Molecular Mechanisms of Cardioprotective Actions of Tanshinones

Tanshinones are lipophilic compounds derived from Salvia miltiorrhiza (Danshen) that has been widely used to treat coronary heart diseases in China. The cardioprotective actions of tanshinones have been extensively studied in various models of myocardial infarction, cardiac ischemia reperfusion injury, cardiac hypertrophy, atherosclerosis, hypoxia, and cardiomyopathy. This review outlines the recent development in understanding the molecular mechanisms and signaling pathways involved in the cardioprotective actions of tanshinones, in particular on mitochondrial apoptosis, calcium, nitric oxide, ROS, TNF-α, PKC, PI3K/Akt, IKK/NF-κB, and TGF-β1/Smad mechanisms, which highlights the potential of these compounds as therapeutic agents for treating cardiovascular diseases.


Introduction
Danshen, also called red sage root, is dried roots of Salvia miltiorrhiza that is cultivated widely in many Asian countries including China, Korea, and Japan.Danshen and Danshen related products have long been used as traditional Chinese medicine to treat cardiovascular dysfunctions such as angina pectoris, stroke, and hypertension [1][2][3][4].For example, a recent systematic review indicates that Danshen dripping pill is more effective than isosorbide dinitrate in treating angina pectoris [5].Although with its long history of medicinal use, the mechanism of actions of Danshen has only been intensively investigated in recent years, with the progress in elucidating its phytochemistry and active compounds.The identified chemical constituents of Danshen can be classified into lipophilic and hydrophilic components.The main lipophilic constituents are tanshinones which are diterpene quinones.There are more than 40 tanshinones that have been isolated [6].The main tanshinones include tanshinone I (TI), tanshinone IIA (TIIA), tanshinone IIB (TIIB), tanshinone VI (TVI), cryptotanshinone (CT), and dihydrotanshinone (DHT) as illustrated in Figure 1.
Tanshinones have been demonstrated with various pharmacological activities including antioxidant, anti-inflammatory, antibacterial, antineoplastic, immunomodulatory, cardioprotective, and neuroprotective actions and have been used in treating various conditions, including cardiovascular diseases, cancer, and diabetes [7][8][9][10][11][12][13][14].One of the promising therapeutic actions of tanshinones is their cardioprotective actions [15].For example, TIIA has been shown to dilate coronary vessels [16] and regulate vascular endothelial functions [17] and inhibit myocardial infarction [18], ischemia reperfusion (I/R) injury [19,20], and cardiomyopathy [21].It also reduced atherosclerosis [22].CT has also been shown to reduce atherosclerosis and I/R induced cardiac injuries [23,24], potentially via vasodilatation [25] and anti-inflammatory actions [26].Similarly, TVI has been shown with antihypertrophy activity in cardiomyocytes [27], and DHT has been reported with vasorelaxation and antiplatelet actions [28,29].Tables 1 and 2 summarize cardioprotective actions of major tanshinones in vitro and in vivo.This short review outlines the recent development in understanding the molecular mechanisms of cardioprotective actions of tanshinones, with a focus on their regulation of various signaling pathways.

Effects of Tanshinones on Atherosclerosis
Atherosclerosis is a vascular endothelial dysfunction caused by inflammation promoted by low-density lipoproteins (LDL) [30].Various atherogenic stimuli, including diabetes and oxidative stress, can induce endothelial dysfunction, leading to atherosclerosis [31].Current approaches for reducing atherosclerosis have been focused on antiinflammatory, antioxidant, and vasodilator mechanisms [32].
Studies have demonstrated the antiatherosclerosis actions of various tanshinones and their associations with various signaling pathways.For example, a tanshinone mixture was shown to inhibit proliferation of vascular smooth muscle cells (VSMCs) in vitro, which is associated with reduction of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, cyclin D1, and enhancing p21 waf1/cip1 (cyclindependent kinase inhibitor 1) expressions [33].This is supported by the finding of inhibition of intimal hyperplasia by tanshinone in the ligated mouse carotid artery in vivo [34].
Treatment with total tanshinone reduced the development of intimal thickening of injured vessels and proliferating cell nuclear antigen-(PCNA-) positive vascular smooth muscle cells in mice, indicating that tanshinone may potentially inhibit VSMCs proliferation from arterial injury and reduce occurrence of atherosclerosis [34].TIIA has been shown to increase nitric oxide (NO) generation and inhibiting platelet aggregation/leukocyte adhesion in vitro [73].It also attenuated H 2 O 2 induced endothelial dysfunction by enhancing superoxide dismutase (SOD) activity [38] and reducing caspase 3 activity in human umbilical vein endothelial cell (HUVECs) [39].The antiatherosclerosis action of TIIA was associated with its anti-inflammatory activities by inhibition of cluster of differentiation 40 (CD40) and p53 protein expression [38,39], and inhibition of tumor necrosis factor- (TNF-) induced expressions of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and fractalkine, possibly through inhibition of IKK/NF-B signaling pathway [37,74].TIIA was found to reduce oxidized LDL protein production and superoxide production by enhancing Cu/Zn SOD in rat [70] and also decreased cholesterol content and oxLDL level in apolipoprotein E deficient (ApoE −/− ) mice [22].It also inhibited the expression of inflammatory regulator CD40 and matrix metalloproteinase-2 (MMP-2) in high fat diet induced atherosclerosis in rabbits [71].A recent study in ovariectomized ApoE −/− mice found that TIIA had a phytoestrogenlike activity reducing aortic lipid deposition and serum levels of malonyl dialdehyde (MDA), nuclear factor-kappa B (NF-B), and lipids, involving inhibiting p-ERK 1/2 [75].Further study is required to elucidate the role of NO synthesis and redox signaling (specifically on regulation of mitochondrial anti/prooxidative stress) in the actions of TIIA.
Similarly, CT was found to attenuate TNF- induced endothelial dysfunction in HUVECs by reducing endothelin-1 accumulation, enhancing eNOS/NO, and inhibiting of NF-B pathways [26].In human aortic smooth muscle cells (HASMCs), CT inhibited TNF- induced activation of NF-B and activator protein 1 (AP1, a transcriptional factor that regulates genes of cytokines and growth factors), as well as activation of matrix metalloproteinases-9 (MMP-9), which plays an important role in migration and proliferation of vascular smooth cells and into the intima [23].On the other hand, CT was found to reduce calcium flux and calcium induced contraction in rat coronary artery [25] and inhibit thromboxane A2 analogue U46619-induced constriction of the porcine coronary artery [76].Another tanshinone, DHT, was shown with a vasodilator activity by reducing CaCl 2 elicited contraction in rat coronary artery [28].The calcium regulation effect of DHT was also demonstrated in its inhibition of collagen-induced rabbit platelet aggregation, which involves intracellular calcium mobilization, arachidonic acid liberation, and thromboxane B2 generation mechanisms [29].

Effects of Tanshinones on Myocardial Infarction
Myocardial infarction (MI) is caused by disruption of blood supply to the heart and often accompanied by ventricular remodeling and cardiac hypertrophy, which can lead to cardiac cell death [77].Cardiomyocytes apoptosis is an important mechanism in MI [78], and it is closely associated with cellular inflammatory reaction, intracellular calcium homeostasis, and NO and oxidative stress [79].
There is evidence that tanshinones reduced cardiac infarct size and improved cardiac function in MI via inhibiting intracellular calcium, cell adhesion molecules, apoptotic protein, and inflammation protein expressions.An in vivo study demonstrated that acute myocardial infarction induced by occlusion of rat coronary vessel was decreased by posttreatment of a tanshinone extract [57].This improvement was partially mediated by modulation of expression of genes which regulate intracellular calcium, cell adhesion molecules, and alternative complementary pathways (i.e., neuronal pentraxin 1, intercellular adhesion molecule 1, and C3 convertase) [46,57].The anti-inflammatory and antiapoptotic properties of tanshinones may play an important role in their anti-MI actions [18,46].TIIA was shown to inhibit TNF- induced monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-1 (TGF-1) expression in cardiac fibroblasts in vitro [18].It reduced TNF-, NF-B, MCP-1, and TGF-1 expressions [18] and improved cardiac dysfunction with inhibition of expressions of p-38, serum response factor (SRF: a transcription factor that has a role in modulating muscle proliferation and cell migration and apoptosis), and myocyte enhancer factor 2 (MEF2: a transcription factor that regulates tissue remodeling in cardiac muscle) [46].

Effects of Tanshinones on Ischemia Reperfusion Injuries
Myocardial ischemia reperfusion (I/R) injury is caused by insufficient oxygen supply to the cardiac tissue which can occur in various conditions, such as thrombosis, coronary atherosclerotic plaques, and diabetes complications [80].Sudden oxygen supply during reperfusion after prolonged ischemia can lead to impairment of myocardial cells via reactive oxygen species (ROS) dependent mechanisms [81].
The myocardial I/R injury can be affected by a number of cellular factors/regulators including calcium, pH, ROS, and in particular mitochondrial function.For example, mitochondrial membrane transition pore (MPTP) opening has been a research focus and potential therapeutic target for developing new treatments for I/R injury in recent years [82].I/R injuries are likely to be the main cause of MI and subsequently heart failure due to imbalance in redox signaling [83].Therefore, regulating redox signaling during reperfusion may be an important turning point in treating MI and heart failure [84].
Tanshinones have been well demonstrated with protective actions on reducing ischemia I/R injuries involving inhibition of free radical formation and mitochondrial apoptosis, as well as regulating eNOS/NO and AMPK/PI3K/Akt pathways.In a rat model of I/R injury, TIIA (20 mg/kg) was shown to reduce infarction size, possibly by upregulating eNOS/NO pathway and AMPK/PI3K/Akt pathway [40].TIIA was also shown to enhance glutathione peroxidase and SOD activities and decrease production of free radicals (H 2 O 2 and O 2 − ) [19,44,85].Since glutathione peroxidase, rather than SOD, is the major antioxidant in the cardiomyocytes [86], TIIA may be a promising agent to treat I/R injuries and MI due to its better permeability to cell membranes.
TIIA has been shown to inhibit hydrogen peroxide induced cell apoptosis in neonatal cardiomyocytes in vitro, with a 40% reduction of cell death as evidenced by decreased Hoechst 33342 staining and DNA fragmentation [19].Similar findings were reported for its inhibition of H 2 O 2 induced cardiomyocytes cell apoptosis and intracellular calcium increase [44].However, the exact role of TIIA in I/R injuries still needs to be elucidated due to its complex interactions of H 2 O 2 with other signaling pathways.Additionally, antiapoptotic actions of TIIA have been shown in neonatal cardiomyocytes under hypoxia condition, with reducing p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and miR-1 level [46], and enhanced ERK1/2 phosphorylation [45].Our study has also demonstrated that TIIA (3 M) reduced hypoxia induced late apoptosis in H9C2 cells, the effect linked to decreasing hypoxia inducible factor-1 (HIF-1) translocation, balancing in Bcl-2/Bax ratio, reducing cytochrome c release and caspase 3 activity [36].A recent study also found that the protection by TIIA on hypoxic ischemia-induced injury could be enhanced by the JAK2/STAT3 inhibitors [87].Additionally, the antiapoptosis effects of TIIA were associated with regulation of intracellular calcium, ATP, mitochondrial superoxide, and SOD levels but not intracellular H 2 O 2 [35].Studies in vivo found that TIIA inhibited I/R induced apoptosis (about 50% reduction of TUNEL staining), reduced caspase 3 activity, increased SOD level and Bcl-2/Bax ratio [19], and restored I/R induced diminished eNOS/NO production and coronary dysfunction [40].Thus, it is likely that the protection of TIIA on myocardial I/R injuries involves multiple mechanisms including inhibition of intrinsic apoptosis pathway and anti-inflammatory and antioxidant activities.Interestingly, recent studies on cerebral I/R injury also indicate that TIIA may act by inhibiting intrinsic death pathway, inflammatory response, and ROS [7,88].
TVI has also been demonstrated with protective action on hypoxia/reoxygenation injury and improvement of cardiac function [89].TVI treatment improved myocardial contractility in reoxygenated rat heart, accompanied by reduced release of ATP metabolites and decrease in cellular calcium contents, but further study is required to elucidate its actions on ROS mechanisms [89].

Effects of Tanshinones on Cardiac Hypertrophy
Cardiac hypertrophy is defined as an enlargement of cardiac tissue or myocardium, resulting in a decrease in size of ventricle chambers.The pathological cardiac hypertrophy is associated with increased interstitial fibrosis, cell death, and cardiac dysfunction and influenced by many factors/conditions such as hypertension, ischemic injury, and genetic polymorphism [90,91].The main cellular and molecular regulators of cardiac hypertrophy include calcineurin/nuclear factor of activated T-cells (NFAT), G protein-coupled receptors (GPCR), MAPKs and apoptosis pathways, and cytokines activated by various stress stimuli including AngII, endothelin-1, and insulin-like growth factor-1 (IGF-1) [91,92].Pathological cardiac hypertrophy has been shown to be more susceptible to proapoptotic changes, including modulations of Fas, Bcl-2 family proteins and caspases [93], and apoptosis occurring during hypertrophy may act as a cardiac adaptive response [94,95].Additionally, hypertrophic stimuli (i.e., angiotensin II (AngII) and endothelin-1 (ET-1) and catecholamines) have also been shown to induce hypertrophy by stimulating ROS production (via NADPH oxidase, xanthine oxidase, and nitric oxide synthase) [96].It has been suggested that NADPH oxidase plays an importance role regarding redoxsignaling involvement in pathological hypertrophy [92].
In addition, there is evidence for the involvement of microRNAs in the cardioprotection of tanshinones.It was found that TIIA increased the expressions of MiR-133 and ERK1/2 in hypoxic neonatal cardiomyocytes [45].In rats with acute myocardial infarction, TIIA inhibited postinfarct cardiac fibrosis and improved impaired left ventricular function, with downregulation of expression of TGF-1 and upregulation of expression of miR-29b.The antifibrotic effect of TIIA was blocked by a miR-29b inhibitor and Smad3 siRNA, indicating that miR-29 may play an important role in the action of TIIA in postinfarct cardiac remodeling, possibly mediated by TGF--Smad3 signaling pathway [97].

Effects of Tanshinone IIA on Adriamycin Induced Cardiomyopathy
Adriamycin (ADR), also called doxorubicin, is an effective anticancer drug.However, its clinical usage is limited by its cardiomyopathy which may lead to heart failure [98].ADRinduced cardiomyopathy is related to increased oxidative stress [99].Increasing ROS can reduce mitochondria oxidative phosphorylation, which in turn depletes cellular ATP, resulting in cardiac cell impairment [100].Other possible mechanisms for ADR-induced cardiomyopathy include activation of mitogen-activated protein kinases (MAPKs) and necrosis and extrinsic pathways [101].TIIA has been demonstrated to prevent ADR-induced cardiomyocytes death in vitro by preventing DNA fragmentation (60% compared to positive control), suppressing ROS production, and increasing Bcl-2/Bax ratio [43].Hong et al. also showed that TIIA prevented ADR-induced cell apoptosis and ROS production by 33% and 37%, respectively, with enhanced Bcl-xl protein expression, inhibited cytochrome c release and caspase 3 activity, and restored Akt phosphorylation [21].Thus, TIIA and other tanshinone analogues may potentially be useful in preventing ADR-induced cardiomyopathy.

Effects of Tanshinone IIA on Endotoxin-Induced Cardiac Dysfunction
Tanshinone IIa has recently been shown to attenuate endotoxin-induced cardiac dysfunction in septic mice via inhibiting NADPH oxidase 2-related signaling pathway.It inhibited LPS-induced production of TNF-, IL-1, and ROS and decreased Nox2, p-ERK1/2, and p38 MARK [72].

Synthetic Tanshinone Derivatives and Structure Optimization
Natural tanshinones have a unique structure and bioactivity but also some limitations such as low water solubility and poor bioavailability [12].Approaches have been made to develop synthetic derivatives of tanshinones to optimize their structures for potential clinical applications.For example, sodium tanshinone IIA sulfonate was synthesized as a water soluble derivative of tanshinone IIA, which has been used in China for the treatment of angina pectoris and myocardial infarction [102].Several tanshinone IIA derivatives with better vasodilatation activity have been synthesized by the reactions of tanshinone IIA with aromatic aldehydes, and the structure-activity relationship indicated that the methyoxylsubstituting group in the meta-position of aromatic aldehydes presented better activity than that of nitro substitution derivatives [103].A number of tanshinone derivatives have been synthesized with modification of tanshinones' four rings with fatty acids, N-containing substituent, and other function groups, resulting in analogues with potential better bioactivity and bioavailability [102].

Conclusion
In conclusion, tanshinones have been demonstrated with significant cardiac protective actions, which may be potentially useful as therapeutic agents to treat cardiac diseases.The molecular mechanisms of cardioprotective actions of tanshinones involve anti-inflammatory, antioxidant, and antiapoptosis actions although regulating various signaling pathways, in particular TNF-alpha signaling, mitochondrial apoptosis, and calcium and NO signaling pathways, as illustrated in Figure 2. Further studies on tanshinones and tanshinone analogues may lead to development of novel drugs for treating cardiovascular diseases.

Table 1 :
Pharmacological actions of tanshinones in vitro.
0.1-10 M A n t i fi b r o s i sa c t i o na g a i n s tH 2 O

Table 2 :
Pharmacological actions of tanshinones in vivo.