Molecular Signatures of Beneficial Class Effects of Statins on Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes
Statins prevent cardiac diseases via inhibition of cholesterol biosynthesis and exert pleiotropic effects on the cardiovascular system.1 Statins may also act as antihypertrophic and antiapoptotic agents to prevent cardiomyocyte injury.
Statins prevent cardiac diseases via inhibition of cholesterol biosynthesis and exert pleiotropic effects on the cardiovascular system.1 Statins may also act as antihypertrophic and antiapoptotic agents to prevent cardiomyocyte injury. However, the effects of clinically relevant concentrations of statins (ie, serum peak concentration) on cardiomyocytes remain largely unknown. In the current study, we investigate the class effects of atorvastatin, lovastatin, simvastatin, and fluvastatin applied at their respective serum peak concentration, on the transcriptome and functional properties of human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs).2
Human iPSCs from 2 male (Lines 1 and 2) and female (Lines 3 and 4) control individuals were differentiated to iPSC-CMs (Figure A) with higher than 80% efficiency, as measured by expression of cardiac troponin (data not shown). Statins mediated no effect on the viability (2 and 7 days) and contractile properties of iPSC-CMs (7 days) when tested in a dose-dependent manner (data not shown). To examine the transcriptional effects of statins, we performed comprehensive RNA sequencing analysis of each iPSC-CM line after treatment with the serum peak concentration of each statin, corresponding to the clinical dosage of 40 mg. We focused on significantly differentially expressed genes compared with vehicle-treated cells (false discovery rate adjusted P≤0.05). We found that fluvastatin (F1–F4) mediated the most potent effects on the iPSC-CM transcriptome, followed by atorvastatin (A1–A4), simvastatin (S1–S4), and lovastatin (L1–L4; Figure B, upper panel). Thirty-three common differentially expressed genes were significantly affected (FC≥2) by atorvastatin, simvastatin, and fluvastatin (Figure B, lower panel). Many of the commonly regulated genes were related to cholesterol biosynthesis, such as methylsterol monooxygenase-1 (MSMO1), stearoyl-CoA desaturase (SCD), 3-hydroxy-3-methylglutaryl-CoA synthase-1 (HMGCS1), low-density lipoprotein receptor (LDLR), 24-dehydrocholesterol reductase (DHCR24), and 7-dehydrocholesterol reductase (DHCR7; Figure B, lower panel). These results suggest that statins induce a molecular signature in a drug-specific manner independent of genetic background.
Figure. Statin class effects on the transcriptome and functional properties of induced pluripotent stem cell–derived cardiomyocytes.A, Schematic depiction of study design. Atorva indicates atorvastatin; CM, cardiomyocyte; Fluva, Fluvastatin; iPSC, induced pluripotent stem cell; Lova, lovastatin; Pt1–4, iPSC lines; Simva, simvastatin. B, Upper panel: Heatmap of differentially expressed gene (DEG) fold-changes (FCs) from RNA sequencing analysis. Red boxes (left side) indicate genes significantly affected by fluvastatin, atorvastatin, and simvastatin, blue indicates genes significantly affected by any 2 statins, and black indicates genes affected by only 1 drug. Genes with false discovery rate adjusted P≤0.05 under likelihood ratio test were considered as significant. Lower panel: Heatmap of FCs of commonly affected DEGs by all drugs. A indicates atorvastatin; C, control; F, fluvastatin; L, lovastatin; S, simvastatin; and 1–4, Lines 1–4. C, Upper panel: Heatmap of AmpliSeq data showing FCs of DEGs in statin-treated iPSC-CMs from Line 2. Lower panel: Heatmap of Ampli-seq data showing FCs of DEGs regulated in common by atorvastatin, fluvastatin, simvastatin, and lovastatin in iPSC-CMs. Genes with false discovery rate adjusted P≤0.05 were considered as significant. A1–A3 indicates atorvastatin; C1–C3, control; F1–F3, fluvastatin; L1–L3, lovastatin; and S1–S2, simvastatin. D,Representative Western blot analysis of HMGCS1 protein expression in iPSC-CMs following statin treatment. E,Bubble chart showing the top signaling pathways and cellular processes with the largest number of affected DEGs, according to transcriptomic analysis. F,Quantitative real-time–polymerase chain reaction quantification of hypertrophic cardiomyopathy (HCM) pathway genes in HCM MYBPC3 p.Val321Met iPSC-CMs treated with statins. G, Cell size analysis of HCM MYBPC3 p.Val321Met iPSC-CMs after treatment with statins. Error bars indicate 95% CIs and square dots represent mean values. Dash lines show 95% CI in the control group. H, Cell death analysis (apoptosis) of healthy iPSC-CMs (Lines 2 and 3) treated with each statin. *P≤0.05, †P≤1e-6, ‡P≤1e-33 under paired t test. DOX indicates Doxorubicin.
Additional transcriptomic profiling of 3 different iPSC-CM batches derived from a single donor (Line 2) was performed to ensure reproducibility of the observed effects, independent of technical variability. Comprehensive assessment of these iPSC-CM transcriptomes after drug treatment was performed using the Ion AmpliSeq Human Transcriptome Kit. In accordance with the RNA sequencing analysis, fluvastatin mediated the most potent effects on iPSC-CM transcriptome, followed by atorvastatin, simvastatin, and lovastatin (Figure C, upper panel). Further analysis revealed that fluvastatin, atorvastatin, and simvastatin regulate a common set of 6 differentially expressed genes related to cholesterol biosynthesis (MSMO1, SCD, LDLR, DHCR24, HMGCS1, and DHCR7; Figure C, lower panel). This set of commonly regulated genes represents the core molecular signature of the effects of fluvastatin, atorvastatin, and simvastatin in iPSC-CMs. Our results correlate with the clinical efficacy of statins, except for fluvastatin, which may have cardiac specific effects and needs to be further investigated in the future.
The increased protein levels of HMGCS1 were confirmed by Western blot analysis in all iPSC-CMs after statin treatment (Figure D). Atorvastatin and fluvastatin mediated the strongest effects on HMGCS1 protein expression, followed by simvastatin and lovastatin. These results are in accordance with both RNA sequencing and AmpliSeq analysis, further suggesting that statins regulate the expression of key regulators of the metabolic properties of iPSC-CMs in a drug-specific manner. Subsequently, we performed functional enrichment analysis to uncover the signaling pathways and cellular processes affected by statins in iPSC-CMs. All statins affected signaling pathways/cellular processes that are primarily involved in cholesterol metabolism, secondary alcohol biosynthesis, and sterol biosynthetic pathway (Figure E). Although the hypertrophic cardiomyopathy (HCM) signaling pathway was not significantly enriched, several genes were found to be significantly regulated. Significant downregulation of 2 HCM pathway genes, TPM2 and MYL3, was observed in iPSC-CMs differentiated from a HCM patient (MYBPC3 p.Val321Met [c.961G≥A])3 after treatment by atorvastatin, simvastatin, and lovastatin (Figure F). In addition, simvastatin and lovastatin significantly reduced the cell size of HCM iPSC-CMs, corroborating the antihypertrophic effects at the cellular level (Figure G). Finally, we also tested the antiapoptotic effects of statins in 2 healthy iPSC-CM lines (Lines 2 and 3) after treatment with Doxorubicin (0.1 umol/L), as previously described.4 Our data showed that simvastatin and lovastatin significantly reduced the number of DOX-induced apoptosis in iPSC-CMs (Figure H), demonstrating prosurvival effects for each of these drugs.
Overall, our study reveals that fluvastatin mediates the strongest effects on the transcriptome of healthy iPSC-CMs. On the other hand, simvastatin and lovastatin exerted antihypertrophic effects in HCM iPSC-CMs, and prosurvival effects in Doxorubicin-treated iPSC-CMs. When applied at physiologically relevant concentrations, statins primarily affect genes related to cholesterol and fatty acid homeostasis, which is in accordance with previous reports implicating the long-chain polyunsaturated fatty acids in statins’ cardioprotective effects.5 In addition, statins exert antihypertrophic and antiapoptotic cellular processes in human iPSC-CMs in a drug-specific manner. These effects might be independent from the clinical action of statins on atherosclerosis, although further work is needed to confirm this observation.
Sources of Funding
Dr Oikonomopoulos was supported by American Heart Association (AHA) grant 17SDG33660794. Dr Kitani was supported by the Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. Dr Liu was supported by AHA 16POST30960020. Dr Ong was supported by NIH R00 HL130416. Mr Smeets was supported by the Dutch Heart Association and the Michaël Fonds. Dr Karakikes was supported by NIH R01 HL139679. Dr Sayed was supported by National Institutes of Health (NIH) grant K01 HL135455 and Stanford TRAM scholar award. Dr Wu was supported by NIH R01 HL123968, R01 HL126527, R01 HL113006, R01 HL146690, R01 HL130020, and P01 HL141084.
Dr Wu is a cofounder of Khloris Biosciences but has no competing interests, because the work presented here is completely independent. The other authors report no conflicts.
*Drs Tian and Oikonomopoulos contributed equally.
The data supporting the findings of this study are available from the corresponding author upon request. RNA sequencing data is publicly available with GEO accession number GSE113546.
Joseph C. Wu, MD, PhD, 265 Campus Drive, Rm G1120B, Stanford, CA 94305-5454. Email firstname.lastname@example.org
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