NASH triggers cardiometabolic HFpEF in aging mice

Abstract

Both heart failure with preserved ejection fraction (HFpEF) and non-alcoholic fatty liver disease (NAFLD) develop due to metabolic dysregulation, has similar risk factors (e.g., insulin resistance, systemic inflammation) and are unresolved clinical challenges. Therefore, the potential link between the two disease is important to study. We aimed to evaluate whether NASH is an independent factor of cardiac dysfunction and to investigate the age dependent effects of NASH on cardiac function. C57Bl/6 J middle aged (10 months old) and aged mice (24 months old) were fed either control or choline deficient (CDAA) diet for 8 weeks. Before termination, echocardiography was performed. Upon termination, organ samples were isolated for histological and molecular analysis. CDAA diet led to the development of NASH in both age groups, without inducing weight gain, allowing to study the direct effect of NASH on cardiac function. Mice with NASH developed hepatomegaly, fibrosis, and inflammation. Aged animals had increased heart weight. Conventional echocardiography revealed normal systolic function in all cohorts, while increased left ventricular volumes in aged mice. Two-dimensional speckle tracking echocardiography showed subtle systolic and diastolic deterioration in aged mice with NASH. Histologic analyses of cardiac samples showed increased cross-sectional area, pronounced fibrosis and Col1a1 gene expression, and elevated intracardiac CD68⁺ macrophage count with increased Il1b expression. Conventional echocardiography failed to reveal subtle change in myocardial function; however, 2D speckle tracking echocardiography was able to identify diastolic deterioration. NASH had greater impact on aged animals resulting in cardiac hypertrophy, fibrosis, and inflammation.

Keywords: Fatty liver; Inflammation; Liver fibrosis; Metabolic dysfunction; Strain rate analysis.

Fig. 1

CDAA diet induces key hepatic features of NASH. Study design (A). Body and liver weight, (n = 5–10) (B, C). Picrosirius red staining and its macroscopic quantification (n = 5–10). The representative images were capture at × 20 magnification (D). Quantitative real-time PCR of pro-fibrotic and pro-inflammatory genes (n = 4–6) (E, F). CON, control diet; NASH, non-alcoholic steatohepatitis; MID, middle aged. One-way ANOVA followed by Fischer’s LSD post hoc test or Kruskal–Wallis test followed by uncorrected Dunn’s post hoc test, P < 0.05 was considered significant difference, * shows difference between age-matched cohorts, # shows difference between control animals, ϕ shows difference between animals with NASH

Fig. 2

Conventional and two-dimensional speckle tracking echocardiography. Heart weight (n = 5–10) (A). Bar graphs of conventional echocardiographic parameters with representative images of parasternal short axis M-mode (n = 5–10) (B). Bar graphs of two-dimensional speckle tracking echocardiographic parameters with representative images of strain rate analysis (n = 5–10) (C). The dotted lines represent average values of young animals. CON, control diet; NASH, non-alcoholic steatohepatitis; MID, middle aged; LV, left ventricle; LVESV, left ventricular end-systolic volume; LVEDV, left ventricular end-diastolic volume; ESD, end-systolic diameter, EDD, end-diastolic diameter; GCS, global circumferential strain; CSrE, early diastolic strain rate of circumferential fibers. One-way ANOVA followed by Fischer’s LSD post hoc test or Kruskal–Wallis test followed by uncorrected Dunn’s post hoc test, P < 0.05 was considered significant difference, * shows difference between age-matched cohorts, # show difference between control animals, ϕ shows difference between animals with NASH

Fig. 3

Characterization of cardiac morphology in mice with NASH. Lectin histochemistry (n = 5–10). Blue shows nuclei, red shows cardiac endothelial cells, and green shows the cell membrane of cardiomyocytes. Quantification of cross-sectional area and microvascular density (A). Bar graphs of cardiac hypertrophy markers (n = 4–6) and BNP serum level (n = 6–10) (B). Cardiac picrosirius red staining and its macroscopic quantification. Bar graphs of pro-fibrotic genes (n = 5–10) (C). Immunostaining of CD68⁺ macrophages and its quantification (n = 5–10) (D). Gene expression of pro-inflammatory cytokines in the heart and serum IL-1β level (n = 4–8) (E). CON, control diet; NASH, non-alcoholic steatohepatitis; MID, middle aged; BNP, b-type natriuretic peptide. One-way ANOVA followed by Fischer’s LSD post hoc test or Kruskal–Wallis test followed by uncorrected Dunn’s post hoc test, * shows difference between age-matched cohorts, # shows difference between control animals, ϕ shows difference between animals with NASH