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.

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.

https://www.ahajournals.org/journal/circ

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.

他汀类药物通过抑制胆固醇生物合成来预防心脏病，并对心血管系统发挥多效作用。¹他汀类药物还可以作为抗肥大和抗凋亡药物来预防心肌细胞损伤。然而，临床相关浓度的他汀类药物（即血清峰值浓度）对心肌细胞的影响仍然很大程度上未知。在目前的研究中，我们研究了在各自的血清峰值浓度下应用阿托伐他汀、洛伐他汀、辛伐他汀和氟伐他汀对人诱导多能干细胞衍生心肌细胞 (iPSC-CM) 的转录组和功能特性的类别效应。²

来自 2 名男性（第 1 和 2 行）和女性（第 3 和 4 行）对照个体的人类 iPSCs 分化为 iPSC-CMs（图 A），其效率高于 80%，通过心肌肌钙蛋白的表达测量（数据未显示） . 当以剂量依赖性方式测试时，他汀类药物对 iPSC-CMs 的活力（2 天和 7 天）和收缩特性（7 天）没有影响（数据未显示）。为了检查他汀类药物的转录作用，我们在用每种他汀类药物的血清峰值浓度治疗后对每个 iPSC-CM 细胞系进行了全面的 RNA 测序分析，对应于 40 mg 的临床剂量。与载体处理的细胞相比，我们专注于显着差异表达的基因（错误发现率调整后的P≤0.05)。我们发现氟伐他汀 (F1-F4) 介导对 iPSC-CM 转录组最有效的影响，其次是阿托伐他汀 (A1-A4)、辛伐他汀 (S1-S4) 和洛伐他汀 (L1-L4；图 B，上图) . 33 个常见的差异表达基因受到阿托伐他汀、辛伐他汀和氟伐他汀的显着影响（FC≥2）（图 B，下图）。许多常见的调控基因与胆固醇生物合成有关，如甲基甾醇单加氧酶-1（MSMO1）、硬脂酰辅酶A去饱和酶（SCD）、3-羟基-3-甲基戊二酰辅酶A合酶-1（HMGCS1）、低密度脂蛋白受体 ( LDLR )、24-脱氢胆固醇还原酶 ( DHCR24 ) 和 7-脱氢胆固醇还原酶 ( DHCR7); 图 B，下面板）。这些结果表明，他汀类药物以与遗传背景无关的药物特异性方式诱导分子特征。

数字。 他汀类药物对诱导多能干细胞衍生心肌细胞转录组和功能特性的影响。A，研究设计的示意图。Atorva 表示阿托伐他汀；CM，心肌细胞；Fluva，氟伐他汀；iPSC，诱导多能干细胞；洛伐他汀；Pt1-4，iPSC 线；辛伐他汀。B，上图：来自 RNA 测序分析的差异表达基因 (DEG) 倍数变化 (FC) 的热图。红色框（左侧）表示受氟伐他汀、阿托伐他汀和辛伐他汀显着影响的基因，蓝色表示受任何 2 种他汀类药物显着影响的基因，黑色表示仅受 1 种药物影响的基因。错误发现率调整后的基因P在似然比检验下≤0.05被认为是显着的。下图：所有药物通常影响的 DEG 的 FC 热图。A表示阿托伐他汀；C、控制；F、氟伐他汀；L、洛伐他汀；S、辛伐他汀；和 1-4，第 1-4 行。C，上图：AmpliSeq 数据的热图，显示来自第 2 行的他汀类药物处理的 iPSC-CM 中 DEG 的 FC。下图：Ampli-seq 数据的热图，显示由阿托伐他汀、氟伐他汀、辛伐他汀和洛伐他汀共同调节的 DEG 的 FC iPSC-CM。错误发现率调整P≤0.05的基因被认为是显着的。A1-A3 表示阿托伐他汀；C1-C3，控制；F1-F3，氟伐他汀；L1-L3，洛伐他汀；S1-S2，辛伐他汀。D, 他汀类药物治疗后 iPSC-CMs 中 HMGCS1 蛋白表达的代表性蛋白质印迹分析。E，根据转录组分析，气泡图显示受影响的 DEG 数量最多的顶级信号通路和细胞过程。F ，用他汀类药物治疗的 HCM MYBPC3 p.Val321Met iPSC-CM中肥厚型心肌病 (HCM) 通路基因的定量实时聚合酶链反应定量。G ，用他汀类药物治疗后 HCM MYBPC3 p.Val321Met iPSC-CMs的细胞大小分析。误差线表示 95% CI，方点表示平均值。虚线显示对照组中的 95% CI。H，用每种他汀类药物治疗的健康 iPSC-CM（第 2 和 3 行）的细胞死亡分析（细胞凋亡）。* P ≤0.05 , ^† P ≤1e ^-6 , ^‡ P ≤1e ^-33在配对t检验下。DOX 表示多柔比星。

对源自单个供体（第 2 行）的 3 个不同 iPSC-CM 批次进行了额外的转录组分析，以确保观察到的效果的可重复性，与技术可变性无关。使用 Ion AmpliSeq Human Transcriptome Kit 对药物治疗后的这些 iPSC-CM 转录组进行综合评估。根据 RNA 测序分析，氟伐他汀对 iPSC-CM 转录组的影响最大，其次是阿托伐他汀、辛伐他汀和洛伐他汀（图 C，上图）。进一步分析表明，氟伐他汀、阿托伐他汀和辛伐他汀调节一组共同的与胆固醇生物合成相关的 6 个差异表达基因（MSMO1、SCD、LDLR、DHCR24、HMGCS1和DHCR7；图 C，下面板）。这组共同调控的基因代表了 iPSC-CM 中氟伐他汀、阿托伐他汀和辛伐他汀作用的核心分子特征。我们的研究结果与他汀类药物的临床疗效相关，但氟伐他汀可能具有心脏特异性作用，未​​来需要进一步研究。

在他汀类药物治疗后，所有 iPSC-CM 中的蛋白质印迹分析证实了 HMGCS1 蛋白水平的增加（图 D）。阿托伐他汀和氟伐他汀对 HMGCS1 蛋白表达的影响最强，其次是辛伐他汀和洛伐他汀。这些结果与 RNA 测序和 AmpliSeq 分析一致，进一步表明他汀类药物以药物特异性方式调节 iPSC-CM 代谢特性的关键调节因子的表达。随后，我们进行了功能富集分析，以揭示 iPSC-CM 中受他汀类药物影响的信号通路和细胞过程。所有他汀类药物都会影响主要参与胆固醇代谢、二级醇生物合成和甾醇生物合成途径的信号通路/细胞过程（图 E）。尽管肥厚型心肌病 (HCM) 信号通路没有显着丰富，但发现有几个基因受到显着调控。2个HCM通路基因显着下调，在用阿托伐他汀、辛伐他汀和洛伐他汀治疗后，在与 HCM 患者 (MYBPC3 p.Val321Met [c.961G≥A]) ^{3分化的 iPSC-CM 中观察到}TPM2和MYL3 （图 F）。此外，辛伐他汀和洛伐他汀显着降低了 HCM iPSC-CM 的细胞大小，证实了细胞水平的抗肥大作用（图 G）。最后，我们还测试了他汀类药物在用多柔比星 (0.1 umol/L) 治疗后在 2 条健康 iPSC-CM 系（第 2 和第 3 行）中的抗凋亡作用，如前所述。⁴我们的数据显示辛伐他汀和洛伐他汀显着降低了 iPSC-CM 中 DOX 诱导的细胞凋亡的数量（图 H），证明了这些药物中的每一种都具有促存活作用。

总体而言，我们的研究表明氟伐他汀对健康 iPSC-CM 的转录组具有最强的影响。另一方面，辛伐他汀和洛伐他汀在 HCM iPSC-CM 中发挥抗肥大作用，在多柔比星处理的 iPSC-CM 中发挥促存活作用。当以生理相关浓度应用时，他汀类药物主要影响与胆固醇和脂肪酸稳态相关的基因，这与之前的报道一致，即长链多不饱和脂肪酸对他汀类药物的心脏保护作用。⁵此外，他汀类药物以药物特异性方式在人 iPSC-CM 中发挥抗肥大和抗凋亡细胞过程。这些影响可能独立于他汀类药物对动脉粥样硬化的临床作用，尽管需要进一步的工作来证实这一观察结果。

Oikonomopoulos 博士得到了美国心脏协会 (AHA) 拨款 17SDG33660794 的支持。Kitani 博士得到了日本心脏基金会/Bayer Yakuhin Research Grant Abroad 的支持。刘博士得到了 AHA 16POST30960020 的支持。Ong 博士得到了 NIH R00 HL130416 的支持。Smeets 先生得到了荷兰心脏协会和 Michaël Fonds 的支持。Karakikes 博士得到了 NIH R01 HL139679 的支持。Sayed 博士得到了美国国立卫生研究院 (NIH) 授予的 K01 HL135455 和斯坦福 TRAM 学者奖的支持。Wu 博士得到 NIH R01 HL123968、R01 HL126527、R01 HL113006、R01 HL146690、R01 HL130020 和 P01 HL141084 的支持。

吴博士是 Khloris Biosciences 的联合创始人，但没有相互竞争的利益，因为这里介绍的工作是完全独立的。其他作者报告没有冲突。

* Tian 博士和 Oikonomopoulos 博士的贡献相同。

https://www.ahajournals.org/journal/circ

支持本研究结果的数据可根据要求从相应作者处获得。RNA 测序数据可通过 GEO 登录号 GSE113546 公开获得。