TIMP3 interplays with apelin to regulate cardiovascular metabolism in hypercholesterolemic mice

Abstract

Objective: Tissue inhibitor of metalloproteinase 3 (TIMP3) is an extracellular matrix (ECM) bound protein, which has been shown to be downregulated in human subjects and experimental models with cardiometabolic disorders, including type 2 diabetes mellitus, hypertension and atherosclerosis. The aim of this study was to investigate the effects of TIMP3 on cardiac energy homeostasis during increased metabolic stress conditions.

Methods: ApoE(-/-)TIMP3(-/-) and ApoE(-/-) mice on a C57BL/6 background were subjected to telemetric ECG analysis and experimental myocardial infarction as models of cardiac stress induction. We used Western blot, qRT-PCR, histology, metabolomics, RNA-sequencing and in vivo phenotypical analysis to investigate the molecular mechanisms of altered cardiac energy metabolism.

Results: ApoE(-/-)TIMP3(-/-) revealed decreased lifespan. Telemetric ECG analysis showed increased arrhythmic episodes, and experimental myocardial infarction by left anterior descending artery (LAD) ligation resulted in increased peri-operative mortality together with increased scar formation, ventricular dilatation and a reduction of cardiac function after 4 weeks in the few survivors. Hearts of ApoE(-/-)TIMP3(-/-) exhibited accumulation of neutral lipids when fed a chow diet, which was exacerbated by a high fat, high cholesterol diet. Metabolomics analysis revealed an increase in circulating markers of oxidative stress with a reduction in long chain fatty acids. Using whole heart mRNA sequencing, we identified apelin as a putative modulator of these metabolic defects. Apelin is a regulator of fatty acid oxidation, and we found a reduction in the levels of enzymes involved in fatty acid oxidation in the left ventricle of ApoE(-/-)TIMP3(-/-) mice. Injection of apelin restored the hitherto identified metabolic defects of lipid oxidation.

Conclusion: TIMP3 regulates lipid metabolism as well as oxidative stress response via apelin. These findings therefore suggest that TIMP3 maintains metabolic flexibility in the heart, particularly during episodes of increased cardiac stress.

Keywords: Apelin; Arrhythmia; Heart; Lipid metabolism; Oxidative stress; TIMP3.

Graphical abstract

Figure 1

ApoE^−/−TIMP3^−/− animals reveal decreased lifespan, arrhythmias and susceptibility to increased cardiac stress. A) Combined retro- and prospective survival analysis in a cohort of 64 male and female mice (n = 32 for ApoE^−/−TIMP3^−/− M/F = 12/20 and n = 32 for ApoE^−/− M/F = 12/20) over a time period of 12 months (p = 0.0002, Cox Test). B) Telemetry 24 h ECG in 32-weeks-old ApoE^−/−TIMP3^−/− and ApoE^−/− mice. We observed a higher amount of arrhythmic episodes in ApoE^−/−TIMP3^−/− as well as sudden deaths in 4 out of 6 male mice during the 7-day registration (p = 0.06, Fisher's exact test). C) Increased peri-operative mortality during experimental myocardial infarction by LAD ligation in ApoE^−/−TIMP3^−/− (13/19) compared to ApoE^−/− (3/13) (***p < 0.001 by Chi-square test). D) Representative photomicrographs of Masson-Trichrome stained hearts 4 weeks post-LAD ligation showing increased infarction size as confirmed by morphometric quantification of percent total LV circumference. Data are mean ± SEM (*p < 0.05 by Student t-test, n = 3–4 per group). E) Reduced systolic (dp/dtmax) and diastolic (dp/dtmin) heart function in ApoE^−/−TIMP3^−/− mice 4 weeks after LAD ligation as measured by Millar Catheter. Data are mean ± SEM (*p < 0.05 by Student t-test, n = 4–5 per group).

Figure 2

ApoE^−/−TIMP3^−/− animals show increased deposition of neutral lipids in the heart on normal chow and western diet. A) Representative sections of myocardium of 6–8 month old ApoE^−/−TIMP3^−/− and ApoE^−/− mice on normal chow diet stained with ORO. B) Representative sections and quantification of myocardium from 6 to 8 month old mice fed a Western diet for 8 weeks stained with ORO showing increased lipid deposition in hearts of ApoE^−/−TIMP3^−/− mice. Data are mean ± SEM (*p < 0.05, Student t-test, n = 4 per group). Magnification further displays globular intracellular lipid accumulation and a punctate pattern reminiscent of extracellular lipid deposition in ApoE^−/−TIMP3^−/− animals.

Figure 3

Metabolic characterization of ApoE^−/−TIMP3^−/− mice reveals impaired lipid oxidation. A) Analysis of fasting glucose levels reveals significant reduction in ApoE^−/−TIMP3^−/− mice (***p < 0.001, Student's t test, n = 14 for ApoE^−/− and n = 6 for ApoE^−/−TIMP3^−/− per group). B) Intraperitoneal Glucose Tolerance test shows no significant differences between the two groups (n = 14 for ApoE^−/− and n = 6 for ApoE^−/−TIMP3^−/− per group). C) Mild insulin resistance of ApoE^−/−TIMP3^−/− mice at the Insulin Tolerance Test (*p < 0.05, Student's t test, n = n = 4 for ApoE^−/− and n = 5 for ApoE^−/−TIMP3^−/− per group). D) Indirect calorimetry of ApoE^−/−TIMP3^−/− compared to ApoE^−/− mice during 12 consecutive hours of fasting conditions. Data show the interval from 8 to 12 h of fasting. RER was significantly increased and lipid oxidation significantly reduced in ApoE^−/−TIMP3^−/− mice (*p < 0.05, Student's t test, n = 3–4).

Figure 4

ApoE^−/−TIMP3^−/− mice have impairment of fatty acid metabolism. A) Metabolomics analysis of the serum shows a decrease in long-chain monounsaturated FFAs, medium-chain acyl-carnitines and medium-chain fatty acid markers in ApoE^−/−TIMP3^−/− animals (*p ≤ 0.05 and **p < 0.01, Student's t test, n = 6 per group). B) Regulation of MUFA/SUFA balance in mammalian cell lipids by SCD1.

Figure 5

ApoE^−/−TIMP3^−/− mice have loss of defense against oxidative stress. A) Metabolomics analysis of the serum of ApoE^−/−TIMP3^−/− vs. ApoE^−/− animals shows a depletion of dietary antioxidants suggesting a loss of dietary antioxidant and anti-inflammatory chemicals (*p ≤ 0.05, **p < 0.01 and ***p < 0.001, Student's t test, n = 6 per group). B) Glutathione regulation pathway in mammalian cells.

Figure 6

ApoE^−/−TIMP3^−/− mice have disturbed sphingolipid metabolism. A) Altered sphingolipid metabolism in ApoE^−/−TIMP3^−/− mice revealed by metabolomics analysis of the serum (*p ≤ 0.05, **p < 0.01 and ***p < 0.001, Student's t test, n = 6 per group). B) Sphingolipid metabolism in mammalian cells that can be in part connected to increased stearoylcarnitine and its derivatives.

Figure 7

Loss of TIMP3 leads to disrupted apelin secretion from heart while supplementation can correct some of the metabolic abnormalities in the heart. A) Apelin expression by qPCR in heart, muscle, adipose tissue and liver reveals a difference between ApoE^−/−TIMP3^−/− and ApoE^−/− animals only in the heart (*p < 0.05, one-way ANOVA, n = 5 per group). B) Circulating apelin levels measured by ELISA were also found to be reduced in the ApoE^−/−TIMP3^−/− (*p < 0.05, Student's t test, n = 4 per group). C) Single dose of intraperitoneally injected apelin restores impaired lipid oxidation of ApoE^−/−TIMP3^−/− mice during the prolonged fasting state (interval of 8–12 h) measured by indirect calorimetry. Data are mean ± SEM (****p < 0.001, one-way ANOVA, n = 5–6 per group).