Organ dysfunction is common in the elderly with sepsis or endotoxemia [22]. However, it remains unclear why advanced age makes the subject more vulnerable to severe organ dysfunction. In the present study, we found that the more severe and long-lasting cardiac dysfunction in old endotoxemic mice is due to excessive and prolonged myocardial inflammation. Klotho insufficiency in the aging hearts contributes to the mechanism of exaggerated and prolonged inflammation, as well as impaired recovery of cardiac function in old endotoxemic mice. Recombinant Klotho improves the recovery of cardiac function in old endotoxemic mice by down-regulation of the myocardial inflammatory responses and promotion of inflammation resolution. Up-regulation of Klotho production in the aging heart is a novel function of anti-inflammatory cytokine IL-37 and constitutes an important mechanism by which IL-37 promotes myocardial inflammation resolution and cardiac function recovery.
The magnitude and duration of tissue inflammatory change in response to a noxious insult is determined by the level of initial inflammatory response and the ability to resolve the inflammation [23]. When the integrity of anti-inflammatory mechanism is affected by aging, inflammatory response is exaggerated and the ability of resolution of inflammation becomes attenuated, resulting in exacerbated and long-lasting inflammation. Such a phenomenon is termed as “inflamm-aging” [24]. In the present study, we observed myocardial inflamm-aging is present in old endotoxemic mice, reflected as greater magnitude of myocardial inflammation in the early phase of endotoxemia (up to 24 h after exposure to LPS) and the inability to resolve myocardial inflammation in the subsequent phase (from 24 to 96 h). Clearly, the results of the present study demonstrate that inflamm-aging exacerbates myocardial inflammation following endotoxemia by elevating the inflammatory response and attenuating the ability of clearing the inflammation.
Inflammatory cytokines, such as TNF-α, IL-1β and IL-6, are cardiodepressants and contribute to cardiac dysfunction via their direct effects on the myocardium and/or though activation of other detrimental mechanisms in the myocardium, including generation of toxic oxygen species, mitochondrial dysfunction, the loss of calcium homeostasis and expression of adhesion molecules [6, 7, 25, 26]. In the present study, we observed that in comparison to young adult mice, old mice display greater myocardial production of inflammatory mediators, i.e. IL-6, ICAM-1 and VCAM-1 examined, following an exposure to LPS, and inflammation resolution is impaired in the hearts of old mice as levels of inflammatory mediators in the myocardium remain elevated 4 days after the exposure to LPS. The exacerbated and prolonged myocardial inflammation in old endotoxemic mice is a characteristic manifestation of myocardial inflamm-aging, and it is closely associated with the more severe and long-lasting cardiac dysfunction in old endotoxemic mice. Ample evidence demonstrates the mechanistic role of inflammatory mediators in cardiac dysfunction and support the notion that inflamm-aging contributes to the mechanism underlying the more severe and persistent cardiac dysfunction caused by noxious insults in the old subjects. Currently, the mechanism of myocardial inflamm-aging is unclear.
Klotho is an anti-aging protein present in multiple tissues, and its level declines with aging. Decreased Klotho protein level correlates with reduced lifespan and aging-related disorders, such as osteoporosis, coronary artery disease, and stroke [27]. Indeed, we observed significantly lower levels of myocardial Klotho in old mice in comparison to young adult mice. Interesting observations of the present study include greater reduction of myocardial Klotho levels by endotoxemia in old mice and inability of aging hearts to restore their Klotho levels 4 days after in endotoxemia. A low level of inflammation associated with aging is one of the mechanisms of Klotho down-regulation [28]. The greater reduction of myocardial Klotho level and failure to restore the Klotho level after 4 days are likely due to exaggerated production of inflammatory mediators and persistent elevation of these mediators in the myocardium as inflammatory cytokines have been found to down-regulate Klotho expression in mice [29]. Several studies demonstrate that Klotho has an anti-inflammatory function[7, 13, 30]. Particularly, recombinant Klotho has been found to inhibit TNF-α-induced NF-κB activation and attenuate the expression of ICAM-1 and VCAM-1 in HUVECs [31]. Based on the information, it is reasonable to hypothesize that Klotho insufficiency in aging hearts and further reduction myocardial Klotho level following endotoxemia play a mechanistic role in mediating myocardial hyper-inflammatory response and persistent myocardial inflammation observed in old endotoxemic mice.
We tested this hypothesis by evaluating the effect of recombinant Klotho on myocardial inflammation and cardiac function in old endotoxemic mice. Recombinant Klotho not only reduced the levels of inflammatory mediators in the myocardium at 24 h, but also promoted the resolution of myocardial inflammation, resulting in essentially normalized levels of inflammatory mediators in the myocardium after 4 days into endotoxemia. As a result, cardiac function recovery is markedly improved to a level slightly lower than control. The novel finding that Klotho promotes myocardial inflammation resolution and cardiac function recovery in old endotoxemic mice indicates that Klotho insufficiency is responsible for the impaired myocardial inflammation resolution and delayed cardiac function recovery. This finding also highlights the therapeutic potential of recombinant Klotho for cardiac protection in old subjects affected by endotoxemia or sepsis.
Inflammation associated with aging is believed to be a mechanism by which Klotho expression is down-regulated in old subjects [28]. However, it remains unclear whether Klotho expression in old subjects could be up-regulated. We explored the potential of anti-inflammatory approach for up-regulation of Klotho expression in old mice.
Our previous study demonstrates a potent anti-inflammatory effect of IL-37 on the cardiovascular tissue [19, 32]. Interestingly, we observed in the present study that administration of recombinant IL-37 to naïve old mice increases Klotho protein levels in the heart and kidney in a time-dependent fashion, with the maximal increase at 4 days. Further, treatment of old endotoxemic mice with recombinant IL-37 results in the restoration of Klotho levels in the heart and kidney at 4 days into endotoxemia. With the restoration of Klotho level in the heart, myocardial inflammation is completely resolved, and cardiac function is fully recovered in IL-37-treated old endotoxemic mice at 4 days into endotoxemia. It is noteworthy that old endotoxemic mice treated with recombinant IL-37 have better cardiac function than those treated with recombinant Klotho. It is likely that other mechanisms utilized by IL-37 in addition to the mechanism through up-regulation of Klotho have a moderate contribution to the full recovery of cardiac function.
Anti-inflammation might be the main mechanism by which IL-37 up-regulates Klotho levels. The effect IL-37 on Klotho level appears to be systemic, not selective to the heart, as kidney Klotho levels are also increased in old mice treated with IL-37. Up-regulation of Klotho by IL-37 occurs in both naïve old mice and old endotoxemic mice, demonstrating a novel mechanism for protection of the heart against aging-related inflammatory conditions. The beneficial effect of recombinant IL-37 on Klotho expression in naïve old mice is especially significant as it indicates that this approach may have a broader application for anti-aging and prevention of aging-related health problems.
Noticeably, myocardial inflammation is completely resolved, and cardiac function is fully recovered in young adult mice 4 days after endotoxemia while myocardial inflammation is still present and cardiac function remains low in old mice at this time. However, cardiac dysfunction in old endotoxemic mice appears to be reversible as the recovery is markedly improved by recombinant Klotho or IL-37. It is unclear from the present study how many more days are needed for the complete inflammation resolution and full cardiac function recovery in old endotoxemic mice without intervention. Our preliminary experiment indicates that approximately 10 days are required. Future study will determine the time course of natural recovery from endotoxemia in old mice and the days shortened by therapeutic interventions.