Early pathological mechanisms in a mouse model of heart failure with preserved ejection fraction

Figures

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  Figure 1.. Diastolic dysfunction with preserved ejection fraction after 3- and 6-wk exposure to excess mineralocorticoid and a Western diet. C57BL/6J control mice were given a chow diet. The DOCA+WD group denotes mice subjected to DOCA, high-fat diet (HFD), and saline challenges for 3 and 6 wk. A: representative tissue Doppler echocardiographic images 3 and 6 wk after challenge showing peak myocardial relaxation velocity at early diastole (e’) and peak myocardial contraction velocity (s’). B: ejection fraction (EF). C: peak myocardial relaxation velocity (e’). D: ratio between mitral E wave and e’. E: isovolumetric relaxation time (IVRT). F: peak myocardial contraction velocity (s’). G: stroke volume (SV). H: left atria (LA) diameter. I: relative wall thickness (RWT = posterior wall + interventricular septum at end-diastole)/left ventricular internal diameter at end-diastole). Data from both male and female mice were presented together. n = 10–28. Data presented as means ± SE. Data analyzed with ANOVA *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Figure 2.. Signs of elevated pulmonary pressure and organ congestion following 3 and 6 wk of exposure to excess mineralocorticoid and a Western diet. Control C57BL/6J mice were assigned to a standard chow diet. The group labeled as DOCA+WD indicates mice exposed to DOCA, high-fat diet (HFD), and saline challenges for 3 and 6 wk. A: representative flow Doppler echocardiographic images of pulmonary systolic velocity (PSV). Pulmonary acceleration time (PAT) represents the duration from the onset of acceleration to the attainment of peak velocity. Pulmonary ejection time (PET) is assessed from the onset of acceleration until the return to baseline. Cardiac cycle length (CL) denotes the interval from the initiation of acceleration in one cycle to the initiation of acceleration in the subsequent cycle. B: mean PSV. C: PAT/PET ratio. D: PAT/CL ratio. E: ratio of heart weight (HW, g) and tibia length (TL, mm). F: ratio of lung weight (LW, g) and tibia length (TL, mm). G: ratio of spleen weight (g) and tibia length (TL, mm). Data from both male and female mice were presented together. n = 8–26. Data presented as means ± SE. Data analyzed with ANOVA *P < 0.05; **P < 0.01; ***P < 0.001.

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  Figure 3.. Metabolic phenotype of mice after 3 wk of exposure to excess mineralocorticoid and a Western diet. Control C57BL/6J mice were given a standard chow diet, while the DOCA+WD group signifies mice exposed to DOCA, high-fat diet (HFD), and saline challenges for 3 wk. A: weekly body weight (BW) progression of female and male mice. B and C: % fat mass and % lean mass measured by NMR of female and male mice, separately. Gonadal white adipose tissue (gWAT) (g) (D) and subcutaneous (sc) WAT weights (g) (E) of female and male mice, separately. F: interscapular brown adipose tissue (iBAT) weight (g) of female and male mice, separately. G: fasting blood glucose levels (mg/dL), intraperitoneal glucose tolerance test (GTT), and area under the curve (AUC) of the GTT of male and female mice together. H: total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol of male and female mice together (mg/dL). n = 9–18. Data presented as means ± SE. A–G analyzed with Student’s t test. H analyzed with two-way ANOVA *P < 0.05; ***P < 0.001; ****P < 0.0001.

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  Figure 4.. Inflammation, oxidative stress, downregulation of nitric oxide signaling, and impaired coronary flow reserve after 3 wk of exposure to excess mineralocorticoid and a Western diet. C57BL/6J control mice were fed a standard chow diet for 3 wk, while the DOCA+WD group indicates mice exposed to DOCA, high-fat diet (HFD), and salt challenges for the same duration. A: C-reactive protein and interleukin-6 (IL-6) detected by ELISA. B: Western blot representative images of Gp91(phox) regulatory subunit of nicotinamide dinucleotide phosphate (NADPH) oxidase in females (left) and males (right) from whole heart homogenates, and summarized quantification of Gp91(phox) abundance over GAPDH. C: quantification of the production of reactive oxygen species (ROS) in isolated ventricular myocytes using 2′,7′ dichlorofluorescein diacetate (DCFH). D: representative Western blot images of females (left) and males (right) from whole heart homogenates, alongside summarized quantification of phospho-specific antibodies targeting AKT threonine 308 and AKT serine 473 over total AKT (E), including summary data for AKT abundance over GAPDH. F: Western blot images representing females (left) and males (right)…

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  Figure 5.. Dysregulation in calcium regulatory proteins and calcium-handling properties following a 3-wk challenge. Control C57BL/6J mice were fed a standard chow diet, whereas the DOCA+WD group consisted of mice exposed to challenges involving DOCA, a high-fat diet (HFD), and saline during a 3-wk period. A: representative plots showing ΔF/F0 obtained from isolated ventricular cardiomyocytes (left), calcium transient (CaT) amplitude (middle), and the decay constant of CaT, tau (τCaT) (right). B: representative plots illustrating ΔF/F0 obtained from isolated ventricular cardiomyocytes post-caffeine treatment (left), caffeine-induced calcium transients (CaTcaff) amplitude (middle), and the decay constant of the caffeine transient (τCaff) (right). C: representative Western blot images in females (left) and males (right) and summarized quantification of phosphorylation of phospholamban (PLB) at serine 16 and threonine 17 over total PLB and total PLB abundance over GAPDH in whole heart homogenates (D). E–G: representative Western blot images in females (left) and males (right) and summarized quantification of the phosphorylation and abundance of CaMKII, PKA-c (catalytic subunit), and SERCA over…

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  Figure 6.. Exposure to excessive mineralocorticoids and a Western diet for 3 wk affected myofilament proteins phosphorylation. Control C57BL/6J mice were provided with a standard chow diet for 3 wk, while the DOCA+WD group refers to mice subjected to DOCA, high-fat diet (HFD), and saline challenges over the same period. Representative images of ProQ diamond-stained SDS-PAGE gel of phosphorylated myofilament proteins (A) and Coomassie R-250-stained gel (B). C: summarized analysis showing the phosphorylation of titin, cardiac myosin binding protein-C (cMyBP-C), and myosin light chain 2 (MLC2). D: representative images in females (left) and males (right) and Western blotting quantification of cMyBP-C at Serine 273, 282, 302 over total cMyBP-C, and total cMyBP-C quantification over actin. E: representative Western blot images in females (left) and males (right) and quantification of PKG over GAPDH. F: representative images in females (left) and males (right) of vasodilator-stimulated phosphoprotein (VASP) phosphorylation, VASP abundance, and GAPDH. Histograms displaying the quantification of VASP phosphorylation relative to VASP abundance, as well as VASP phosphorylation and VASP abundance…

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  Figure 7.. The 3-wk challenge results in significant atrophy in the large intestine. C57BL/6J mice were fed chow or challenged with DOCA + Western diet for 3 wk. A: representative images of cecum and colon. B: colon length (mm). C: colon weight (g). D: fecal content (g). E: histological images of colon by H&E staining. Scale bars, 200 µm. F: colon crypt length, as shown by a double-headed yellow arrow, was measured. G: the thickness of the mucus layer, as indicated with yellow lines and double-headed arrow, was measured in samples stained with Periodic Acid-Schiff (PAS). Scale bars, 60 µm. H: mucus layer thickness, as shown by double-headed yellow arrow, was measured. I: lipopolysaccharide (LPS) levels in plasma (ng/mL). Data from both male and female mice were presented together. n = 8. Data presented as means ± SE. Data analyzed with Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Figure 8.. Scheme illustration of the proposed early underlying mechanisms of heart failure after 3 wk of excess mineralocorticoids, high salt intake, and a diet rich in fat and low in fiber. Challenges induce a proinflammatory state, resulting in increased levels of reactive oxidative species (ROS). This elevation in ROS may lead to eNOS uncoupling and a reduction in eNOS activity. This effect is compounded by decreased eNOS phosphorylation through reduced AKT activity. The resultant decline in eNOS activity leads to diminished titin phosphorylation via the downregulation of the NO-cGMP-PKG pathway, contributing to cardiac stiffness and diastolic dysfunction, as indicated by echocardiography. In addition, reduced NO and vasodilator-stimulated phosphoprotein (VASP) phosphorylation may suggest endothelial dysfunction. We also observed decreased coronary flow reserve suggesting coronary dysfunction. There are also alterations in calcium regulatory proteins, such as increased phospholamban-mediated inhibition of SERCA affecting calcium dynamics. These initial pathological mechanisms contribute to heart failure, which can subsequently lead to intestinal hypoperfusion and increased gut venous…