Effect of Cardiotonic Steroid Marinobufagenin on Vascular Remodeling and Cognitive Impairment in Young Dahl-S Rats

Abstract

The hypertensive response in Dahl salt-sensitive (DSS) rats on a high-salt (HS) diet is accompanied by central arterial stiffening (CAS), a risk factor for dementia, and heightened levels of a prohypertensive and profibrotic factor, the endogenous Na/K-ATPase inhibitor marinobufagenin (MBG). We studied the effect of the in vivo administration of MBG or HS diet on blood pressure (BP), CAS, and behavioral function in young DSS rats and normotensive Sprague-Dawley rats (SD), the genetic background for DSS rats. Eight-week-old male SD and DSS rats were given an HS diet (8% NaCl, n = 18/group) or a low-salt diet (LS; 0.1% NaCl, n = 14-18/group) for 8 weeks or MBG (50 µg/kg/day, n = 15-18/group) administered via osmotic minipumps for 4 weeks in the presence of the LS diet. The MBG-treated groups received the LS diet. The systolic BP (SBP); the aortic pulse wave velocity (aPWV), a marker of CAS; MBG levels; spatial memory, measured by a water maze task; and tissue collection for the histochemical analysis were assessed at the end of the experiment. DSS-LS rats had higher SBP, higher aPWV, and poorer spatial memory than SD-LS rats. The administration of stressors HS and MBG increased aPWV, SBP, and aortic wall collagen abundance in both strains vs. their LS controls. In SD rats, HS or MBG administration did not affect heart parameters, as assessed by ECHO vs. the SD-LS control. In DSS rats, impaired whole-heart structure and function were observed after HS diet administration in DSS-HS vs. DSS-LS rats. MBG treatment did not affect the ECHO parameters in DSS-MBG vs. DSS-LS rats. The HS diet led to an increase in endogenous plasma and urine MBG levels in both SD and DSS groups. Thus, the prohypertensive and profibrotic effect of HS diet might be partially attributed to an increase in MBG. The prohypertensive and profibrotic functions of MBG were pronounced in both DSS and SD rats, although quantitative PCR revealed that different profiles of profibrotic genes in DSS and SD rats was activated after MBG or HS administration. Spatial memory was not affected by HS diet or MBG treatment in either SD or DSS rats. Impaired cognitive function was associated with higher BP, CAS, and cardiovascular remodeling in young DSS-LS rats, as compared to young SD-LS rats. MBG and HS had similar effects on the cardiovascular system and its function in DSS and SD rats, although the rate of change in SD rats was lower than in DSS rats. The absence of a cumulative effect of increased aPWV and BP on spatial memory can be explained by the cerebrovascular and brain plasticity in young rats, which help the animals to tolerate CAS elevated by HS and MBG and to counterbalance the profibrotic effect of heightened MBG.

Keywords: Dahl salt-sensitive rats; aortic pulse wave velocity; arterial stiffness; behavioral tests; cardiovascular remodeling; echocardiography; fibrosis; genes; hypertension; marinobufagenin; spatial memory; steroidal inhibitor of Na/K-ATPase; vascular dementia.

Figure 1

Schematic presentation of the experimental design and the group description. Sprague–Dawley (SD) and Dahl salt-sensitive (DSS) rats received a low-salt (LS) diet for 8 weeks, a high-salt (HS) diet for 8 weeks, or marinobufagenin (MBG), which was administered via osmotic minipumps for 4 weeks. The MBG-treated rats received an LS diet for the duration of the study.

Figure 2

Morris water maze (MWM) behavioral test results. MWM test was performed in four-month-old Sprague–Dawley (SD; n = 8; blue line) rats and four-month-old Dahl salt-sensitive (DSS; n = 7; red line) rats, which were kept on a low-salt (LS) diet. Values are expressed as mean ± SEM. (a) Distance to the platform in affected MWM test. Visible platform: trials 1–9, 11, 13, 15, and 17; invisible platform: trials 10, 12, 14, and 16. (b) Path efficiency in MWM. The differences between trials 9 (visible platform) and 10 (invisible platform) are presented. Two-way ANOVA with false discovery rate (FDR) correction: * p < 0.01, for trial 9 vs. trial 10; # p < 0.05, for SD vs. DSS. The DSS rats traveled longer distances to find the invisible platform vs. visible platform in comparison to the SD rats.

Figure 3

Neuronal density in the hippocampus of Sprague–Dawley (SD-LS) and Dahl salt-sensitive (DSS-LS) rats which were kept on a low-salt (LS) diet. (a) Neuronal density in CA1 and CA3 regions of hippocampus is presented as estimated number of neurons per mm² in a particular hippocampus region. Bars are presented as mean ± SEM; unpaired Student’s t-test: ** p < 0.01, DSS-LS rats (n = 5) vs. SD-LS rats (n = 5). (b) Representative image of a sagittal section of rat brain stained by Luxol/Cresyl violet stain; scale bar is 200 μm (right bottom corner). The quantified CA1 and CA3 regions which were used for the analysis of the neuronal density are schematically outlined by the dotted lines. Abbreviations for hippocampus and the surrounding regions: alv, alveus layer of CA; CA1 and CA3, Cornu Ammonis fields 1 and 3; DG, dentate gyrus; fi, fimbria; Hil, Hilus of DG; Th, thalamus. (c–f) Representative photomicrographs of CA fields stained by Luxol/Cresyl violet stain. Neurons are stained dark blue to purple. Scale bars, 50 μm (right bottom corner). (c) SD-LS CA1 area; (d) SD-LS CA3 area; (e) DSS-LS CA1 area, (f) DSS-LS CA3 area.

Figure 4

Effect of administration of MBG and high-salt (HS) dietary intervention on systolic blood pressure (a), pulse wave velocity (b), and collagen abundance in aortic wall (c). Bars represent mean ± SEM. Two-way ANOVA followed by Holm-Sidak’s multiple comparison post hoc test: * p < 0.05, ** p < 0.01 vs. LS groups for both SD and DSS rats; # p < 0.01 DSS vs. SD; & 0.05 < p < 0.1, vs. SD-LS. Two-tail unpaired t-test: † p < 0.05, DSS-LS vs. SD-LS (pulse wave velocity). Experimental group: LS, low salt for 8 weeks (blue bars); MBG, administration of MBG for 4 weeks (green bars); HS, high salt for 8 weeks (red bars); SD, Sprague–Dawley rats; DSS, Dahl salt-sensitive rats.

Figure 5

(a–f) Representative photomicrographs of aortae from each experimental group stained with Masson’s trichrome stain. Collagen in the aortic wall is stained blue; muscle and intracellular fibers are magenta. SD-LS (a), SD-MBG (b), SD-HS (c), DSS-LS (d), DSS-MBG (e), DSS-HS (f). SD, Sprague–Dawley rats; DSS, Dahl salt-sensitive rats; LS, low-salt diet; MBG, marinobufagenin; HS, high-salt diet. Scale bar is 50 μm.

Figure 6

Venn diagrams of the expression of profibrotic and AD-related genes. (a) The effect of marinobufagenin (MBG) and high-salt diet (HS) in comparison to low-salt diet (LS) in Dahl salt-sensitive (DSS) rats. (b) The effect of the HS in comparison to LS in DSS vs. Sprague–Dawley (SD) rats. (c) The effect of MBG and HS in comparison to LS in SD rats. (d) The effect of MBG in comparison to LS in DSS vs. SD rats. The graphic presentation is based on the qPCR data (Table 4 and Table 5). The genes that are downregulated in comparison to the LS control are presented in blue and the genes that are upregulated in comparison to LS control are shown in red. The genes in the intersecting areas are shared between the compared groups.

Figure 7

Morris water maze (MWM) test results. (a) Distance to the platform in MWM test. (b) Path efficiency in MWM test. MWM test was performed in four-month-old Sprague-Dawley (SD) rats and four-month-old Dahl salt-sensitive (DSS) rats which received low-salt (LS) diet (SD, n = 8; DSS, n = 7), MBG (SD, n = 8; DSS, n = 7) and high-salt (HS) diet (SD, n = 7; DSS, n = 8). Values are expressed as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test: * p < 0.05, ** p < 0.01, for trial 9 vs. trial 10 in the corresponding group. Two-tailed t-test: † p < 0.05 for trial 9 vs. trial 10 in DSS-HS. Dotted lines, SD groups; solid lines, DSS groups; blue, LS; green, MBG; red, HS.

Figure 8

Schematic presentation of the MBG and HS effects on profibrotic signaling in Sprague–Dawley (SD) rats (a) and Dahl salt-sensitive (DSS) rats (b). HS diet had a similar effect on cardiovascular fibrosis in SD and DSS rats with an activation of TGFβ signaling, whereas MBG administration had a different effect on the genes participating in profibrotic pathways. In SD rats, MBG activated both Fli-1 and TGFβ dependent pathways, whereas in DSS rats, MBG activated only Fli1-dependent profibrotic signaling. In addition, MBG downregulated expression of APOE and APP genes in DSS rats. HS, high salt; MBG, marinobufagenin; NKA, Na/K-ATPase; Fli-1, Freund leukemia integration 1 transcription factor; TGFβ, transforming growth factor beta; APOE, apoliporpotein E; APP, amyloid precursor protein; a, the factor which participates in the activation of TGFβ pathway by an HS diet; x, the factor, which participates in APOE pathway downstream of MBG; y, the factor which participates in APP pathway downstream of MBG; z, the factor which participates in Fli-1 pathway activation and possibly blocks TGFβ pathway. The hypothetical factors a, x, y and z require additional investigation; factor a may be present in both SD and DSS rats, and it is regulated by HS intake; factors x, y and z may be activated by MBG in DS rats.