Atorvastatin impairs liver mitochondrial function in obese Göttingen Minipigs but heart and skeletal muscle are not affected

Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.


Supplementary figures
At study start, minipigs were of equal BW. Minipigs fed a high fat and high cholesterol diet (HFD, n=7) and minipigs fed a high fat diet and treated with atorvastatin (HFD+ATO, n=7) increased their BW considerably compared to minipigs fed a standard diet (CON,n=6). At termination of the study, HFD and HFD+ATO had significantly higher BW than CON (P=0.0034). Data are means, n=6-7 in each group. The figure is modified from [1].

Substrate inhibitor titration protocols
The mitochondrial respiratory capacity was compared between the three groups (CON, HFD and HFD+ATO) in liver samples and in permeabilized muscle fibers from heart and skeletal muscle. Titration protocols were used to stimulate state 3 respiration (respiration with adenylates) with substrates supporting electron transport through complex I, complex II and convergent electron flow through complex I+II.
Moreover, uncoupled respiration and complex IV stimulated respiration were included in the protocols. In heart and skeletal muscle, state 3 respiration supported with the fatty acid substrate palmitoyl carnitine delivering electrons through the electron transfer flavoprotein (ETF) was also determined.
High resolution respirometry and high resolution fluorometry were done with the following respiration buffers: Three substrate and inhibitor protocols (Protocols A, B and C) were used in the study.
No cytochrome c effect was seen in any of the protocols.
Protocol A. Evaluating complex I and complex I+II linked respiratory capacity in liver and H 2 O 2 release in heart and skeletal muscle (Fig S3a) Protocol A was applied to liver and measured in respiration medium Mir05.
In heart and skeletal muscle, H 2 O 2 release was measured simultaneously with HRR in buffer Z with the addition of 25 µM blebbistatin [5] (Fig S3b).
In the permeabilized fibers from these tissues the following was added: Amplex Red  Protocol B: evaluating complex I; complex I+II linked respiratory capacity and COX activity Protocol B was applied to permeabilized skeletal muscle, heart muscle and to liver using Mir05 as respiration medium: State 2 respiration (absence of adenylates) was assessed with malate (2 mM) and glutamate (10 mM) followed by state 3 respiration (5 mM ADP) and then cytochrome c (10 µM). Simultaneous electron input into complex I + II (maximal OXPHOS capacity) was assessed with succinate (10 mM). Then rotenone (0.5 µM) was added to inhibit complex I, and atractyloside (0,05 mM) to inhibit mitochondrial ADP transport. Then antimycin A (2.5 µM) was added to inhibit complex III (ROX).
Finally ascorbate and N,N,N´,N´-Tetramethyl-p-phenylenediamine (TMPD) was added (2 mM and 0.5 mM, respectively) to activate complex IV (COX). Figure S4 shows a representative trace of oxygraphic measurements using protocol B in heart  This was followed by the addition of palmitoyl carnitine (0.075 mM) and thereafter ADP (4.5 mM) to obtain maximal coupled respiration with convergent electron input to complex I and electron transfer flavoprotein (ETF) Integrity of the outer mitochondrial membrane was tested by adding cytochrome c (10 µM). Representative trace of protocol C applied in heart muscle is shown in Figure S5.