Effect of pNaKtide on body weight, tissue weight, energy expenditure, locomotor activity, and oxygen consumption in C57B16 aging mice

We evaluated the effects of Na/K-ATPase signaling and pNaKtide in vivo using a mouse model of aging and a western diet (WD) regimen to induce oxidative stress. Our results showed that body weight, visceral fat, and subcutaneous fat weights were increased in the old mice and further increased in old mice fed a WD (Table 1). These increases were significantly decreased by pNaKtide treatment (Table 1). There were no significant differences in any of these measures between young mice and young mice treated with pNaKtide.

Table 1 Effect of pNaKtide on body weight; visceral fat, subcutaneous fat, and heart weight; energy expenditure; locomotor activity and oxygen consumption in C57Bl6 old mice. Full size table

There were no significant differences in heart weight between any of the groups (Table 1). Energy expenditure was determined as heat production in kcal/kg/hr21. The results showed that there was a significant increase in energy expenditure in pNaKtide treated old mice fed a WD as compared to old mice fed a WD (Table 1). Locomotor activity was also measured based on the ambulatory count over a 24 hr period21. Old mice showed decreased activity as compared to young mice, and old mice fed a WD showed decreased activity as compared to young mice, old baseline, and old mice. Administration of pNaKtide to old mice significantly increased their levels of locomotor activity compared to old baseline and old mice. Administration of pNaKtide to old mice fed a WD significantly increased their levels of locomotor activity compared to old mice fed a WD (Table 1). Oxygen consumption was measured in terms of mL/kg/hr. The results showed that there was a significant increase in oxygen consumption in pNaKtide treated old mice fed a WD as compared to old mice fed a WD (Table 1).

Effect of pNaKtide on adipocyte phenotype, senescence, and Na/K-ATPase signaling in C57Bl6 aging mice

Adipose tissue dysfunction occurs with aging22,23. Hematoxylin and eosin (H&E) staining of visceral fat tissues showed increased adipocyte size in adipose tissue of old mice with further increases in old mice fed a WD (Fig. 1A and B). Crown-like structures, representing an accumulation of macrophages around adipocytes, were apparent in adipose tissues of old mice with further increases noted in old mice fed a WD (Fig. 1A). In both groups, these increases were significantly attenuated with pNaKtide treatment. The TUNEL assay demonstrated that apoptosis was significantly increased in the adipose tissue of old mice and further increased in old mice fed a WD. These increases were also attenuated with pNaKtide treatment (Fig. 1C). The expression level of peroxisome proliferator-activated receptor gamma (PPAR-γ) is upregulated in diet-induced obesity, indicative of adipocyte hypertrophy and physiologic dysregulation of adipose tissue24,25. Apolipoprotein J (ApoJ) and p21 are known to increase in stress-induced cellular senescence25,26. Our results showed that ApoJ, p21, and PPARγ levels were increased in the old mice and further increased in old mice fed a WD (Fig. 1D,E and F). The magnitudes of these increases were significantly decreased with pNaKtide treatment. Protein carbonylation is well-known indicator of oxidative stress15. Old mice had demonstrable increases in protein carbonylation in adipose tissues, and these increases were amplified with a WD (Fig. S1A). The pSrc levels were also increased in the adipose tissues of old mice with further increases with a WD (Fig. S1B). Again, these changes were negated by treatment with pNaKtide. Levels of TNFα, a pro-inflammatory cytokine, and thiobarbituric reactive substances (TBARS), another marker of oxidative stress, increased significantly in old mice and old mice fed a WD as compared to the young mice (Fig. S1C and D). These increases were again negated by treatment with pNaKtide.

Figure 1 Effects of pNaKtide on adipocyte phenotype, senescence, and apoptosis in C57Bl6 aging mice. (A) H&E staining in visceral adipose tissue. Images taken with 40X objective lens; scale bar represents 100 µm. Arrows mark “crown like structures” indicative of inflammation. (B) Quantitative analysis of adipocytes area in visceral adipose tissue. (C) Representative images of TUNEL assay with quantification in C57B16 aging mice. Images taken with 40X objective lens; scale bar represents 25 µm. (D–F) qRT-PCR analysis of ApoJ, p21 and PPARγ in C57Bl6 aging mice with GAPDH as a loading control. Y, young; Y + P, young + pNaKtide; OB, old baseline; O, old; O + P, old + pNaKtide; O + WD, old + western diet; O + WD + P, old + western diet + pNaKtide. N = 8/group, *p < 0.05, **p < 0.01 vs Y, #p < 0.05, ##p < 0.01 vs O, &p < 0.05, &&p < 0.01 vs O + WD. Full size image

Effect of pNaKtide on heart senescence, apoptosis, and Na/K-ATPase signaling in C57Bl6 aging mice

Sirius red staining was performed to quantify fibrosis in heart tissues. Sirius red staining demonstrated a significant increase in fibrosis of the heart tissue in the old mice with further increases in old mice fed a WD (Fig. 2A and B). Treatment with pNaKtide decreased the fibrosis in both of these groups. H&E staining of cardiac tissues demonstrated remnants of degenerating myofibers in the old mice and old mice fed a WD (data not shown). Again, these changes were attenuated by pNaKtide treatment.

Figure 2 Effect of pNaKtide on fibrosis, senescence markers, and apoptosis in heart tissue of C57Bl6 aging mice. (A and B) Sirius red staining for detection of cardiac fibrosis with quantitative analysis. Images taken with 20X objective lens; scale bar represents 100 µm. (C–E) qRT-PCR analysis of Fibronectin, p21 and ApoJ in C57B16 aging mice. GAPDH was used as a loading control. (F,G) Representative images of TUNEL assay with quantification in C57B16 aging mice. Images taken with 40X objective lens; scale bar represents 25 µm. N = 8/group, *p < 0.05, **p < 0.01 vs Y, #p < 0.05, ##p < 0.01 vs O, &p < 0.05, &&p < 0.01 vs O + WD. Full size image

Aging resulted in cardiac hypertrophy and diastolic dysfunction as assessed by echocardiographic methods, summarized in Table 2. Specifically, old mice and old mice fed a WD had increased left ventricular wall thickness (anterior wall thickness (AWT), posterior wall thickness (PWT), relative wall thickness (RWT)), and left ventricular mass index (LVMI)). In addition, impaired function was noted in these groups with the myocardial performance index (MPI). These changes were significantly attenuated by pNaKtide treatment (Table 2). A significant increase in fibronectin, p21, and ApoJ levels were seen in both the old mice and old mice fed a WD (Fig. 2C,D and E). The magnitudes of these increases were significantly decreased with pNaKtide treatment. The TUNEL assay demonstrated that apoptosis was significantly increased in the hearts of old mice and further increased in old mice fed a WD (Fig. 2F and G). These increases were attenuated with pNaKtide treatment. There was significant increase in protein carbonylation in old mice with further increases in old mice fed a WD. These increases were attenuated with pNaKtide (Fig. S2A). TBARS levels followed a similar trend (Fig. S2B).

Table 2 Summary of transthoracic echocardiograph results. Full size table

Effect of pNaKtide on H 2 O 2 -induced senescence in human dermal fibroblasts (HDFs)

Administration of H 2 O 2 results in oxidative stress and DNA damage, which can lead to senescence27,28. Dose concentration curves were generated to determine the optimal dose of pNaKtide, which is effective in attenuating senescence (Fig. S3A). Our results showed that H 2 O 2 treated HDF exhibited morphological changes indicative of cellular senescence that included enlargement, flattening, and elongation of the cells along with decreased cell number, when viewed under light microscopy (Fig. S3A). We also noted that expression of senescence genes, ApoJ, p21, and fibronectin29 exhibited significant upregulation after subjection to H 2 O 2 treatment compared to untreated, control cells (Fig. S3B, S3C, S3D). Treatment with 1 µM pNaKtide significantly attenuated this effect when administered to H 2 O 2 treated cells. Elevated levels of beta-galactosidase (SA β-Gal), senescence-associated marker30, and phosphorylated histone H2AX (γ-H2AX), a marker of double-stranded DNA breaks31, were noted in the H 2 O 2 group and these changes were also attenuated by pNaKtide (Fig. 3A and B). Using a TUNEL assay to detect levels of fragmented and degraded DNA32, we found that apoptosis levels were elevated in cells treated with H 2 O 2 , which was attenuated by pNaKtide treatment. Caspase-9 is an essential initiator caspase required for apoptosis signaling33. Caspase-9 activity was elevated in H 2 O 2 treated cells and this was decreased by treatment with pNaKtide (Fig. 3C and D). No significant differences between the control group and the control group treated with pNaKtide were noted.

Figure 3 Effects of pNaKtide on H 2 O 2 -induced senescence markers, apoptosis, and Na/K-ATPase signaling in human dermal fibroblasts (HDFs). (A) Qualitative and quantitative analysis of ß-galactosidase levels. (B) Representative images and quantification of γ-H2AX levels. Images taken with 40X objective lens; scale bar represents 100 µm. (C) Representative images and quantification of the TUNEL assay (D) Quantitative analysis of Caspase-9 activity in cell lysates. (E) pATM, (F) pCHK2, and (G) p53 immunoblot analysis. Data shown are the mean band density normalized to ATM, CHK2, and actin, respectively. (H) Protein carbonylation levels with Coomassie staining as a loading control. (I) pSrc immunoblot analysis with data shown as mean band density normalized to Src. Data are displayed as “scatter plots” showing data points and “box plots” showing the distribution of a continuous variable as described in the Methods section. All gels were cropped above and below the band. N = 6/group, *p < 0.05, **p < 0.01 vs. control, #p < 0.05, ##p < 0.01 vs. H 2 O 2 . Full size image

Effect of pNaKtide on the p53 senescence pathway and Na/K-ATPase signaling in H 2 O 2 -treated HDFs

Cell senescence can be induced and maintained through the p53 pathway34,35,36. The effects of oxidative stress, such as double-strand breaks in DNA, trigger activation of ATM and CHK2, which then activates p537,26,37,38,39. Treatment of HDFs with H 2 O 2 significantly increased levels of pATM, pCHK2, and p53 protein compared with untreated controls. This effect was significantly attenuated following pNaKtide treatment (Fig. 3E,F and G). Further, the pNaKtide-treated group had significantly lower p21 mRNA expression compared with the H 2 O 2 -treated group (Table 3). Ki-67 is a well-established marker of cell proliferation40,41. The H 2 O 2 -treated group had significantly lower expression of Ki-67 mRNA and expression levels were restored in the pNaKtide-treated group (Table 3). Further, mRNA levels of other senescence markers, including ApoJ, matrix metalloproteinase 9 (MMP9), fibronectin, and collagenase were measured. All four were significantly decreased in the pNaKtide-treated group compared with the H 2 O 2 -treated group (Table 3). Protein carbonylation increased in H 2 O 2 -treated cells, and this increase was negated by pNaKtide treatment (Fig. 3H). Treatment with pNaKtide also blocked Na/K-ATPase-regulated Src activation in H 2 O 2 -treated HDFs (Fig. 3I). Lactate dehydrogenase (LDH) release, a marker of cytotoxicity42, was increased in the H 2 O 2 -treated group, and this was attenuated by concomitant treatment with pNaKtide (Table 3).

Table 3 Effects of pNaKtide on senescence genes, and LDH assay in H 2 O 2 induced senescence in HDF. Full size table

Effect of anti-oxidants on H 2 O 2 -induced senescence in HDFs

NAC and Vitamin E are known antioxidants, which have been shown to be effective in attenuating cellular senescence43,44,45,46,47. Dose concentration curves were generated to determine the optimal doses of NAC and Vitamin E, which were somewhat effective in attenuating senescence in our H 2 O 2 model (Fig. 4A). Cells exposed to H 2 O 2 and treated with 5 mM concentration of NAC or 50uM concentration of Vitamin E attenuated the morphological or biochemical changes of senescence, but less so than pNaKtide (Fig. 4A,C–E). However, the reductions in cellular oxidant stress as assessed by TBARS achieved with NAC and Vit E were virtually identical to that seen with pNaKtide (Fig. 4B).

Figure 4 Effect of anti-oxidants on H 2 O 2 induced cellular senescence in HDFs. (A) Representative images of HDFs exposed to H 2 O 2 and treated with varying concentrations of NAC and Vitamin E respectively. Images taken with 20X objective lens; scale bar represents 100 µm. (B) Quantitative analysis of TBARS. (C) Representative images and quantification of γ-H2AX levels. Images taken with 40X objective lens; scale bar represents 100 µm. qRT-PCR analysis of (D) p21 and (E) MMP9 in HDFs exposed to H 2 O 2 . GAPDH was used as a loading control. N = 6/group. **p < 0.05 vs. CTR, ##p < 0.01 vs. H 2 O 2 , &&p < 0.01 vs. H 2 O 2 + pNaKtide. Full size image

Effect of pNaKtide on UV radiation-induced senescence in HDFs

HDFs were treated with UV radiation, another mechanism for triggering oxidative-stress–mediated senescence48. Levels of SA β-Gal and TBARS were significantly higher in the UV-treated group compared with the control group (Fig. S4A and B). These increases were attenuated in the pNaKtide-treated group. The mRNA expression levels of p21, ApoJ, MMP9, and fibronectin were all significantly higher in the UV-treated group, and these increases were negated when cells were also treated with pNaKtide (Fig. S4C, S4D, S4E, and S4F).

Effect of pNaKtide on ouabain-induced senescence in HDFs

Since H 2 O 2 -mediated ROS initiated Na/K-ATPase signaling and induced senescence, we next studied whether ouabain, a specific receptor for Na/K-ATPase signaling activation, could also induce cellular senescence. A concentration curve was generated to determine the optimal dose of ouabain, and it was found that treatment with a 50 nM concentration significantly exhibited morphological changes indicative of cellular senescence (Fig. S5A). Additionally, LDH release increased significantly in the ouabain-treated group compared with the control group (Fig. S6B). Our results showed a significant increase in levels of γ-H2AX (Fig. S5C), and pATM, pCHK2, p53 protein (Fig. S6A,B and C) in the ouabain treated group that were attenuated with pNaKtide treatment. Also, pNaKtide decreased p21 mRNA expression when administered with ouabain (Fig. S6D). Caspase 9 activity increased significantly in the ouabain-treated group; this was also significantly attenuated by concomitant treatment with pNaKtide (Fig. S5D). The ouabain treated group also had significantly decreased Ki-67 levels as compared to the control group. This decrease was negated with pNaKtide treatment (Fig. S6E). Ouabain, of course, increased the pSrc/Src ratio, and this was also significantly decreased by pNaKtide treatment (Fig. S6F).

Effect of glucose oxidase (GO) and pNaKtide on oxidative stress, cell proliferation, and apoptosis in HDFs

GO generates moderate amounts of H 2 O 2 which results in a steady increase in intracellular ROS in HDFs49. Concentrations of GO ranging from 0 to 10 mU/ml were administered to HDFs. Our result showed that TBARS level increased with increasing concentrations of GO (Fig. 5A). Treatment with pNaKtide attenuated this increase; however it is important to note that TBARS concentration in the pNaKtide treated cells was still demonstrably elevated with the high concentrations of GO. To determine the role of Na/K-ATPase signaling in maintaining cell function and proliferation, MTT and CyQUANT assays were performed. Based on the MTT assay, 1 and 3 mU/ml of GO increased cell proliferation compared with control group, whereas 5 and 10 mU/ml of GO decreased cell count (Fig. 5B). Based on these observations, we hypothesized that some activation of the NKAL, demonstrated with 1 and 3 mU/ml GO may actually result in enhanced cell proliferation. Along these lines, we saw that concomitant treatment with pNaKtide allowed for enhanced cell proliferation in the groups exposed to 5 and 10 mU/ml of GO. The CyQUANT assay showed that compared with controls, the number of cells increased with GO 1 mU/ml and GO 3 mU/ml respectively; whereas 5 and 10 mU/ml of GO decreased cell count (Fig. 5C). Our results further showed that pNaKtide treatment did not increase cell proliferation in the groups exposed to 1, 3, and 5 mU/ml of GO. However, cell counts were increased in cells treated with 10 mU/ml GO with pNaKtide compared with 10 mU/ml GO alone. Based on caspase 9 activity, 5 and 10 mU/ml of GO increased apoptosis significantly compared with the control group. Treatment with pNaKtide negated this effect (Fig. 5D). These results support the concept that oxidants at low concentrations can stimulate cell proliferation whereas at higher concentrations, cell apoptosis results50. Interestingly, pNaKtide not only decreased the net amount of oxidant stress as assessed by the accumulation of TBARS but had more profound effects on cell proliferation and apoptosis than one would anticipate from the antioxidant effect alone.

Figure 5 Effects of glucose oxidase (GO) and pNaKtide on oxidative stress, cell proliferation, and apoptosis in HDFs. Concentrations of GO ranging from 0 to 10 mU/ml were administered to HDFs. (A) Quantitative analysis of TBARS (B) Quantitative analysis of the MTT assay (C) Cell count by CyQUANT assay (D) Caspase-9 activity assay. N = 16/group, *p < 0.05, **p < 0.01 vs. corresponding GO concentration groups without pNaKtide. Full size image

Effect of GO and pNaKtide on protein carbonylation in HDFs

Immunofluorescence of protein carbonylation using 2,4-dinitrophenylhydrazine (DNP) showed that 3, 5, and 10 mU/ml of GO significantly increased evidence for carbonylation, more so in the nucleus, compared with the control group (Fig. 6A and B). Furthermore, treatment with pNaKtide significantly reduced the carbonylation levels, especially in the nucleus, compared with GO alone. Immunoblotting of whole cell lysates with anti-DNP antibody showed that 10 mU/ml of GO significantly increased protein carbonylation compared with the control group (Fig. 6C). Conversely, this effect was negated when cells were treated with pNaKtide. Further, a double-staining immunofluorescence study was performed to determine the co-occurrence of protein carbonylation and DNA damage and fluorescent probes for DNP and γ-H2AX were visualized with confocal microscopy (Fig. 6D). HDFs exposed to GO 10 mU/ml concentration showed increased DNA damage along with increased intra-nuclear carbonylation when compared to control showing a positive co-relation between protein carbonylation and DNA damage (Fig. 6E). These increases with GO were attenuated with concomitant pNaKtide treatment.