Mitochondrial protection by simvastatin against angiotensin II-mediated heart failure

Abstract

Background and purpose: Mitochondrial dysfunction plays a role in the progression of cardiovascular diseases including heart failure. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins), which inhibit ROS synthesis, show cardioprotective effects in chronic heart failure. However, the beneficial role of statins in mitochondrial protection in heart failure remains unclear.

Experimental approach: Rats were treated with angiotensin II (1.5 mg·kg^-1 ·day^-1 ) or co-administered simvastatin (oral, 10 mg·kg^-1 ) for 14 days; and then administration was stopped for the following 14 days. Cardiac structure/function was examined by wheat germ agglutinin staining and echocardiography. Mitochondrial morphology and the numbers of lipid droplets, lysosomes, autophagosomes, and mitophagosomes were determined by transmission electron microscopy. Human cardiomyocytes were stimulated, and intracellular ROS and mitochondrial membrane potential (ΔΨ_m ) changes were measured by flow cytometry and JC-1 staining, respectively. Autophagy and mitophagy-related and mitochondria-regulated apoptotic proteins were identified by immunohistochemistry and western blotting.

Key results: Simvastatin significantly reduced ROS production and attenuated the disruption of ΔΨ_m . Simvastatin induced the accumulation of lipid droplets to provide energy for maintaining mitochondrial function, promoted autophagy and mitophagy, and inhibited mitochondria-mediated apoptosis. These findings suggest that mitochondrial protection mediated by simvastatin plays a therapeutic role in heart failure prevention by modulating antioxidant status and promoting energy supplies for autophagy and mitophagy to inhibit mitochondrial damage and cardiomyocyte apoptosis.

Conclusion and implications: Mitochondria play a key role in mediating heart failure progression. Simvastatin attenuated heart failure, induced by angiotensin II, via mitochondrial protection and might provide a new therapy to prevent heart failure.

Figure 1

Simvastatin attenuates Ang II‐induced cardiac hypertrophy in vivo. Male Sprague–Dawley rats were treated with Angiotensin II (Ang II, 1.5 mg·kg⁻¹·day⁻¹) or Ang II + simvastatin (SIM, oral, 10 mg·kg⁻¹) for 28 days. Cardiac cachexia was determined by (a) body weights (n = 6 per group) and (b) gastrocnemius muscle weight (n = 6 per group). Left ventricle hypertrophy was determined by (c) H&E staining (n = 6 per group) and (d) WGA staining (n = 6 per group). (d) Relative folds were determined by comparing with the control (Con) group. *P < .05, significantly different from Con

Figure 2

Simvastatin suppresses Ang II‐induced heart failure in vivo. Cardiac function was examined by M‐mode echocardiography: (a) ejection fraction (EF; n = 6 per group), (b) fractional shortening (FS; n = 6 per group), (c) left ventricular mass (LVM; n = 6 per group), (d) left ventricular mass contractility (LVMc; n = 6 per group), (e) left ventricular internal diameter in diastole (LVIDd; n = 6 per group), (f) left ventricular internal diameter in systole (LVIDs; n = 6 per group), (g) heart weight normalized to body weight (n = 6 per group), (h) lung weight normalized to body weight (n = 6 per group), and (i) gastrocnemius muscle (GM) weight normalized to body weight (n = 6 per group). *P < .05, significantly different from control (Con)

Figure 3

Simvastatin suppresses Ang II‐induced cardiac mitochondrial damage in vivo. (a) Scoring criteria and examples of Ang II‐damaged mitochondria (n = 6 per group). To determine whether simvastatin has mitochondrial protective effects in Ang II‐damaged mitochondria, the (b) morphological appearance (n = 6 per group), (c) mitochondrial length (n = 6 per group), (d) number of swollen mitochondria (n = 6 per group), and (e) number of mitochondria with vacuolization (n = 6 per group) were measured by FE‐TEM analysis. *P < .05, significantly different from control (Con)

Figure 4

Simvastatin inhibits Ang II‐induced ROS, ΔΨ _m disruption, and mitochondrial‐mediated apoptosis. Cultured HCMs were treated with Ang II (10 μM for 1.5 hr) or Ang II + simvastatin (pretreatment, 0.5 μM for 2 hr), and then the (a) mitochondrial superoxide (MitoSOX Red; n = 5) and (b) intracellular ROS (DCFH‐DA) production were determined by flow cytometry (n = 5). Data are representative of three independent experiments. Cultured HCMs were treated with Ang II (10 μM for 2 hr) or Ang II + simvastatin (pretreatment, 0.5 μM for 2 hr), and then the HCM ΔΨ _m was determined by measuring changes in JC‐1‐derived fluorescence from red (high potential, J‐aggregrates) to green (low potential, monomeric) using confocal microscopy. Data are representative of three independent experiments, and (c) the scale bar in each image is 20 μm (n = 5). Cultured HCMs were treated with Ang II (10 μM for 24 hr) or Ang II + simvastatin (pretreatment, 0.5 μM for 24 hr), and then the (d) mitochondrial outer membrane protein: Bcl‐2 (green; nucleus, blue), mitochondrial intermembrane/intercristae spaces protein: cytochrome c (cyto c; cleavage from, red; nucleus, blue), and pro‐apoptotic protein caspase‐3 (red; nucleus, blue) expression were measured by confocal microscopy (n = 5). In vivo, simvastatin attenuation of Ang II‐induced apoptosis was confirmed by (e) TUNEL staining (green; nucleus, blue; n = 6 per group), (f) immunohistochemistry, and (g) western blot analysis (n = 6 per group). (g) Relative folds were determined by comparing with the control (Con) group. Qualitative data shown are representative of three independent experiments. *P < .05, significantly different from Con

Figure 5

Simvastatin regulates LDs and lysosome levels in myocardial tissue. To explore the simvastatin increased LD formation in myocardial tissue, (a) Oil Red O staining (n = 6 per group) and FE‐TEM were used to examine and quantify (b) LD content (n = 6 per group). (a) Relative fold‐changes were determined by comparing with the control (Con) group. Red arrows indicate LDs, and (c) lysosome (L) numbers and distribution are identified and quantified by FE‐TEM analysis (n = 6 per group). Red arrows indicate lysosomes in part (c). *P < .05, significantly different from Con

Figure 6

Simvastatin promotes mitophagy against Ang II‐induced mitochondrial damage in vivo. To explore mechanisms of protection by simvastatin, (a) autophagosomes (red arrows) and (c) mitophagosomes (red arrows) were identified and quantified by FE‐TEM analysis (n = 6 per group). Representative images are shown in (a) and (c), and quantitative plots are shown in (b) and (d); n = 6 per group. *P < .05 versus control (Con). Expressions of mitophagy‐associated proteins LC3‐I/II, p62, PINK1, and Parkin were confirmed by (e) immunohistochemistry (n = 6 per group) and (f) western blot analysis (n = 6 per group). *P < .05, significantly different from control

Figure 7

Summary scheme of the mitochondrial protection mechanism of simvastatin in Ang II‐induced HF. Simvastatin could reduce ROS generation, regulate LDs and lysosome levels to provide energy to maintain ΔΨ _m, regulate mitochondrial quality control to promote mitophagy, and prevent mitochondrial‐regulated apoptosis