Up-regulation of Myocardial Klotho Expression to Promote Cardiac Functional Recovery in Old Mice following Endotoxemia

Objective: Endotoxemic cardiac dysfunction contributes to greater morbidity and mortality in elderly patients with sepsis. This study tested the hypothesis that Klotho insufficiency in aging heart exaggerates and prolongs myocardial inflammation to hinder cardiac function recovery following endotoxemia. Methods: Endotoxin (0.5 mg/kg, iv) was administered to young adult (3–4 months) and old (18–22 months) mice with or without subsequent treatment with recombinant interleukin-37 (IL-37, 50 μg/kg, iv) or recombinant Klotho (10 μg/kg, iv). Cardiac function was analyzed using a microcatheter 24, 48 and 96 h later. Myocardial levels of Klotho, ICAM-1, VCAM-1 and IL-6 were determined by immunoblotting and ELISA. Results: In comparison to young adult mice, old mice had worse cardiac dysfunction accompanied by greater myocardial levels of ICAM-1, VCAM-1 and IL-6 at each time point following endotoxemia and failed to fully recover cardiac function by 96 h. The exacerbated myocardial inflammation and cardiac dysfunction were associated with endotoxemia-caused further reduction of lower myocardial Klotho level in old mice. Recombinant IL-37 promoted inflammation resolution and cardiac functional recovery in old mice. Interestingly, recombinant IL-37 markedly up-regulated myocardial Klotho levels in old mice with or without endotoxemia. Similarly, recombinant Klotho suppressed myocardial inflammatory response and promoted inflammation resolution in old endotoxemic mice, leading to complete recovery of cardiac function by 96 h. Conclusion: Myocardial Klotho insufficiency in old endotoxemic mice exacerbates myocardial inflammatory response, impairs inflammation resolution and thereby hinders cardiac functional recovery. IL-37 is capable of up-regulating myocardial Klotho expression to improve cardiac functional recovery in old endotoxemic mice.


Introduction
Sepsis management is a major challenge for the healthcare systems worldwide. In the United States, sepsis accounts for a signi cant portion of all hospital deaths [1]. Sepsis occurs mainly in people of advanced age, and old septic patients have greater comorbidities and worse prognoses [2]. With the prolongation of life expectancy, the impact of sepsis is becoming greater. Investigation of the mechanisms underlying the pathobiology of sepsis in the elderly will provide information for the development of therapeutic approaches.
Septic cardiomyopathy is a common complication of severe sepsis and worsens the prognosis of patients [3]. Accumulating evidence indicates that myocardial injury caused by sepsis is due to excessive in ammation [3], and several in ammatory mediators induced by bacterial products, such as endotoxin (lipopolysaccharide, LPS), are cardiodepressants [4]. Thus, LPS contributes to the mechanism underlying the pathobiology associated with sepsis [5]. Our previous studies demonstrated that a low dose of LPS induces rapid cardiac contractile depression [6]. Further, we observed that old endotoxemic mice develop more severe cardiac dysfunction than young adult mice at 6-24 h following an exposure to LPS, along with greater elevation of cytokines and chemokines in the plasma and myocardium [6,7]. A study found that old mice had a lower survival rate and exhibited excessive multi-organ dysfunction, as well as exaggerated in ammation during sepsis induced by cecal ligation and puncture (CLP) [8]. Observations made in laboratory animals are comparable to the clinical manifestation of older patients with sepsis. Therefore, the excessive in ammatory response may be the key risk factors for multi-organ dysfunction, particularly cardiac dysfunction, in old septic subjects.
Anti-aging protein Klotho was initially identi ed in the kidney [9] and has been found subsequently in the parathyroid gland, skeletal muscle and large blood vessels [7,10,11]. We have found that the myocardium and heart valve tissue also express Klotho, and the expression of this anti-aging protein in these cardiovascular tissues is down-regulated with aging [7,12]. Several studies indicated that Klotho has an anti-in ammatory function [7,13,14]. In this regard, Klotho knockout mice display a pre-mature aging phenotype and have greater in ammation and more severe organ injury than wild-type mice when being subjected to infection [15]. More importantly, a clinical study found that renal Klotho levels were markedly reduced in septic patients [16], indicating a weakened anti-in ammatory capacity in patients with sepsis. Our previous study found that recombinant Klotho suppresses myocardial in ammatory response in the early phase of endotoxemia, up to 24 h [7]. Thus, Klotho insu ciency in aging heart likely exacerbates myocardial in ammation caused by endotoxemia and sepsis. Such pro-in ammatory effect of aging-related Klotho insu ciency may lead to worse and long-lasting cardiac dysfunction following endotoxemia or sepsis. Pharmacological up-regulation of Klotho levels in aging heart could be a feasible approach for cardiac protection in old subjects with endotoxemia or sepsis.
IL-37, a member of the IL-1 family of cytokines, was identi ed as an inhibitor of innate immunity [17]. Its expression has been identi ed in most human cell types, including monocytes, dendritic cells, endothelial cells and epithelial cells, and it acts as a natural regulator of the in ammatory responses [17]. IL-37 has repressive effects on LPS-stimulated cells, such as macrophages and endothelial cells [17]. A clinical study found that the level of IL-37 is increased in response to in ammation in old patients with sepsis [18]. In addition, our previous study found that IL-37 improves cardiac function in old mice at 24 h after an exposure to LPS [19]. Currently, the effect of IL-37 on cardiac functional recovery in the late phase of endotoxemia, at 48 to 96 h, is unclear, and the interaction of IL-37 with Klotho has not been investigated.
The purposes of this study were to determine the mechanistic role of Klotho insu ciency in myocardial in ammation and its resolution following endotoxemia in old mice and to explore the potential of IL-37 for up-regulation of myocardial Klotho level. Speci cally, this study determined whether Klotho insu ciency in aging hearts leads to prolonged myocardial in ammation in endotoxemia and whether prolonged myocardial in ammation is responsible for long-lasting cardiac dysfunction. Further, this study examined whether IL-37 is capable of up-regulating Klotho level to promote myocardial in ammation resolution and cardiac function recovery in old endotoxemic mice.

Animals and treatment
Male young adult (3 to 4 months) and old (18 to 22 months) C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, Maine) and National Institute on Aging (Bethesda, MD), respectively.
Mice were acclimated for 14 days in a 12:12 h light-dark cycle with free access to water and a regular chow diet.
Young adult mice were assigned to LPS group or saline group. Old mice were assigned to saline, LPS + saline, LPS + IL-37 or LPS + Klotho group. All treatments were performed in the morning through tail vein injection. LPS (0.5 mg/kg) was delivered in 0.1 ml of sterile normal saline. Recombinant Klotho (10 µg/kg) and recombinant IL-37 (50 µg/kg), in 0.1 ml of sterile normal saline, was administered 30-60 min after administration of LPS. Control treatment was performed using the same volume of sterile normal saline. Experiments were terminated at 24, 48 and 96 h (n ≥ 6 for each time point) after administration of LPS or saline. Blood, myocardium and kidney were collected following analysis of left ventricle (LV) function using a pressure-volume micro-catheter.

Measurement of cardiac function
Cardiac function was assessed as described previously [20]. Brie y, mice were anesthetized with iso urane inhalation (5% for induction, 2% for maintenance) and anticoagulated with heparin (Elkins-Sinn, Cherry Hill, NJ; 1,000 units/kg, IP). Animals were laid supine on a heating blanket and core body temperature was maintained at 37 ± 0.5℃. A pressure-volume microcatheter (Millar Instrument, Houston, TX; 1.0 F) was inserted into the left ventricle (LV) through the right common carotid artery. The pressurevolume loop and heart rate were recorded using the MPVS-400 system with the aid of PVAN software (Millar Instruments, Houston, TX). Heart rate, LV developed pressure, end-systolic and end-diastolic volume, ejection fraction, and cardiac output was analyzed.

Immunoblotting
Immunoblotting was applied to determine Klotho protein levels in the kidney and myocardium, as well as ICAM-1 and VCAM-1 levels in the myocardium. Proteins in tissue homogenate were separated on gradient (4%-20%) mini-gels and transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA).
The membranes were blocked with 5% skim milk solution for 1 hour at room temperature. The blocked membranes were incubated with a primary antibody against a protein of interest overnight at 4℃. After washing with phosphate-buffered saline (PBS) containing 0.05% Tween 20, the membranes were incubated with a peroxidase-linked secondary antibody (speci c to the primary antibody) for 1 hour at room temperature. After further washing, the membranes were treated with enhanced chemiluminescence reagents. The membrane was then exposed to X-ray lms. Image J (Wayne Rasband, National Institutes of Health, Bethesda, MD) was used to analyze band density. ELISA ELISA kits were utilized to quantify IL-6 and Klotho in myocardial tissue homogenate. Samples and standards were prepared according to manufacturers' instructions. The absorbance of standards and samples was determined spectrophotometrically at 450 nm, using a microplate reader (Bio-Rad Laboratories, Inc, Hercules, CA). Results were plotted against the linear portion of a standard curve.

Statistical analysis
Data are present as mean ± standard error (SE). Statistical analysis was performed using StatView software (Abacus Concepts, Calabasas, CA, USA). Analysis of variance (ANOVA) with Fisher post hoc test was used to analyze differences between experimental groups, and differences were con rmed using the Mann-Whitney U-test. Statistical signi cance was de ned as P ≤ 0.05.

Result
Persistent cardiac dysfunction in old endotoxemic mice is associated with exacerbated and prolonged myocardial in ammation (in amm-aging) In previous studies on the acute effect of endotoxemia, we observed that endotoxemia induces greater myocardial in ammatory responses and worse cardiac dysfunction at 24 h in old mice compared to young adult mice [7]. To understand the relationship of cardiac functional recovery with myocardial in ammation resolution, we prolonged the observation time to 96 h in the present study. We found that both young adult and old mice displayed cardiac dysfunction at 24 h after LPS challenge. As shown in Fig. 1A and Table 1, old mice had much lower ejection fraction (EF: 31 ± 4% vs. 49 ± 5%; p < 0.05) and LV developed pressure (DP: 50 ± 5 mmHg vs. 62 ± 7 mmHg; p < 0.05) at 24 h in comparison to young adult mice. EF and DP had recovered to baseline levels at 96 h after LPS challenge in young adult mice. However, these cardiac functional parameters remained signi cantly lower than baseline levels in old mice (EF: 43 ± 3% vs. 73 ± 8%; DP: 70 ± 7 mmHg vs. 85 ± 9 mmHg; p < 0.05). These results show that endotoxin induces worse cardiac dysfunction in old mice and that the recovery of cardiac function is incomplete by 96 h in old mice.
Interestingly, we found the more severe and persistent cardiac dysfunction in old endotoxemic mice is accompanied by myocardial "in amm-aging", a term specifying exacerbated in ammation associated with aging [21]. As shown in Fig. 1B, myocardial IL-6 levels increased in endotoxemic young adult and old mice at 24 h. Compared with young adult mice at 24 h, myocardial IL-6 levels in old mice were about 2 folds of that in young adult mice (505 ± 67 vs. 260 ± 30 ng/mg; p < 0.05). Myocardial ICAM-1 and VCAM-1 levels were also much higher in old mice in comparison to young adult mice (Fig. 1C). The levels of IL-6, ICAM-1 and VCAM-1 in both young adult and old mice decline at 48 and 96 h. While the levels of these pro-in ammatory mediators returned to the baseline levels at 96 h in the hearts of young adult mice, they remained elevated in hearts of old mice ( Fig. 1B and 1C). These results demonstrate that exaggerated myocardial in ammatory response and impaired ability of resolution of myocardial in ammation are components of myocardial in amm-aging, and both contribute to the mechanism of persistent cardiac dysfunction in old endotoxemic mice. IL-37 suppresses in ammation and promotes in ammation resolution to improve the recovery of cardiac dysfunction in old endotoxemic mice IL-37 is a member of the interleukin family and plays an anti-in ammatory effect in several in ammatory diseases. A recent study found that IL-37 improves cardiac function in adult endotoxemia mice via inhibition of the in ammatory NF-κB signaling pathway at 6 h [19]. In the present study, we found that IL-37 administration markedly improved left ventricle function in old endotoxemic mice (Table 2). Compared with the LPS only group, the developed pressure was elevated by 38%, ejection fraction was increased by 55% and cardiac output was increased by about 63% in the LPS + IL-37 group at 24 h in old mice (Table 2)  IL-37 up-regulates myocardial expression of Klotho in old mice and preserves myocardial Klotho level following endotoxemia Figure 3A shows that Klotho levels were much lower in the heart and kidney of old mice compared with adult mice. With an exposure to endotoxin, Klotho levels markedly decreased at 24 h in the heart and kidney of both adult and old mice, and the reduction of Klotho level was greater in old mice. Interestingly, Klotho levels in the heart and kidney of young adult mice recovered to the baseline at 96 while they remained low in the heart and kidney of old mice at this time point.
To determine the effect of recombinant IL-37 on the expression of Klotho in old mice, we treated old mice with recombinant IL-37 for 24, 48 and 96 h in the absence of exposure to LPS. Surprisingly, Klotho levels in the heart and kidney were markedly elevated over time. Klotho levels in both heart and kidney at 96 h were about 5 times higher than the levels in the saline-treated control group (Fig. 3B). As LPS decreases Klotho levels in the heart and kidney of old mice at 24, 48, and 96 h, we administered recombinant IL-37 to the old endotoxemic mice. Interestingly, treatment with IL-37 prevented the decline of Klotho levels in the heart and kidney, resulting in the preservation and elevation of Klotho levels in old mice following endotoxemia (Fig. 3C).

Klotho promotes myocardial in ammation resolution and improves the recovery from cardiac dysfunction in old mice
To determine the effect of Klotho on the myocardial in ammation resolution in old mice, we evaluated and compared the levels of ICAM-1, VCAM-1 and IL-6 levels in LPS alone group and LPS + Klotho group at 24, 48 and 96 h after administration of LPS. Although the levels of these in ammatory mediators decreased over time from 24 to 96 h, they remained signi cantly higher than the baseline at 96 h in LPS only group. In contrast, Klotho treatment resulted in the returning to the baseline levels for myocardial ICAM-1, VCAM-1 and IL-6 at 96 h (Fig. 4). Thus, recombinant Klotho not only attenuates myocardial in ammation response in old endotoxemic mice, but also promotes their myocardial in ammation resolution.
The representative pressure-volume loops in Fig. 5 show LV function at 24, 48, and 96 h after the exposure to LPS. Compared to the saline-treated old mice, old mice treated with LPS alone displayed greater reduction in LV function at 24 h. Although LV function was improved over time, it remained signi cantly lower at 96 h than the control level. However, old endotoxemic mice treated with Klotho had better LV function at all time points (Fig. 5 and Table 3). At 96 h, these mice displayed signi cantly improved developed pressure (75 ± 6 mmHg vs. 68 ± 7 mmHg in LPS alone group; P < 0.05, n ≥ 6 ), ejection fraction (56 ± 7% vs. 43 ± 3% in LPS alone group; P < 0.05, n ≥ 6 ) and cardiac output (5.1 ± 0.6 ml/min vs. 4.0 ± 0.5 ml/min in LPS alone group; P < 0.05, n ≥ 6 ) in comparison to old mice treated with LPS alone (Fig. 5 and Table 3).

Discussion
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 in ammation. Klotho insu ciency in the aging hearts contributes to the mechanism of exaggerated and prolonged in ammation, 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 in ammatory responses and promotion of in ammation resolution. Up-regulation of Klotho production in the aging heart is a novel function of antiin ammatory cytokine IL-37 and constitutes an important mechanism by which IL-37 promotes myocardial in ammation resolution and cardiac function recovery.
The magnitude and duration of tissue in ammatory change in response to a noxious insult is determined by the level of initial in ammatory response and the ability to resolve the in ammation [23]. When the integrity of anti-in ammatory mechanism is affected by aging, in ammatory response is exaggerated and the ability of resolution of in ammation becomes attenuated, resulting in exacerbated and longlasting in ammation. Such a phenomenon is termed as "in amm-aging" [24]. In the present study, we observed myocardial in amm-aging is present in old endotoxemic mice, re ected as greater magnitude of myocardial in ammation in the early phase of endotoxemia (up to 24 h after exposure to LPS) and the inability to resolve myocardial in ammation in the subsequent phase (from 24 to 96 h). Clearly, the results of the present study demonstrate that in amm-aging exacerbates myocardial in ammation following endotoxemia by elevating the in ammatory response and attenuating the ability of clearing the in ammation.
In ammatory 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 in ammatory mediators, i.e. IL-6, ICAM-1 and VCAM-1 examined, following an exposure to LPS, and in ammation resolution is impaired in the hearts of old mice as levels of in ammatory mediators in the myocardium remain elevated 4 days after the exposure to LPS. The exacerbated and prolonged myocardial in ammation in old endotoxemic mice is a characteristic manifestation of myocardial in amm-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 in ammatory mediators in cardiac dysfunction and support the notion that in amm-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 in amm-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 signi cantly 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 in ammation 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 in ammatory mediators and persistent elevation of these mediators in the myocardium as in ammatory cytokines have been found to down-regulate Klotho expression in mice [29]. Several studies demonstrate that Klotho has an anti-in ammatory 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 insu ciency in aging hearts and further reduction myocardial Klotho level following endotoxemia play a mechanistic role in mediating myocardial hyper-in ammatory response and persistent myocardial in ammation observed in old endotoxemic mice.
We tested this hypothesis by evaluating the effect of recombinant Klotho on myocardial in ammation and cardiac function in old endotoxemic mice. Recombinant Klotho not only reduced the levels of in ammatory mediators in the myocardium at 24 h, but also promoted the resolution of myocardial in ammation, resulting in essentially normalized levels of in ammatory 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 nding that Klotho promotes myocardial in ammation resolution and cardiac function recovery in old endotoxemic mice indicates that Klotho insu ciency is responsible for the impaired myocardial in ammation resolution and delayed cardiac function recovery. This nding also highlights the therapeutic potential of recombinant Klotho for cardiac protection in old subjects affected by endotoxemia or sepsis.
In ammation associated with aging is believed to be a mechanism by which Klotho expression is downregulated in old subjects [28]. However, it remains unclear whether Klotho expression in old subjects could be up-regulated. We explored the potential of anti-in ammatory approach for up-regulation of Klotho expression in old mice.
Our previous study demonstrates a potent anti-in ammatory 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 in ammation 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-in ammation 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 agingrelated in ammatory conditions. The bene cial effect of recombinant IL-37 on Klotho expression in naïve old mice is especially signi cant as it indicates that this approach may have a broader application for anti-aging and prevention of aging-related health problems.
Noticeably, myocardial in ammation is completely resolved, and cardiac function is fully recovered in young adult mice 4 days after endotoxemia while myocardial in ammation 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 in ammation 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. VCAM-1 (C) were assessed using ELISA and immunoblotting, respectively. Levels of these in ammatory mediators increased in the myocardium of young adult mice at 24 and 48 h, and they declined to the control level at 96 h. In old mice, myocardial levels of these in ammatory mediators were much higher at each time point than those in young adult mice, and at 96 h, they remained above control levels. Data are expressed as mean ± SE, n≥6 , *P< 0.05 vs. control, # P< 0.05 vs. young adult at the same time point.

Figure 2
Recombinant IL-37 suppresses myocardial in ammatory responses and promotes myocardial in ammation resolution in old endotoxemic mice. Old mice were treated with LPS (0.5 mg/kg, iv) alone or  Recombinant Klotho down-regulated myocardial in ammatory responses and promotes myocardial in ammation resolution. Old mice were treated with LPS (0.5 mg/kg, iv) alone or LPS followed by recombinant Klotho (10 μg/kg, iv) 30 min later. Control mice were treated with normal saline. ICAM-1 and VCAM-1 levels in myocardial tissue at 24, 48, and 96 h post-treatment were determined by immunoblotting and densitometry, and IL-6 levels were assessed by ELISA. Treatment with recombinant Klotho reduced myocardial levels of these in ammatory mediators in old mice at 24 and 48 h following the exposure to LPS and restored their levels to the baseline by 96 h. Data are expressed as mean ± SE, n≥6 , *P< 0.05 vs. control, # P< 0.05 vs. LPS alone at the same time point.