Plin5 alleviates myocardial ischaemia/reperfusion injury by reducing oxidative stress through inhibiting the lipolysis of lipid droplets

Abstract

Myocardial ischaemia-reperfusion (I/R) injury is a complex pathophysiological process. Current research has suggested that energy metabolism disorders, of which the abnormal consumption of fatty acids is closely related, compose the main pathological basis for myocardial I/R injury. Lipid droplets (LD) are critical regulators of lipid metabolism by LD-associated proteins. Among the lipid droplet proteins, the perilipin family members regulate lipolysis and lipogenesis through different mechanisms. Plin5, an important perilipin protein, promotes LD generation and lowers fatty acid oxidation, thus protecting the myocardium from lipotoxicity. This study investigated the protective effects of Plin5 in I/R myocardium. Our results indicated that Plin5 deficiency exacerbated the myocardial infarct area, aggravated left ventricular systolic dysfunction, reduced lipid storage, and elevated free fatty acids. Plin5-deficient myocardium exhibited severely damaged mitochondria, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreased superoxide dismutase (SOD) activity. Furthermore, the decreased phosphorylation of PI3K/Akt in Plin5-null cardiomyocytes might contribute to I/R injury aggravation. In conclusion, Plin5, a new regulator of myocardial lipid metabolism, decreases free fatty acid peroxidation by inhibiting the lipolysis of intracellular lipid droplets, thus providing cardioprotection against I/R injury and shedding new light on therapeutic solutions for I/R diseases.

Figure 1. Plin5 deficiency exacerbated the I/R injury of myocardium.

(A) Mice were sacrificed at the end of reperfusion, and the hearts removed and stained with TTC to measure the myocardial infarct area. (B) The infarct size was expressed as a percentage of area at risk (n = 6). (C) LV tissues were retrieved at the end of reperfusion, and paraffin sections were prepared and subjected to the H&E staining (n = 4). Representative H&E staining images are shown. Scale bar = 20 μm. (D) Myocardial cell size was quantified by using the ipwin32 software. The columns and errors bars represent means ± SEM. *P < 0.05; **P < 0.01.

Figure 2. Plin5 deficiency aggravates heart dysfunction following I/R injury.

(A) Echocardiography was performed at the end of reperfusion, and representative M-mode echocardiograms were recorded in all groups. Mice without LAD occlusion served as basal controls (Sham group). (n = 6). (B–E) Cardiac function was examined by echocardiography after I/R surgery. The EF (B) and FS (C) values of Plin5-null mice decreased more significantly than those in wild-type mice after I/R surgery, whereas LV Vol;s (D) and LVID;s (E) also increased significantly in Plin5-null mice after I/R surgery. (n = 6). The columns and errors bars represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. EF, ejection fraction; FS, fractional shortening; LV Vol;s, left ventricular contraction volume; LVID;s, left ventricular internal diameter at end-systole.

Figure 3. Plin5 deficiency enhanced proliferation of mitochondria in myocardium with I/R injury.

(A) Electron microscopy images of mouse myocardium with or without I/R injury (n = 4). Mitochondrial sizes were analysed using Image pro plus 6.0 software in four electron microscopic images of the myocardium from wild-type and Plin5-null mice. Scale bar = 1 μm. (B) Relative mitochondrial DNA (mtDNA) contents (normalised to the single-copy nuclear gene Ndufv1) in the myocardium of wild-type and Plin5-null mice (n = 4). (C) Immunoblotting analysis of the levels of mitochondria markers in the myocardium of mice (n = 4). (D) Quantitative PCR analysis of the mRNA levels of mitochondria markers in the myocardium with or without I/R injury (n = 4). The columns and errors bars represent the means ± SEM. *P < 0.05, **P < 0.01.

Figure 4. Plin5 deficiency reduced lipid storage and elevated the cytosolic fatty acid levels in myocardium with I/R injury.

(A) Oil Red O staining showed that Plin5 deficiency led to reduced cardiac lipid accumulation in mice with either sham or I/R surgery (n = 4). Scale bar = 20 μm. (B–D) Quantitative analysis of serum TG levels (B), myocardial TG contents (C), and FFA contents (D) (n = 4). Serum TG levels were obviously increased after I/R injury, but no difference was observed between wild-type and Plin5-null mice. (C) The amounts of TG storage decreased slightly, whereas the levels of intracellular FFA (D) increased in Plin5-deficient myocardium. The columns and errors bars represent the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. FFA, free fatty acid.

Figure 5. Plin5 deficiency increased the ROS levels in I/R myocardium.

Cardiac levels of ROS (A), MDA (B) and SOD (C) (n = 4). The ROS and MDA levels of the I/R myocardium increased more so than in wild-type mice, whereas the SOD activity decreased in Plin5-null mice. The columns and errors bars represent the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase.

Figure 6. Plin5 played a protective role following ischaemia reperfusion in the myocardium by activating the PI3K/Akt signalling pathway.

The levels of total PI3K, Akt and their phosphorylated forms (p-PI3K, p-Akt) were assessed using Western blot (n = 4). (A) Representative results for western blot analysis. (B,C) Semi-quantitative analysis of the relative expression levels of PI3K and p-PI3K, Akt and p-Akt in each group of mice were normalized against those of β-tubulin and are presented as a ratio between p-PI3K/PI3K (B) and p-Akt/Akt (C). The columns and errors bars represent the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.