Bacopa monnieri extract increases rat coronary flow and protects against myocardial ischemia/reperfusion injury

Background This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points. Methods In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 μg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca2+-currents (ICa, L) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion. Results In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 μg/ml) and 216 ± 21% (100 μg/ml), compared to control (5 ± 3%) (n = 8–10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 μg/ml), 82 ± 10% (100 μg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 μg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 μg/ml) reduced ICa, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50–1000 μg/ml) B. monnieri. Conclusions B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1637-z) contains supplementary material, which is available to authorized users.


Background
Disease of the myocardial vasculature including the coronary artery is a major cause of disability and death in spite of widespread application of highly efficacious cardiovascular drugs. Myocardial infarction (MI) most commonly arises from an unstable atheroma forming fragments and clots that occlude downstream vessels culminating in regional myocardial ischemia [1]. The resultant energy failure has numerous cytopathological consequences, including changes of pH and Na + levels, subsequently leading to reversal of Na + /Ca 2+ exchange, which in turn results in cytosolic Ca 2+ loading and activation of proteolytic enzymes mediating necrosis [2]. Mitochondrial Ca 2+ homeostasis also fails, leading to disruption of the electron transport chain, cytochrome C release and initiation of apoptosis. However, these changes are slow during sustained occlusion [2] but most damage begins on reperfusion. Glucose is shunted into glycolysis producing lactic acid and mitochondrial beta-fatty acid oxidation [2]. Early reperfusion is accompanied by excessive superoxide production [3] and consequently leads to other reactive species which increase cell damage and to further cytosolic Ca 2+ loading [4]. Fragments of damaged cells then activate resident immune cells and immune receptors on endothelial cells, which in turn secrete inflammatory cytokines and adhesion proteins, respectively. Endothelial cells generate further superoxide via adherent xanthine oxidase, cytokine activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and oxidised nitric oxide (NO) synthase [5]. Endothelial cells not only lose their vasorelaxant influence over vascular smooth muscle, but they release vasoconstrictors, especially endothelin-1 [6], which further exacerbates the conditions causing MI. Endothelial injury manifests as cell blebbing, swelling and plugging culminates in "no-reflow" [7]. Thus we have a potent cytotoxic combination of Ca 2+ , superoxide, inflammation and vasoconstriction, all of which conspire over the ensuing minutes-hours in myocardial cell death.
Targeting these pathologies individually produces improved recoveries in experimental animals provided the protectant is present before ischemia begins, but they have failed in translation. Thus at present, the standard treatments focus on atherosclerosis by using thrombolytics, anticoagulants or angioplasty. Clinical applications targeting mitochondria have met with some limited positive outcomes [2,8]. Nevertheless, it is clear that for any protectant to be useful, it would have to be administered prophylactically to high risk patients, act against the major reperfusion pathologies, be cheap and have low toxicity and ideally, help to reduce the initial risk of MI. It is unlikely that any classical single substance or drug could perform in this manner, but several multifunctional herbals have the potential to fulfill such a role.
They include, a medicinal Ayurvedic herb, which is thought to improve cardiovascular function. Thus, oral B. monnieri given to rats for three weeks reduced biochemical and histopathological perturbations caused by ischemia/reperfusion (I/R), while increasing myocardial antioxidant enzymes and reducing myocardial apoptotic signaling proteins [9]. Over-dosing rats with isoproterenol reduced endogenous antioxidant enzymes and increased lipid peroxidation, but less so in those animals which had been chronically treated with B. monnieri [10]. Necrosis and markers of myocardial injury were also reduced.
There is also some evidence that B. monnieri improves systemic vascular function. It relaxes a wide range of arteries [11], partly through endothelial NO release and in vascular smooth muscle by inhibiting Ca 2+ influx and Ca 2+ release from the sarcoplasmic reticulum [11]. Nevertheless, chronic oral administration of B. monnieri to rats for 12 weeks increased cerebral blood flow without changing blood pressure [12]. This is particularly important because B. monnieri has been traditionally used to enhance memory and several clinical trials suggest that it does have nootropic actions [13,14]. Furthermore, it is neuroprotective [15,16] and the mechanism of action is mainly related to its antioxidant and antiinflammatory properties [16][17][18]. Recently, we have shown that B. monnieri had no signs of toxicity in either acute or chronic oral toxicity tests of both male and female rats, indicating that it is relatively safe [19].
Clearly, several lines of evidence point to B. monnieri having potentially favorable cardiovascular actions arising from the hemodynamic changes and protective effects in vulnerable tissues as well as reducing some of the pathological changes contributing to I/R injury. Nevertheless, it is unclear whether these effects of B. monnieri translate directly to improving the function of the myocardium or whether they could ameliorate the damage following cessation and restoration of blood flow, i.e., I/R injury. To answer this, we aimed to show that an extract of B. monnieri could improve myocardial perfusion in normally beating isolated rat hearts and to show that it produces an independent acute recovery in cardiac function after I/R while at the same time not creating cellular toxicity in isolated ventricular myocytes.

Preparation of Langendorff heart perfusion and protocols
Effect of B. monnieri on normal and I/R hearts Rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) followed by heparin (250 UI/kg, intravenously). The level of anesthesia was assessed by toe pinch and corneal reflex before the surgery. The hearts were then rapidly removed and immersed in ice-cold KHB to stop contractions, and the myocardium was perfused retrogradely with KHB using the Langendorff method at a constant perfusion pressure of 80 cm of water (7.8 kPa). The perfusate was bubbled with 95% oxygen and 5% carbon dioxide and maintained at 37°C. To measure left ventricular end diastolic pressure (LVEDP), a water-filled latex balloon (4x8 mm, EMKA Technologies, Paris, France) was inserted into the left ventricle and connected to a pressure transducer (682002, Becton Dickinson, Sandy, Utah, USA) and a Gould TA240 Physiograph (Gould Instrument Systems, Courtaboeuf, France). The pressure within the balloon was adjusted to 2-8 mmHg, i.e., equivalent to the normal LVEDP [22].
The intraventricular pressure and heart rate (HR) were continuously recorded. The left ventricular developed pressure (LVDP) calculated as left ventricular systolic pressure minus LVEDP, was used as an indicator of left ventricular systolic function. Total coronary flow was measured by collecting aliquots of coronary venous effluent. All hemodynamic parameters were measured at 2 min intervals. The hearts were stabilized for 10-20 min and then the following two experimental protocols (Table 1) were carried out (54 rats, n = 8-10/group).
The concentrations of B. monnieri extract were based on earlier in vitro studies where the IC 50 was around 100 μg/ml for inducing vasorelaxation in small resistance arteries [11].

Effect of B. monnieri on myocardial infarct size
Rat hearts were isolated and perfused using the Langendorff method as described above. The hearts were preperfused with KHB (control) or KHB supplemented with 100 μg/ml B. monnieri for 30 min prior to 30 min global ischemia followed by 2 h reperfusion, sufficient to allow histological changes to develop [23] (14 rats, n = 8 for control, n = 6 for treatment). After 2 h of reperfusion, hearts were perfused for 5 min with 10 ml of 1% (w/v) triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO, USA), then removed and placed in 1% TTC solution at 37°C for 10 min [23]. The hearts were incubated in 2.5% glutaraldehyde for 1 min, and set in 5% agarose before sectioning in 750 μm thick slices. All slices were incubated in 10% (v/v) formaldehyde overnight at room temperature before re-hydration overnight with 2) 30 μg/ml B. monnieri extract (n = 10) 3) 100 μg/ml B. monnieri extract (n = 8) Protocol: Isolated hearts were perfused with KHB alone or KHB containing B. monnieri extract for 30 min (Fig. 1).
phosphate buffer saline (PBS) at 4°C. The heart sections were scanned and planimetry was carried out and surface area of the whole, and TTC-negative, myocardium was transformed to volume [23]. The TTCnegative infarction volume was expressed as a percentage of heart volume. All analyses of infarct size were done by an investigator, who was blinded with regard to the group assignments.
Electrophysiological measurements of the L-type Ca 2+ current Effect of B. monnieri (100 μg/ml) on L-type Ca 2+ current (I Ca, L ) was studied in HL-1 cells (kind gift from Prof. Dr. William C. Claycomb, Louisiana State University Medical Center, LA, USA). This cell line, derived from an atrial tumor lineage of the AT-1 mouse, spontaneously contracts and retains the phenotypic characteristics of cardiomyocytes [24], including the expression of L-type Ca 2+ channels. Inhibition of I Ca, L by nifedipine was previously demonstrated in HL-1 cells as shown in Additional file 1: Figure S1. The whole-cell patch clamp technique was used for recording of I Ca, L . The experimental protocol was adopted from a previous study [25].

Determination of cell viability
Cell viability was assessed by trypan blue exclusion assay [26]. After cell plating, the isolated cardiac myocytes were replenished with modified M199 culture medium (control) or medium containing B. monnieri extract (10-1000 μg/ml) for 1 h. Then the supernatants were removed, the cells were resuspended in 50 μl 0.4% trypan blue solution and incubated for 5 min. After that, supernatants were removed and replaced with PBS solution. One hundred cells in each well (n = 3) were randomly counted under a light microscope. The cell viability was calculated as number of unstained cells divided by total number of cells and expressed as a percentage.

B. monnieri increased coronary flow in normal hearts
In normally perfused isolated rat hearts, during stabilization, all hemodynamic parameters including coronary flow (Fig. 1a), LVDP and HR remained constant and similar in all treatment groups. Application of 30 or 100 μg/ml B. monnieri for 30 min produced a concentration-dependent increase in coronary flow (Fig. 1b) and LVDP (Fig. 1c). The coronary flow was 1.6fold increased in the presence of B. monnieri at 30 μg/ ml (mean = 168 ± 13%, n = 10), and 3.1-fold at 100 μg/ml (mean = 321 ± 21%, n = 8) compared to control (mean = 105 ± 3%, n = 9) (Fig. 1e). There were also small increases in LVDP (Fig. 1c, e), but no change in HR (Fig. 1d, e) in response to both concentrations of B. monnieri. Fig. 1 B. monnieri improved hemodynamics in isolated rat hearts. a coronary flow (ml/min) using normal perfusate (control) and perfusate containing 30 or 100 μg/ml B. monnieri extract for 30 min, b coronary flow, %, c LVDP, %, d HR, %: b-d expressed as percentage of mean data from the same heart compared to the 10 min stabilization using normal perfusate and e mean expressed as the average of each parameter after application of normal perfusate or perfusate containing B. monnieri extract for 15 min onward. Data are expressed as mean ± SEM (n = 8-10 hearts) and *p < 0.05, **p < 0.001 for 30, 100 μg/ml B. monnieri compared to control, ## p < 0.001 for 30 μg/ml compared to 100 μg/ml B. monnieri B. monnieri improved recovery after I/R B. monnieri extract was added to the perfusate for 10 min whereupon coronary flow increased as above.
The perfusion was then stopped for 30 min to emulate global ischemia after which perfusion was restored for a further 30 min (Fig. 2a). In the control group (n = 9), all the hemodynamic parameters were substantially reduced compared to before ischemia (Fig. 2b-e). However, pretreatment with 30 or 100 μg/ml B. monnieri improved cardiac function in I/R heart. Coronary flow was immediately restored to 90 ± 10% (n = 9), and 111 ± 7% (n = 9) with pre-exposure to 30 and 100 μg/ml B. monnieri, respectively (Fig. 2b). Although coronary flow declined, this was always higher in the B. monnieri pre-treated groups at every time point compared to those without B. monnieri (p < 0.001). LVDP as a measure of myocardial contractile force, gradually increased in B. monnieritreated groups and maintained after reperfusion for 20 min onwards (52 ± 6%, 84 ± 10%, 82 ± 10%, for control, 30 μg/ml and 100 μg/ml B. monnieri, respectively, p < 0.001 for B. monnieri compared to control) (Fig. 2c). Likewise, functional recovery showed a sustained increase (Fig. 2e). HR was depressed on initial reperfusion, but there was no clear change during the recovery period (Fig. 2d).

Pretreatment of B. monnieri reduced myocardial infarction
An ex vivo global ischemia for 30 min followed by 2 h reperfusion resulted in the myocardial infarction up to 51 ± 2% of ventricular volume (n = 8) (Fig. 3, control). Pretreatment with 100 μg/ml B. monnieri substantially B. monnieri reduced I Ca, L in HL-1 cardiac cells Ca 2+ is an important mediator of ischemic cell damage and in cardiac muscle, an important Ca 2+ influx pathway is through L-type Ca 2+ channels and blockade of these channels can offer some protection [27,28]. Whole-cell voltage clamping of HL-1 cardiac cells showed transient inward currents of −71.4 ± 19.6 pA previously identified as I Ca, L [25] ('control' in Fig. 4a). The extract decreased the peak current amplitude of I Ca, L to about one third i.e., to 37.1 ± 4.1% compared to control (p < 0.001, n = 4, Fig. 4b).
Cytotoxicity of B. monnieri extract Direct effect of B. monnieri on cell viability of freshly isolated rat ventricular myocytes was determined using the trypan blue exclusion assay. After 1 h incubation of ventricular myocytes with various concentrations of B. monnieri extract (10-1000 μg/ml), cell survival was decreased in those cells exposed to concentrations of 50 μg/ml and above (Fig. 5). High concentrations of the extract (≥500 μg/ml) produced more than 50% cell death (Fig. 5). Fig. 3 The sensitivity to myocardial infarction in rat hearts subjected to global ischemia/reperfusion, in the presence or absence of 100 μg/ml B. monnieri. The data shown is the normalised infarction volume of isolated rat hearts in the presence of 100 μg/ml B. monnieri extract for 30 min before the onset of 30-min global ischemia followed by 2 h of reperfusion. The bar graphs represent means ± SEMs (n = 8 for control and n = 6 for B. monnieri extract, **p < 0.0001 vs control)

Discussion
The key findings of the present study were that B. monnieri extract induced a concentration-dependent improvement in coronary flow, promoted cardiac function, and reduced infarct area resulting from ischemia and reperfusion in isolated perfused hearts. Previously, B. monnieri was shown to dilate various isolated rat arteries by (i) stimulating endothelial NO release, and (ii) acting directly on vascular smooth muscle cells by inhibiting extracellular Ca 2+ influx and Ca 2+ release from sarcoplasmic reticulum [11]. Also, B. monnieri inhibits Ca 2+ influx in various tissues including several types of isolated rat arteries [11,29], guinea pig ileum and trachea [29,30] and rat trachea [31]. Similar mechanisms may well operate in the coronary vasculature. Likewise, chronic oral administration of B. monnieri increased cerebral blood flow in normal rats [12].
Coronary blood flow is normally mediated by demands of myocardial contraction [32]. However, in the presence of B. monnieri coronary flow was substantially greater than the increased LVDP, which suggests that the vasculature dilated independently of myocardial needs. Nevertheless, there was a slightly increased myocardial contractility with B. monnieri, which might be facilitated by the augmented blood flow as seen in the canine and rat hearts [33,34]. Moreover, the positive ionotropic effect of B. monnieri suggests the extract had no adverse action on the intact myocardium.
However, there was a clear disconnect between blood flow and contractility during post-ischemic reperfusion: restoration of blood flow was immediate and slowly declined thereafter while LVDP developed over 15 min (Fig. 2). This implies that contractility was not solely a direct consequence of better blood flow. One explanation for this disconnect could involve Ca 2+ influx, which we show was inhibited in isolated myocytes. On the one hand, inhibition of vascular smooth muscle L-type Ca 2+ maximises the vasodilatation through the lingering action of B. monnieri, thus providing metabolic resources to re-establish the extraand intracellular milieu. At the same time, this same action on cardiac myocytes inhibits their contraction until the accumulated Ca 2+ is cleared after which they can re-establish sinus rhythm. This Ca 2+ overloading of cardiac myocytes has been emphasised in many studies and various drugs targeting this overload including Ca 2+ -channel blockers improve post-IR recovery [27,28].
Our present protocol was short-term but B. monnieri may well have more lasting beneficial effects in vivo [9,10]. For example, in coronary I/R, there is inflammation beginning with endothelial cell blebbing and swelling followed by platelet and leucocyte adhesion leading to microvascular block and a "no flow" condition lasting many hours [7].
With B. monnieri, the initial flow was good but gradually decreased with continued reperfusion for all three treatment groups although the pretreated ones maintained superior flow rates (Fig. 2). The prolonged effects of inflammation do not apply to the present protocol, but our data suggest that the coronary vasculature enters this critical period with a distinct functional advantage. These antiinflammatory effects of B. monnieri may be mediated through one of its constituents, betulinic acid [35,36] by potently blocking the inflammatory transcription factor NF-kB or p38/ERK MAPK pathways [35] thereby preventing the augmented cytokine production [36].
Antioxidant actions are universally claimed to be an action of most herbals including B. monnieri [9,17]. Yet, whatever of this antioxidant remained in the tissue on reperfusion without extract would have little real impact on the superoxide onslaught. Nevertheless, polyphenols and other plant biologicals stereospecifically cause Nrf-2 nuclear translocation thereby increasing expression of some anti-oxidant enzymes [37][38][39][40] and their activation can provide protection against I/R [41].
Our B. monnieri extract appeared to be cytotoxic at a threshold concentration of~50 μg/ml but, nevertheless, it increased LVDP suggesting that toxicity was absent in the intact myocardium. The explanation for this discrepancy may be a combination of: (i) toxicity was studied in culture where myocytes are directly exposed to the extract, while in the perfused heart the concentration of the extract within the extracellular space is likely to be lower, (ii) the incubation time of cultured myocytes in the extract was longer (1 h) than the 10 min perfusion. Importantly, the intriguing notion is that if those components of B. monnieri having beneficial and having cytotoxic actions are different, the cardioprotective components could display a greater efficacy for postischemic recovery.
This study has revealed an improved coronary flow, increased myocardial function and contracted infarct area. The additional anti-inflammatory, antioxidant defenses and reduced Ca 2+ -overload demonstrated in other systems, suggest that B. monnieri might provide a multipronged defense against ischemic cell death. Nevertheless, the model used here, and most of the animal models for I/R poorly replicate the human condition where the underlying vascular pathology develops over a generation and is influenced by 'conditioning'. Thus, major impacts in the treatment of I/R will only become translatable when accurate animal models for the human condition emerge.

Conclusion
B. monnieri protected against I/R injury in rat heart as judged by its ability to recover coronary flow, contractile force and function, and reduced infarct volume. As such, it could form the basis of a promising cardioprotectant (and cerebroprotectant) for patients at risk from infarcts. In pursuing this, the protectant and cytotoxic components need to be identified and plant strains or extraction protocols showing more efficacious chemical profiles selected.

Additional file
Additional file 1: Figure S1.