Myocardial Structural and Biological Anomalies Induced by High Fat Diet in Psammomys obesus Gerbils

Abstract

Background: Psammomys obesus gerbils are particularly prone to develop diabetes and obesity after brief period of abundant food intake. A hypercaloric high fat diet has been shown to affect cardiac function. Here, we sought to determine whether a short period of high fat feeding might alter myocardial structure and expression of calcium handling proteins in this particular strain of gerbils.

Methods: Twenty Psammomys obesus gerbils were randomly assigned to receive a normal plant diet (controls) or a high fat diet. At baseline and 16-week later, body weight, plasma biochemical parameters (including lipid and carbohydrate levels) were evaluated. Myocardial samples were collected for pathobiological evaluation.

Results: Sixteen-week high fat dieting resulted in body weight gain and hyperlipidemia, while levels of carbohydrates remained unchanged. At myocardial level, high fat diet induced structural disorganization, including cardiomyocyte hypertrophy, lipid accumulation, interstitial and perivascular fibrosis and increased number of infiltrating neutrophils. Myocardial expressions of pro-apoptotic Bax-to-Bcl-2 ratio, pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α], intercellular (ICAM1) and vascular adhesion molecules (VCAM1) increased, while gene encoding cardiac muscle protein, the alpha myosin heavy polypeptide (MYH6), was downregulated. Myocardial expressions of sarco(endo)plasmic calcium-ATPase (SERCA2) and voltage-dependent calcium channel (Cacna1c) decreased, while protein kinase A (PKA) and calcium-calmodulin-dependent protein kinase (CaMK2D) expressions increased. Myocardial expressions of ryanodine receptor, phospholamban and sodium/calcium exchanger (Slc8a1) did not change.

Conclusions: We conclude that a relative short period of high fat diet in Psammomys obesus results in severe alterations of cardiac structure, activation of inflammatory and apoptotic processes, and altered expression of calcium-cycling determinants.

Fig 1. Representative haematoxylin and eosin-stained myocardial sections from Psammomys obesus gerbils fed with normal diet (A) or high fat dieting (B, C, D, E) during 16 weeks.

Myocardial sections were obtained at 400-fold (A, B, C, D; Scale bars: 50μm) and 1000-fold (E; Scale bar: 20μm) magnification. Cardiomyocyte hypertrophy (assessed by cardiomyocyte area in square micrometer) was exacerbated in the heart from Psammomys obesus gerbils fed with high fat diet (n = 10; black bars) compared to Psammomys obesus gerbils fed with normal diet (n = 10; white bars) during 16 weeks (F). Values are expressed as mean ± SEM. **0.001<P<0.01 high fat versus normal diet fed Psammomys obesus gerbils.

Fig 2. Representative Masson Trichrome stained myocardial sections from Psammomys obesus gerbils fed with normal diet (A) or high fat diet (B, C, D, E) during 16 weeks.

Trichrome Masson staining was performed to detect fibrotic areas (in green; indicated by arrows). Sections were obtained at 400-fold. Scale bars: 50μm. Relative myocardial gene expressions of pro-alpha 1 chains of collagen type I (Col1A1) and 3 (Col3A1) in 16-week normal (n = 10; white bars) and high fat (n = 10; black bars) diet fed Psammomys obesus gerbils (F). Values are expressed as mean ± SEM. *0.01<P<0.001 high fat versus normal diet fed Psammomys obesus gerbils.

Fig 3. Characterization of inflammatory cells infiltrating the myocardium in 16-week high fat diet-fed Psammomys obesus gerbils.

Neutrophil [myeloperoxidase (MPO)-positive stained cells] immunostaining in myocardial sections from Psammomys obesus gerbils fed with normal (A) or high fat diet (B) during 16 weeks. Arrows indicate MPO-positive cells. Scale bars: 50 μm. The number of extravascular MPO-positive cells was determined in myocardial sections from 16-week normal diet (n = 10; white bars) and high fat (n = 10; black bars) diet fed Psammomys obesus gerbils (C). Values are expressed as mean ± SEM. **0.001<P<0.01 high fat versus normal diet fed Psammomys obesus gerbils.

Fig 4. Myocardial relative expression of genes implicated in the activation of inflammatory processes (A, B), including the interleukin-1beta (IL-1β), the interleukin-6 (IL-6), the tumor necrosis factor-alpha (TNF-α), the intercellular adhesion molecule 1 (ICAM1) and the vascular cell adhesion molecule 1 (VCAM1); of apoptotic pathways (C), including the pro-apoptotic Bax and the anti-apoptotic Bcl-2 mitochondrial members and the resulting pro-apoptotic Bax/Bcl-2 ratio; and in the tissue response to hypoxia exposure (D), including the hypoxia-inducible factor-alpha (HIF-1α) in 16-week normal (n = 10; white bars) versus high fat (n = 10; black columns) diet fed Psammomys obesus gerbils.

Values are expressed as mean ± SEM. * 0.01versus normal diet-fed Psammomys obesus gerbils.

Fig 5. Myocardial relative expressions of genes implicated in calcium (Ca²⁺) handling implicated in cardiac contraction/relaxation, including the sarco/endoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2), the type 2 ryanodine receptor (RYR2), the voltage-dependent L-type Ca²⁺ channel alpha1c subunit (Cacna1c), the solute carrier family 8 (Na⁺/Ca²⁺ exchanger) member 1 (Slc8A1), the Ca²⁺/calmodulin-dependent protein kinase 2 δ (CaMK2D) and the myosin heavy chain isoform 6 (MYH6 or α-MHC) in 16-week normal (n = 10; white bars) and high fat (n = 10; black bars) diet fed Psammomys obesus gerbils (A).

Myocardial expressions of proteins regulating sarco/endoplamic calcium (Ca²⁺) handling implicated in cardiac contraction/relaxation cycle, including the sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2; B), phospholamban (PLN) pentamer (P) and monomer (M) (C) and PKA catalytic subunits (C-α; C) protein contents in 16-week high fat (n = 10; black bars) versus normal (n = 10; white bars) diet fed animals. Values are expressed as mean ± SEM. * 0.01<P<0.05; ** 0.001<P<0.01 high fat versus normal diet fed Psammomys obesus gerbils.