Cardioprotective Properties of Kaempferol: A Review

Cardiac diseases, such as myocardial infarction and heart failure, have become a major clinical problem globally. The accumulating data demonstrate that bioactive compounds with antioxidant and anti-inflammatory properties have favorable effects on clinical problems. Kaempferol is a flavonoid found in various plants; it has demonstrated cardioprotective properties in numerous cardiac injury models. This review aims to collate updated information regarding the effects of kaempferol on cardiac injury. Kaempferol improves cardiac function by alleviating myocardial apoptosis, fibrosis, oxidative stress, and inflammation while preserving mitochondrial function and calcium homeostasis. However, the mechanisms of action of its cardioprotective properties remain unclear; therefore, elucidating its action could provide insight into directions for future studies.


Introduction
Cardiovascular diseases are the principal cause of mortality globally, claiming approximately 18 million lives each year [1]. Cardiac disease refers to conditions affecting the heart, including coronary artery disease and arrhythmia. In coronary artery disease, the blood supply that carries oxygen and nutrients to the heart is interrupted, which could be due to atherosclerotic plaque buildup. This may precipitate an ischemic condition in the heart, leading to myocardial infarction. In animal studies, the condition can be mimicked by the models of ischemia/reperfusion (I/R) produced by the ligation of coronary artery [2] or aorta [3], while in in vitro studies, this may be achieved by employing anoxia/reperfusion (or anoxia/reoxygenation) [4] and hypoxia/reperfusion models [5]. Myocardial infarction may precipitate the development of cardiac remodeling, which later could progress to heart failure. Cardiac remodeling can also be triggered by the administration of angiotensin II (Ang II) [6] and cardiotoxic drugs, including doxorubicin [7], cisplatin [8], 5-fluorouracil [9], and clozapine [10], in animal studies. Phenylephrine [3] and isoprenaline [11], which cause myocardial overstimulation, are also used to develop myocardial infarction and heart failure depending on the dose and duration of exposure [12]. Another complication of myocardial infarction is cardiac arrhythmia. Diabetes may also cause detrimental changes to the heart, known as diabetic cardiomyopathy, evidenced by increased oxidative stress, inflammation, apoptosis, and fibrosis [13].
Plants have a crucial role in human life and well-being. Humans use plants for food, clothing, furniture, and many other things. Plants have been used for medicinal purposes since ancient times. They produce secondary metabolites, such as flavonoids and terpenoids, for their self-defense [14]. Flavonoids are found abundantly in vegetables and fruits. The compounds are responsible for the pigmentation of yellow and red, as well as since ancient times. They produce secondary metabolites, such as flavonoids and terpenoids, for their self-defense [14]. Flavonoids are found abundantly in vegetables and fruits. The compounds are responsible for the pigmentation of yellow and red, as well as other colors in plants. They are divided into seven subclasses: flavonols (e.g., kaempferol and quercetin), flavones (e.g., luteolin and apigenin), flavanols (e.g., catechin and epicatechin), isoflavones (e.g., daidzein and genistein), anthocyanidins (e.g., cyanidin and delphinidin), flavonones (e.g., hesperetin and hesperidin), and chalcones (e.g., butein and naringenin chalcone) [15][16][17].
Flavonoids have been isolated from plants for various therapeutic effects. A study demonstrated that rutin and quercetin reduced Ang-II-induced cardiomyocyte hypertrophy by modulating mitogen-activated protein kinase (MAPK) [18]. On the other hand, kaempferol [11] and luteolin [19] protected against diabetic cardiomyopathy by regulating Kelch-like ECH-associated protein (Keap) and nuclear factor kappa B (NF-B) signaling pathways. Hesperidin also diminishes injury following myocardial infarction in mice by modulating inflammatory response [20]. Therefore, plants have become a promising source of new drugs over the last four decades [21].
Kaempferol (3,4′,5,7-tetrahydroxyflavone) (Figure 1), a yellow crystalline compound, is a flavonol which is rich in various plants such as tea, broccoli, tomatoes [22], and beans (e.g., bitter bean) [23]. Many studies have demonstrated the pharmacological activities of this compound in various pathological conditions, such as cardiovascular disease [6,13] and cancer [24]. It protects against cardiac disease via antiapoptotic, antioxidative, antiinflammatory, calcium regulatory, and antifibrotic mechanisms, as well as maintaining mitochondrial function, resulting in the amelioration of cardiac structure and function [25][26][27]. However, the specific protective mechanisms of kaempferol remain unclear. The current review aims to provide an up-to-date overview of the role of kaempferol in cardiac disease. The information provided could increase the understanding of the cardioprotective effects of kaempferol and could aid in the design of future studies.

Bibliographic Search
A search of the literature was systematically performed using the PubMed, Scopus, and Web of Science databases. The keywords used for the search were "kaempferol" AND "cardiovascular", "kaempferol" AND "cardiac", "kaempferol" AND "heart", "kaempferol" AND "myocardi*", and "kaempferol" AND "cardiomyo*". The search retrieved 41 articles that were published from 2008 to March 2023.

Effects on Cardiac Injury and Structure
Numerous models have been employed to determine the potential effects of kaempferol on cardiac injury. Various agents have been used in experimental animals and cardiomyocytes in vitro to induce cardiac injury, namely Ang II [6], isoprenaline [25], doxorubicin [7], cisplatin [8], 5-fluorouracil [9], phenylephrine [3], and clozapine [10], in addition to I/R [28] and aortic banding [3]. The inducers promote cardiac injury by elevating

Bibliographic Search
A search of the literature was systematically performed using the PubMed, Scopus, and Web of Science databases. The keywords used for the search were "kaempferol" AND "cardiovascular", "kaempferol" AND "cardiac", "kaempferol" AND "heart", "kaempferol" AND "myocardi*", and "kaempferol" AND "cardiomyo*". The search retrieved 41 articles that were published from 2008 to March 2023.

Effects on Cardiac Injury and Structure
Numerous models have been employed to determine the potential effects of kaempferol on cardiac injury. Various agents have been used in experimental animals and cardiomyocytes in vitro to induce cardiac injury, namely Ang II [6], isoprenaline [25], doxorubicin [7], cisplatin [8], 5-fluorouracil [9], phenylephrine [3], and clozapine [10], in addition to I/R [28] and aortic banding [3]. The inducers promote cardiac injury by elevating oxidative stress and inflammation, the culprits in cardiac injury. A burst of reactive oxygen species (ROS) production occurs upon the reintroduction of oxygen (reperfusion). Kaempferol reduces cardiac injury in animal models, as observed by the reduction in the release of cardiac injury markers, including creatine kinase, creatine kinase MB, troponin, and lactate dehydroge-  (Table 1). It also curbs myocardial infarct size in rat hearts that undergo I/R [27] and in rats exposed to isoprenaline [25]. Meanwhile, in rats receiving 5-fluorouracil, kaempferol post-treatment reduced myocardial inflammatory changes, namely hyaline formation, necrosis, and hyperemia [9]. The observed protective effects of kaempferol come from its antioxidant and anti-inflammatory properties, for which the details are discussed later. The antioxidant property of kaempferol is attributable to the presence of hydroxyl group on the B-ring (Figure 1) [29].  Kaempferol decreases levels of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) [3,8,11]. Both peptides are released in response to volumetric stretch of the atrial and ventricular walls [34]. Structurally, it also diminishes myocardial fiber derangement induced by cisplatin [8]. It reduces the detrimental effects of heart inducers by reducing interventricular septal thickness at diastole (IVSD), left ventricular internal diameter (IVIDd), and posterior wall (LVPWd) in diastole and systole [3,30], indicating the ability of kaempferol to decrease left ventricular wall thickening, a common phenomenon in cardiac remodeling. A change in left ventricular geometry triggers its remodeling [35]. The findings propose that kaempferol possesses antihypertrophic activity in the myocardium, confirmed by a reduction in cardiomyocyte size and heart weight-to-body weight ratio [3,6,8,30]. The protective effects are primarily via its blockade of ROS synthesis [3], which prevents subsequent events. Many events are involved in the development of cardiac hypertrophy and remodeling including fibrosis, apoptosis, and altered mitochondrial function, and the effects of kaempferol on the events will be discussed later.
Excessive activity of renin-angiotensin system could predispose to the development of left ventricular hypertrophy [36]. However, studies investigating the effects of kaempferol on the aspect are still lacking. Ang II, a proinflammatory peptide, is a principal substance in the renin-angiotensin system that plays a principal role in the pathogenesis of hypertrophy [37]. Even though many studies have demonstrated the positive effects of kaempferol on Ang-II-induced hypertrophy, the effects of the bioactive compound on the expression of Ang II; the angiotensin-converting enzyme-the Ang II synthesis enzyme; and its receptor, Ang II type 1 receptor, have not been explored. Understanding the mechanisms could shed more light on its potential pharmacological activities.
The beneficial effects of the compound (10 mg/kg on alternate days for 8 weeks) were also observed in diabetic mice with cardiac injury. It decreased disorganization of myofiber and derangement of cellular structures in the diabetic heart [13]. It is unknown whether its protective effects were via reduction in glucose level since it was not measured in the study. However, kaempferol at higher doses (50−200 mg/kg) was reported to reduce the plasma glucose level in streptozotocin-induced diabetic rats after 15 days [38]. Hyperglycemia augments the production of ROS, leading to increased oxidative stress and inflammation, contributing to diabetic complications [39]. Therefore, it is possible that kaempferol protects the diabetic heart by bringing down the glucose level and thereafter preventing hyperglycemia-induced increases in oxidative stress and inflammation.
Taken together, these findings are suggestive of the ameliorative effects of the compound on heart structure, possibly via its antioxidant and anti-inflammatory properties. Via its anticardiac remodeling property, kaempferol could lessen exacerbating economic strain arises from comorbidities experienced by the patients. However, the impact of kaempferol on cardiac structure has not been extensively investigated. To the best of our knowledge, no clinical trial has been conducted to evaluate the effects of kaempferol on cardiovascular disease.

Effects on Cardiac Function
Kaempferol exhibits positive effects on cardiac function in various cardiac injury models. It improves left ventricular fractional shortening (LVFS) and ejection fraction (LVEF) ( Table 2) [3,6,8,33]. Both parameters are used to detect left ventricular systolic function and are reduced in hearts with left ventricular failure [40,41]. Therefore, these parameters are used in the diagnosis of heart failure [42]. Other than the parameters, improvement of the systolic function by kaempferol (15 mM and 20 mg/kg/day) pretreatment are also evidenced by an increase in the maximal rate of rise (+dp/dt max ) and fall (−dp/dt max ) of left ventricular pressure in I/R-and isoprenaline-induced myocardial injury in rats [27,28,31,43], as well as left ventricular systolic pressure and developed pressure [2,27,32] in models of acute myocardial infarction and I/R injury. However, similar positive findings were not observed in left anterior descending coronary artery (LADCA)ligation-induced heart failure in mice receiving 12 mg/kg/day for 3 days [44], possibly due to the shorter duration of kaempferol treatment compared with other studies. The beneficial effects of the flavonoid were also observed in diastolic function. It reduces left ventricular end-diastolic pressure (LVEDP; see Table 2 measures left ventricular preload and diastolic compliance [45], indicating that kaempferol reduces preload, which is useful in angina. The reduction in the ratio of transmitral flow velocity/mitral annular velocity and the increase in left ventricular volume of diastole by kaempferol treatment indicate an improvement in myocardial diastolic function [3,6]. The betterment in diastolic function by kaempferol eventually indirectly improves the systolic function.
Kaempferol possibly protects by preventing the loss of contractile function due to reducing the number of viable cardiomyocytes following an insult to myocardium, thereby hindering the development of cardiac remodeling. Consequently, myocardial inotropic and lusitropic properties are preserved by kaempferol. The restoration of both properties is crucial in maintaining a normal cardiac performance. Cardiac function is partly determined by myocardial cellular and molecular structures, including calcium regulators and mitochondrial function. Ca 2+ is required for myocardial contraction, while mitochondria functions to supply energy for myocardial activities [46]. However, increased production of ROS can perturb the function of the components. Therefore, the capability of kaempferol in scavenging ROS plays a prominent part in its protective role.
Excessive activation of sympathetic nervous and renin-angiotensin systems may augment the risk of cardiac dysfunction [36]. Raised myocardial epinephrine concentration is associated with increased resting heart rate in rats with failing hearts [47]. However, it is unknown whether kaempferol has any effects on epinephrine level or β1-adrenoceptor in the heart that may somewhat contribute to its cardioprotective effects.
Collectively, kaempferol can improve myocardial left ventricular systolic and diastolic function and prevent the development of arrhythmia. However, studies to date have only examined the effect of kaempferol on left ventricular function; no study has investigated its effect on right ventricular function. Future studies should focus on this aspect.
In heart failure, intracellular diastolic Ca 2+ levels are increased [44], preventing ventricular relaxation and blood refill [57]. In various models of cardiac hypertrophy, kaempferol (10 µM) diminished diastolic Ca 2+ waves and sparks [41,44,54]. The findings suggest that kaempferol improves myocardial diastolic function, as demonstrated in other studies [3,6]. However, a similar protective effect of kaempferol (1 µM) was not observed in isolated hearts obtained from mice with thoracic aortic banding following isoprenaline administration [52]. In the study, kaempferol increased the rate of Ca 2+ accumulation in the mitochondria, leading to an increased production of spontaneous Ca 2+ waves, likely due to increased release of the ion from the sarcoplasmic reticulum. Similar findings were observed in primary cardiomyocytes isolated from mice, using 10 µM kaempferol [52]. The discrepancy is unexplainable, and it should be investigated further.
NF-κB is a principal inflammatory regulator that regulates chemokines and proinflammatory cytokines. To function, it needs to be activated prior to its translocation to the nucleus. This activation is strictly governed by the inhibitor of the κB kinase (IκBα and IκBβ) and inhibitor of the NF-κB kinase (IKKα and IKKβ) [66]. Kaempferol downregulates the TNFα and NF-κB expression and upregulates its inhibitory molecules (IκBα and IKKβ) [2,6,8,11,13,28,62] in models of cardiac disease (Table 4 and Figure 2). TNFα is a stimulator of NF-κB. The findings of these studies suggest that kaempferol diminishes pro-inflammatory stimuli, leading to the upregulation of inhibitory molecules for NF-κB, thereby preventing translocation of NF-κB into the nucleus.
Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a calcium-handling protein. It enhances calcium reuptake into the sarcoplasmic reticulum by SERCA [67]. However, under pathological conditions, CaMKII can become oxidized. In mice with Ang-IIinduced sinus node dysfunction, oxidized CaMKII protein expression was significantly elevated; however, oxidation of the protein was inhibited by simultaneous treatment with kaempferol [51], indicating that the flavonoid conserves the activity of the handling protein via its antioxidant mechanism.
Despite the various protective effects of kaempferol against oxidative stress and inflammation, Hamilton et al. [52] reported that kaempferol (10 µmol/L) increased ROS production in the mitochondria of cardiomyocytes isolated from mice with cardiac hypertrophy that underwent arrhythmia induction using isoprenaline. The concentration of kaempferol used was in line with other studies [4,5,10]. The discrepancy is unexplainable until further investigations are performed.
Taken together, kaempferol exerts antioxidant and anti-inflammatory properties in numerous models of cardiac pathology by modulating Nrf2, NF-κB, MAPK, and PI3K/Akt/GSK3β signaling pathways. However, the detailed mechanisms of its antioxidant and anti-inflammatory properties in pathological cardiac events are unclear. The effects of kaempferol on the calcineurin/nuclear factor of activated T cells (NFAT) inflammatory signaling pathway should also be investigated.

Effects on Cardiac Mitochondrial Function Other Organelle Damage
Mitochondria, the cellular powerhouse, are vital in all cells, including cardiomyocytes, for generating energy for cellular functions [68]. The mitochondrial membrane potential plays a crucial role in storing energy during oxidative phosphorylation. Disturbed potential production, such as in the presence of increased ROS production, may cause mitochondrial dysfunction. This results in the opening of the mitochondrial permeability transition pore (mPTP) and ATP loss [69,70].
Human silent information regulator type 1 (SIRT1) is a nuclear protein which has a role in mitochondrial biogenesis and turnover. Together with its substrate, peroxisomeproliferator-activated receptor gamma coactivator-1α (PGC-1α), SIRT regulates mitochondrial energy metabolism. It also governs mitochondrial longevity by promoting the mitophagy of damaged mitochondria [71]. Kaempferol raises the expression of SIRT1 in cardiomyocytes exposed to anoxia/reperfusion [4]. The addition of sirtinol, a SIRT1 inhibitor, impedes the ROS-inhibiting effect, mPTP opening suppression, and mitochondrial membrane potential restoration by kaempferol [4]. The findings confirm the involvement of SIRT1 in the cardioprotection conferred by kaempferol, which then boosts the metabolic function and longevity of mitochondria.
Exposure to stress such as I/R causes disproportioned proteostasis at the endoplasmic reticulum (ER) that results in an accumulation of nonfunctional misfolded proteins. This will trigger ER stress via the unfolded protein response (UPR) signaling pathway to promote cellular repair. Activating transcription factor 6α (ATF6α), protein kinase RNA-like ER kinase (PERK), and inositol-requiring transmembrane kinase endoribonuclease-1α (IRE1α) are ER stress sensors which interact with glucose-regulated protein 78 (GRP78) [72]. The accumulation of misfolded proteins causes dissociation of GRP78 from the stress sensors [73]. Kaempferol protects against the activation of ATF6α, IRE1α, GRP78, X-box binding protein 1 (XBP-1), and eukaryotic initiation factor 2α (eIF2α) in cardiomyocytes exposed to I/R ( Table 5) [32]. XBP-1 is spliced by IRE1α before being translocated to the nucleus, while eIF2α is activated by PERK [73]. Another downstream molecule that is activated in ER stress is C/EBP homologous protein (CHOP), the activation of which is also reduced by kaempferol [32]. Activation of the ER stress proteins will ultimately inhibit the initiation of global protein translation [73]. The findings propose that kaempferol provides protection via its antioxidant and anti-inflammatory activities by diminishing potential stress to ER, thereby preventing the activation of the UPR signaling pathway. The activation of the stress sensors that trigger GRP78 dissociation is then halted by kaempferol. Finally, the synthesis of nonfunctional proteins is decreased. Thus, it can be concluded that kaempferol conserves functional protein synthesis.
Another organelle that is important in cellular function is lysosome. It has a role in cellular protein trafficking via non-classical protein secretion, which is independent of the ER and Golgi apparatus [73], in addition to its degradative function [74]. Kaempferol decreases lysosomal membrane instability [10], in line with the decreased oxidative stress, inflammation, and apoptosis. Indirectly, kaempferol increases lysosomal survivability and, hence, restores the functions of the organelle.
Therefore, kaempferol can protect organelles against oxidative-induced damage. It preserves mitochondrial, lysosomal, and ER membrane integrity, thereby maintaining cellular functions. Other aspects, such as mitochondrial transcription factor A (TFAM), which has a role in mitochondrial replication and energy generation [75]; and fission 1 (FIS1) and optic atrophy 1 (OPA1), which are involved in mitochondrial fission and fusion [76], respectively, could be examined to better comprehend the effects of kaempferol on mitochondrial function.

Effects on Cardiac Apoptosis
Accumulating evidence demonstrates the anti-apoptotic properties of kaempferol in various models of cardiac disease. Apoptosis, a programmed cell death that is elevated in experimental cardiac diseases, has a crucial role in mitochondrial function and is associated with excessive ROS production [4,5,33]. Kaempferol diminishes the expression of major protease effectors caspase 1 and caspase 3, thereby reducing the number of apoptotic cells (Table 6) [2,4,5,7,11,27,28,31,32,51]. The protective effects of the flavonoid are extended to a reduction in apoptotic DNA fragmentation, evidenced by decreased poly(ADP-ribose)polymerase (PAPR) cleavage and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells [2,7,8,11,13,51]. Kaempferol exerts its protective effects by inhibiting the release of cytochrome c into the cytosol [4,7,21], thereby preventing the formation of cytochrome c-apoptotic protease activating factor 1 complex. The complex activates caspase activity (Figure 2) [77]. Caspase activation can be initiated by TNFα binding to its receptors [78], suggesting that the suppressive effects of kaempferol on TNFα expression contribute to caspase inactivation.
Myocardial fibrogenesis is regulated by growth factors such as TGFβ1 and connective tissue growth factor (CTGF), as well as the small mothers against decapentaplegic (Smad) signaling pathway [83]. Cardiac insult triggers the binding of active TGFβ1 to its receptor (TGFR), stimulating collagen synthesis via the Smad signaling pathway [84]. Furthermore, TGFβ1 enhances the conversion of fibroblasts into myofibroblast, marked by the presence of α-smooth actin (α-SMA) [46], the expression of which is downregulated by kaempferol (Table 7) [30]. The expression of the growth factors and phosphorylated Smad was significantly downregulated by kaempferol in various models of cardiac dysfunction [3,6,13,30]. The observed effects of kaempferol indicate that the flavonoid curtails the signaling pathway of collagen synthesis.
MMP degradation is regulated by the tissue inhibitor of metalloproteinase (TIMP); however, studies investigating the effects of kaempferol on TIMP are lacking. Other signaling mechanisms, such as the wingless-related integration site (Wnt)/β-catenin [81,85] and Hippo-Yes-associated protein/transcriptional coactivator with PDZ-binding motif (Hippo-YAP/TAZ) [86], are also involved in the pathogenesis of myocardial fibrosis. Kaempferol may modulate these signaling pathways.

Conclusions and Directions for Future Studies
Kaempferol is a natural antioxidant and anti-inflammatory which can be abundantly found in many plants. Increasing data from animal and in vitro studies have demonstrated the cardioprotective role of kaempferol. It demonstrates beneficial effects on cardiac structure and function. Most of the studies exhibit its prominent anticardiac remodeling. It protects against cardiac hypertrophy and remodeling in various experimental cardiac diseases via its regulation of myocardial calcium level, apoptosis, mitochondrial function, oxidative stress, inflammation, and extracellular matrix assembly. It also displays antiarrhythmic activity, which might be beneficial in patients suffering from myocardial infarction. The findings support its potential as a promising candidate for managing cardiac diseases. However, its effects on congenital cardiac disease and acquired heart diseases in pregnancy are yet to be examined.
Another aspect that could be explored is the impact of kaempferol on autophagy, a mechanism that is also involved in the pathogenesis of diverse cardiac diseases. Clinical studies should be conducted to confirm the protective effects of kaempferol that were observed in laboratories.