal rat cardiomyocytes and heart tissue in CKD-associated left ventricular hypertrophy mouse model [80]. An increase in NOX1, 2 and 4 levels, and induced oxidative stress were also found in calcified vascular smooth muscle cells that were mitigated after administration of exogenous Klotho. Afterward, NOX2-derived ROS production in the aorta of vascular calcification rats was reduced after Klotho injection [81]. There are also several reports confirming the antioxidative role of Klotho, where Klotho targeted oxidative stress during renal or brain injury [6, 35, 59, 69]. Our research has supplemented the knowledge about the antioxidative role of Klotho also in heart injury.