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Comparative Study
. 2019 Oct;6(5):1027-1040.
doi: 10.1002/ehf2.12509. Epub 2019 Sep 14.

Impact of statins on cellular respiration and de-differentiation of myofibroblasts in human failing hearts

Larisa Emelyanova  1 , Amar Sra  1 , Eric G Schmuck  2 , Amish N Raval  2 , Francis X Downey  3 , Arshad Jahangir  3   4 , Farhan Rizvi  1 , Gracious R Ross  1
Affiliations
Comparative Study

Impact of statins on cellular respiration and de-differentiation of myofibroblasts in human failing hearts

Larisa Emelyanova et al. ESC Heart Fail. 2019 Oct.

Abstract

Aims: Fibroblast to myofibroblast trans-differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de-differentiation could mitigate CF-related pathologies. We determined whether 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors-statins, commonly prescribed to patients at risk of heart failure (HF)-can de-differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients.

Methods and results: Either in vitro statin treatment of differentiated myofibroblasts (n = 3-6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4-12). In vitro, hVFs were differentiated by transforming growth factor-β1 (TGF-β1) for 72 h (TGF-72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by α-smooth muscle actin (α-SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P < 0.05, considered significant. In vitro, statins concentration-dependently de-differentiated the myofibroblasts. The respective half-maximal effective concentrations were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 μmol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin). Mevalonic acid (300 μmol/L), the reduced product of HMG-CoA, prevented the statin-induced de-differentiation (α-SMA expression: 31.4 ± 10% vs. 58.6 ± 12%). Geranylgeranyl pyrophosphate (GGPP, 20 μmol/L), a cholesterol synthesis-independent HMG-CoA reductase pathway intermediate, completely prevented the statin-induced de-differentiation (α-SMA/GAPDH ratios: 0.89 ± 0.05 [TGF-72 h + 72 h], 0.63 ± 0.02 [TGF-72 h + simvastatin], and 1.2 ± 0.08 [TGF-72 h + simvastatin + GGPP]). Cellular metabolism involvement was observed when co-incubation of simvastatin (200 nmol/L) with glibenclamide (10 μmol/L), a KATP channel inhibitor, attenuated the simvastatin-induced de-differentiation (0.84 ± 0.05). Direct inhibition of mitochondrial respiration by oligomycin (1 ng/mL) also produced a de-differentiation effect (0.33 ± 0.02). OCR (pmol O2 /min/μg protein) was significantly decreased in the simvastatin-treated hVFs, including basal (P = 0.002), ATP-linked (P = 0.01), proton leak-linked (P = 0.01), and maximal (P < 0.001). The OCR inhibition was prevented by GGPP (basal OCR [P = 0.02], spare capacity OCR [P = 0.008], and maximal OCR [P = 0.003]). Congruently, hVFs from HF showed an increased population of myofibroblasts while HF + statin group showed significantly reduced cellular respiration (basal OCR [P = 0.021], ATP-linked OCR [P = 0.047], maximal OCR [P = 0.02], and spare capacity OCR [P = 0.025]) and myofibroblast differentiation (α-SMA/GAPDH: 1 ± 0.19 vs. 0.23 ± 0.06, P = 0.01).

Conclusions: This study demonstrates the de-differentiating effect of statins, the underlying GGPP sensitivity, reduced OCR with potential activation of KATP channels, and their impact on the differentiation magnitude of hVFs in HF patients. This novel pleiotropic effect of statins may be exploited to reduce excessive CF in patients at risk of HF.

Keywords: Cardiac fibrosis; De-differentiation; Geranylgeranyl pyrophosphate; Mitochondria; Statins.

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Conflict of interest statement

E.G.S. and A.N.R. have a financial interest in Cellular Logistics, Inc. No other authors have any conflicts of interest to report.

Figures

Figure 1
Figure 1
Concentration‐dependent de‐differentiation effect of statins in human ventricular myofibroblasts. Representative immunoblots showing the effect of in vitro treatment for 72 h with (A) atorvastatin (10 nmol/L to 3 μmol/L), (B) rosuvastatin (30 nmol/L to 10 μmol/L), or (C) simvastatin (1 to 500 nmol/L) on differentiated myofibroblasts, as determined by the expression of α‐SMA (α‐smooth muscle actin), a marker of differentiated myofibroblasts. In the right panel, respective non‐linear regression curves show the concentration‐dependent effects of corresponding statin [(D) atorvastatin, (E) rosuvastatin, and (F) simvastatin] on α‐SMA/α/β‐tubulin ratio, normalized to % maximal differentiation. For concentration for half‐maximal inhibition (IC50) analysis of atorvastatin and rosuvastatin, the highest concentration used was considered to have reached a near maximum possible effect, and any further increase in concentration was assumed to have ±5% change from the measured maximum effect (asymptote). The respective IC50 values were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 μmol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin); n = 3 per concentration; four‐parameter logistic fit. Data are mean ± SEM.
Figure 2
Figure 2
Mechanisms of statin‐induced de‐differentiation. (A) Representative immunoblot showing the effect of a single concentration of in vitro statin (simvastatin 200 nmol/L for 72 h) on already‐differentiated myofibroblasts (Diff) isolated from HF patients, with significant decrease (64%) in α‐SMA expression (Diff + Simva). (B) Representative immunocytochemistry showed a higher population of α‐SMA+ (red) cells in the differentiated (TGF) cells compared with control or in vitro treatment of simvastatin (TGF + Simva) (green: vimentin). (C) Representative immunoblots from in vitro differentiation. Simvastatin, applied for 72 h following TGF‐β1‐induced differentiation (5 ng/mL for initial 72 h) in vitro (simulating in vivo differentiated hVFs from HF patients), reproduced similar de‐differentiation, as expression of both α‐SMA and COL III were significantly decreased by 87% and 142%, respectively. Mevalonic acid (MVA, 300 μmol/L) prevented the statin‐induced de‐differentiation, as expression of both α‐SMA (31.4 ± 10% vs. 58.6 ± 12%) and COL III (40 ± 3% vs. 37 ± 27%) did not decrease when simvastatin was co‐administered with MVA. (D) Representative immunoblots depict complete prevention of simvastatin‐induced decreased α‐SMA expression by either GGPP (20 μmol/L) or glibenclamide (10 μmol/L). The effect of statin on the expression of a‐SMA is mimicked by oligomycin (1 ng/mL) in the same time frame. Individual data points column graph displays respective mean ratio of α‐SMA to GAPDH densities. (E) Representative immunoblot depicts significant reduction of atorvastatin‐induced decreased α‐SMA expression by glibenclamide (10 μmol/L). Individual data points column graph displays respective mean ratio of α‐SMA to α/β‐tubulin densities. * P < 0.05 vs. control; *** P < 0.001 vs. control; # P < 0.05 vs. TGF‐72 h + 72 h; * P < 0.05 vs. TGF‐72 h + Ator; considered as significant, n = 3 each; one‐way ANOVA. TGF‐72 h + 72 h = TGF‐β1 (5 ng/mL) treatment for 72 h to differentiate into myofibroblasts, and subsequent 72 h culture was without TGF‐β1. Data are mean ± SEM. Ator, atorvastatin; GGPP, geranylgeranyl pyrophosphate; Gliben, glibenclamide; Oligom, oligomycin; Simva, simvastatin.
Figure 3
Figure 3
Effect of statins on cellular respiration. (A) Graphical representation of pooled data of cellular respiration (OCR) from Seahorse assays of hVFs in the control, differentiated (TGF), the simvastatin‐treated (TGF + Sim), and GGPP/simvastatin co‐administration (TGF + Sim + GGPP) groups. (B) Bar graphs depict that simvastatin reduced the ATP OCR, the proton leak‐linked OCR, and the maximal OCR without any significant effect on spare capacity OCR or non‐mitochondrial OCR. The inhibitory effect of simvastatin on the mitochondrial OCR was reversed by GGPP. (C) Graphical representation of pooled data of extracellular acidification rate (ECAR) from Seahorse assays of hVFs in all the three groups. (D) Simvastatin treatment of the differentiated hVFs (TGF + Sim) in vitro reduced ECAR, after addition of FCCP or AA. GGPP reversed ECAR to a similar level as control differentiated hVFs upon addition of FCCP and AA (TGF + Sim + GGPP); n = 6. Data are mean ± SD. (E) Bar graph depicting that both lipophilic atorvastatin (100 and 300 nmol/L) and hydrophilic rosuvastatin (300 nmol/L and 1 μmol/L) increased the ADP/ATP ratio. (F) Enzymatic activities of mitochondrial OXPHOS complexes in lysates from differentiated (TGF) and simvastatin‐treated hVFs showed significant reduction in Complex V activity by simvastatin (TGF + Simva). n = 3. Data are mean ± SEM. Atorva, atorvastatin; GGPP, geranylgeranyl pyrophosphate; Simva, simvastatin; Rosuva, rosuvastatin.
Figure 4
Figure 4
Effect of either GGPP synthase or Rho kinase inhibition on hVF differentiation and cellular respiration. (A) Representative immunoblot showing the in vitro effect of either GGPP synthase inhibitor digerenyl bisphonate (DGBP, 30 μmol/L) or ROCK inhibitor Y27632 (10 μmol/L) for 72 h on already‐differentiated myofibroblasts (Diff), and corresponding bar graph shows significant de‐differentiation by DGBP without any effect by Y27632. (B) Graphical representation of pooled data of cellular respiration (OCR) from seahorse assays of differentiated hVFs (TGF) and the GGPP synthase‐inhibited (DGBP) groups. Both basal and ATP‐linked OCRs were significantly inhibited by DGBP. (C) Graphical representation of pooled data of OCR showing no significant effect by GGPP or Y27632 on both basal and ATP‐linked respiration. Data are mean ± SD. * P < 0.05; one‐way ANOVA.
Figure 5
Figure 5
Time‐course effect of statins in human ventricular myofibroblasts de‐differentiation and cellular respiration. (A) Representative immunoblots showing the time‐dependent effect of in vitro treatment of simvastatin (200 nmol/L), and corresponding bar graph depicts significant de‐differentiation from 24 h onwards. (B) Graphical representation of pooled data of time‐dependent effect of simvastatin (200 nmol/L) on OCR of differentiated hVFs. Respective bar graphs depict significant reduction in both basal and ATP‐linked OCRs from 18 h onwards. Data mean ± SD. * P < 0.05; one‐way ANOVA.
Figure 6
Figure 6
Effect of statin therapy on differentiation of ventricular fibroblasts in heart failure patients. (A) Representative immunoblot of human ventricular fibroblasts (hVFs) isolated from HF patients displayed a high α‐SMA expression while hVFs from HF + statin showed significantly reduced α‐SMA expression. (B) The individual data points column graph displays each sample value of α‐SMA/GAPDH density ratio with lines depicting mean ± SD; n = 4. (C) Representative immunocytochemistry showed a higher population of α‐SMA+ (green) cells in the HF group vs. HF + statin group (n = 3). (D) Immunoblot shows the expression of SPRY1, a negative regulator of fibrosis, in lysates of hVFs isolated from failing heart patients. (E) The individual data points column graph displays each sample value of SPRY1/GAPDH density ratio with lines depicting mean ± SD, with lower expression in the HF group and significantly higher expression in HF + statin therapy; n = 4. * P < 0.05, ** P < 0.01 considered significant vs. HF; unpaired t‐test.
Figure 7
Figure 7
Reduced mitochondrial respiration in hVFs from HF patients under statin therapy. (A) Graphical representation of pooled OCR data from Seahorse assay of hVFs isolated from failing heart patients. (B) Bar graphs depicting the mean OCR for each group at basal, following oligomycin, FCCP, and antimycin A. The basal, ATP‐linked, maximal, and spare capacity OCRs were significantly reduced in hVFs from HF + statin vs. HF group, without significant effect on the Olig‐insensitive proton leak‐linked‐related OCR or non‐mitochondrial OCR. (C) Graphical representation of pooled data of extracellular acidification rate (ECAR) from Seahorse assays of hVFs isolated from failing heart patients. (D) Bar graph shows ECAR data (mean ± SD) with no significant difference in ECAR between the two groups, following addition of either oligomycin, FCCP, or antimycin A; n = 6; analysed by unpaired t‐test. Data are mean ± SD. (E) Representative immunoblot with total OXPHOS human antibody coScktail and corresponding bar graph (F) showed significantly reduced Complex V subunit following statin therapy; n = 4, analysed by one‐way ANOVA.
Figure 8
Figure 8
Schematic model depicting the potential relationship between statin therapy, mitochondrial energetics, and de‐differentiation of hVFs. Cardiac fibrosis with persistent myofibroblasts is one of the major underlying factors in heart failure (HF) progression. Statin therapy significantly reduced the high level of differentiated myofibroblasts in HF patients. The underlying mechanisms of statin‐induced de‐differentiation of myofibroblasts involve GGPP‐dependent signalling mechanisms, lowered cellular respiration, and KATP channels. GGPP, geranylgeranyl pyrophosphate; KATP, adenosine 5′‐triphosphate‐sensitive potassium channels.
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