Depside salts from Salvia miltiorrhiza improve myocardial microperfusion in rats using laser Doppler flowmetry¹

Introduction

Danshen, the dried roots of the medicinal plant Salvia miltiorrhiza, is a traditional Chinese herbal medicine used for the treatment of coronary heart disease. Depside salts from S miltiorrhiza are novel drugs in which magnesium lithospermate B (MLB, Figure 1) and its analogues are the active components. The pharmacological activities of MLB, which is a major aqueous extract ingredient of Danshen, have been extensively investigated. MLB has been shown to reduce the size of myocardial infarction^[1] as Danshen does^[2]. Further studies investigated the involving mechanisms in several aspects. First, it was speculated that the antioxidative and radical scavenging property of MLB played an essential role in its cardioprotective efficacy^[3–6]. Second, MLB was reported to stimulate the production of nitric oxide in endo-thelial cells^[7–9], possibly via the induction of constitutive NOS (cNOS) expression^[7]. Third, MLB was demonstrated to behave in a Ca²⁺ antagonistic fashion voltage-dependently^[10] and inhibited extracellular calcium influx^[8,9]. In addition, MLB suppressed apoptosis induced by myocardial ischemia/reperfusion^[11,12], probably through the inhibition of the c-Jun N-terminal kinase 3 (JNK 3) activity^[11] and stress-activated protein (SAP) kinase activity^[12]. However, there has been no direct evidence on its effect on coronary micro-circulation.

Figure 1 Structure formula of MLB.

Laser Doppler flowmetry, which offers a continuous real-time measurement of blood cell perfusion in the microcirculatory beds of tissues, is a prospective method to be applied for myocardial microvascular perfusion assessment. Ahn et al^[13] demonstrated that their data of the local myocardium perfusion correlated well with coronary sinus blood flow in the empty-beating hearts of pigs. Sidi et al^[14], who considered movement artifacts from the contraction of myocardium, found that the local changes in flow measured by laser Doppler flowmetry correlated with the regional myocardial blood flow changes obtained by means of radioactive microspheres, but not with global contractility which was evaluated by first-time derivative of left ventricle pressure (LV dp/dt). Klassen et al^[15] proposed a method to overcome the limits in measuring regional phasic myocardial red cell flux during a cardiac cycle using a laser Doppler velocimeter by inserting the probe into the beating heart in various locations. They suggested that the fiber tip moved in concert with the contracting muscle fibers with little tethering of myocardial action, thus only registering movements from the red blood cells. They also confirmed their hypothesis subsequently in the canine myocardium^[16]. Therefore, it appears feasible to apply the laser Doppler technique quantitatively in evaluating the responses of drugs to myocardial microperfusion.

In the present study, we employed the laser Doppler flowmetry system to investigate the effect of S miltiorrhiza depside salts on myocardial microperfusion. We found that S miltiorrhiza depside salts improved coronary microcircula-tion, as well as cardiac output (CO).

Materials and methods

Drugs and reagents S miltiorrhiza depside salts which contain MLB (≥80.0%) were provided by the Department of Phytochemistry, Shanghai Institute of Materia Medica (Shanghai, China). Nifedipine (Sigma, USA) was dissolved in a 5% DMSO/physiological saline solution, and the other drugs involved were dissolved in physiological saline only. Drug concentrations were adjusted to yield an injection volume of 0.1 mL/100 g of body weight.

Experimental preparation Male Sprague-Dawley (SD) rats (Certificate No SCXK 2003–0003, Shanghai SLAC Laboratory Animal Co, Shanghai, China) weighing 250–300 g were housed in cages (2 per cage), maintained at 25 °C with 12 h of light, and were allowed free access to water and standard rat chow. The rats were anesthetized using sodium pentobarbital (40 mg/kg, ip), and additional doses were given to maintain anesthesia when necessary. Body temperature was maintained at 37 °C with a thermostat-controlled operation table (Harvard Apparatus, USA). A catheter (PE-10 fused with PE-50) filled with heparin/saline (2 mg/mL) for measurement of mean arterial pressure (MAP) was introduced into the abdominal aorta through the right femoral artery and connected to a pressure transducer (Powerlab MLT 844, ADInstruments, Australia). A second catheter for drug administration was inserted in the right femoral vein. The trachea was cannulated and connected to a respirator (HX-200, Chengdu Taimeng Technology Co, China). Arterial blood gases (i-STAT Portable Clinical Analyzer, Abbott Labora-tories, Abbott Park, IL, USA) were kept within the normal range by adjusting ventilation volume and/or rate as needed. All experimental protocols involving the use of animals were approved by Animal Care and Use Committee of the Chinese Academy of Sciences.

Determinations of CO A left-sided thoracotomy was performed from the second to the fourth intercostal space to expose the lungs in the thoracic cavity, and the heart was suspended in a pericardial cradle. After the ascending aorta had been gently isolated from the pulmonary artery by blunt dissection, the ultrasonic perivascular flow probe (2SB) of ultrasonic Doppler flowmetry (T206, Transonic Systems Inc, Ithaca, NY, USA) was mounted on the root of the ascending aorta, with the sterile surgilube jelly (E Fougera & Co, USA) filling the probe window to get rid of air bubbles, which permitted the transmission of the ultrasound signal. Additional jelly, loaded by a 20 mL syringe, was injected into the flow probe lumen if necessary.

Determinations of myocardial microperfusion To assess microcirculation, a needle probe (MNP110, length 25 mm, diameter 480 µm) of laser Doppler flowmetry (Powerlab ML191, ADInstruments, Australia) for microcirculation was inserted into a specially-designed holder which was glued to the epicardium of the left ventricular myocardium close to the left anterior descending coronary artery where no obvious large vessels lay. The orientation of probe was appropriately adjusted against the holder until a stable, high-quality signal for laser Doppler flowmetry was obtained. The laser Doppler flowmetry signal was recorded as arbitrary blood perfusion units (BPU), a relative unit scale defined by reference to a controlled motility standard. Due to the flexibility of the optical fiber and the plastic harmonious movement of the light probe accompanying the beating heart, virtually no tethering of the myocardium took place.

Protocol The doses of S miltiorrhiza depside salts were selected based on the pilot study and the literature^[1–4]. After thoracotomy with instrumentation, the rats underwent a recovery period of 30 min for hemodynamics stabilization. The test drugs were injected intravenously, and no animal received more than 1 agent, except those who received vehicle control administration. After each drug was intravenously administered within 15 s, the changes of all para-meters, recorded simultaneously by a computer using the chart software version 5.4.1 (ADInstruments, Australia), were measured for 30 min consistently.

Criteria for an acceptable experiment The rats whose MAP was below 80 mmHg after stabilization were excluded, as well as those who failed to maintain stable within 1 h post-operatively. It was essential that arterial blood gases remained within the physiological range, and standardized respiratory monitoring was performed.

Calculation of hemodynamics The flow-probe cable was connected to a model T206 flow meter (Transonic Systems Inc, USA) to measure CO^[12], which was equal to the ascending aortic flow and was expressed as flow per kg of body weight (CI). MAP was automatically calculated from femoral artery pressure by the software. Systemic vascular resistance (SVR) was determined using the following formula: SVR=MAP/CI. Cardiac inotropy was estimated using the maximal velocity of flow increase (df/dt_max)^[27]. Myocardial microperfusion, detected by laser Doppler flowmetry, was quantified by change percentage due to the temporal and spatial microheterogeneity of the myocardial blood flow^[13], as well as the small region of the myocardium under observation. The mean values for each determination were analyzed over a 10 s period.

Data statistical analysis Results were expressed as mean±SD. Parameters at the same time point were compared with the vehicle control by unpaired Student’s t-test. Student’s paired t-test was used for comparison of the parameters with their corresponding baseline values. A value of P<0.05 was considered significant.

Results

Feasibility of laser Doppler flowmetry for myocardial microperfusion assessment Myocardial microperfusion profile was measured for up to 30 min without intervention. Ascending aortic flow, femoral artery pressure, and myocardial microperfusion were recorded for approximately 10 min while no intervention occurred (Figure 2). When a satisfactory positioning of the probe was made, a distinct, regular, consistent pattern of myocardial microperfusion waveform was observed which remained constant during control and changed correspondingly, following an intervention, as that of other parameters. Ventilation played a pivotal role in the behavior of hemodynamics, and myocardial microperfusion varied correspondingly as well (data not shown). No alteration in either the pattern or value of myocardial microperfu-sion was observed as long as ascending aortic flow and femoral artery pressure were constant (Figure 2).

Figure 2 Representative tracings of ascending aortic flow, femoral artery pressure, myocardial microperfusion, and other hemodynamic parameters recorded in anesthetized SD rats. Cardiac output was calculated from the pulsatile value of ascending aortic flow; Mean artery pressure was calculated from femoral artery pressure; heart rate was calculated from the pulse of femoral artery pressure; myocardial microperfusion measured by laser Doppler flowmetry; and the signals were recorded as blood perfusion units (BPU).

The myocardial microperfusion profile recorded by a laser Doppler flowmetry device behaved in a phasic fashion throughout the cardiac cycle (Figures 2, 3). The relationships between ascending aortic flow, femoral artery pressure, and myocardial microperfusion in cardiac cycles are shown (Figure 3). As we know, diastole originated at the same time point as when the ascending aortic flow returned to the baseline and was indicated by the closure of the aortic valve. The blood perfusion waveform, which increased with the femoral artery pressure and decreased gradually after reaching a peak in diastole, showed a dicrotic notch, as seen in the femoral artery pressure to some extent. The peak of ascending aortic flow from which CO was calculated coincided with the nadir of the myocardial microperfusion waveform where the blood flow started to increase until the early period of diastole (Figure 3). The combined curves showed that the myocardial microperfusion was predominately diastolic and its phase was opposite to the ascending aortic flow.

Figure 3 Combined tracings of ascending aortic flow, femoral artery pressure and myocardial microperfusion, which indicated the relationship between myocardial microperfusion and cardiac cycle.

Representative vasodilators, nifedipine and captopril, were selected for system test and confirmation. The mean basic values of myocardial microperfusion observed in each group for 75 µg/kg nifedipine, 5 mg/kg captopril, and vehicle control were 471±58 BPU, 458±74 BPU, and 445±58 BPU, respectively. Our data showed that intravenous administration of both 75 µg/kg nifedipine and 5 mg/kg captopril exerted an increasing effect on myocardial microperfusion, as well as on CO (Table 1). Intravenous administration of 75 µg/kg nifedipine increased myocardial microperfusion by 7.1%±2.9% (P<0.01) maximally, and 5 mg/kg captopril improved myocardial microperfusion 4 min after drug intervention with a peak increase of 9.5%±4.6% (P<0.01). Typical tracings of CO, artery pressure, and myocardial microperfu-sion after the intravenous administration of 75 µg/kg nifedi-pine are shown in Figure 4.

Table 1 Effects of nifedipine and captopril on CI and MMP in SD rats. CI and MMP refer to cardiac index and myocardial microperfusion respectively. Values are mean±SD. n=7. ^bP<0.05, ^cP<0.01 vs baseline; ^eP<0.05, ^fP<0.01 vs vehicle control.
Full table

Figure 4 Global view of the effect of nifedipine (75 µg/kg) intravenously administered on ascending aortic flow, femoral artery pressure, myocardial microperfusion, and other hemodynamic parameters with a compression ratio of 1000 in anesthetized SD rats. Cardiac output was calculated from the pulsatile value of ascending aortic flow; Mean artery pressure was calculated from femoral artery pressure; heart rate was calculated from the pulse of femoral artery pressure; myocardial microperfusion measured by laser Doppler flowmetry; and the signals were recorded as blood perfusion unit (BPU).

Effects of S miltiorrhiza depside salts on hemodynamics Intravenous administration of S miltiorrhiza depside salts resulted in a significant immediate increase in CO and cardiac inotropy, as well as a fall in SVR (Figure 5). S miltiorrhiza depside salts (30 mg/kg and 60 mg/kg) increased CI by 12.2%±6.3% (P<0.01) and 20.1%±3.5% (P<0.01) from the baseline, respectively, which was in a dose-dependent manner. S miltiorrhiza depside salts (30 mg/kg and 60 mg/kg) caused an increase in cardiac inotropy with a peak value of 21.7%±6.8% (P<0.01) and 50.5%±18.9% (P<0.01) 0.5 min after injection, respectively. However, 30 mg/kg S miltior-rhiza depside salts showed a depressant effect 10–20 min after injection. A slight fall in MAP appeared 10 min after 30 mg/kg S miltiorrhiza depside salts were intravenously administered, with a significant decrease at the 15 min time point (Figure 5C). Conversely, 60 mg/kg S miltiorrhiza depside salts exerted an increasing effect on MAP in the initial period by 17.1%±9.4% (P<0.01 vs baseline) maximally. Cardiac inotropy dropped significantly and reverted to the baseline soon after.

Figure 5 Responses of hemodynamics to intravenous injection of depside salts from S miltiorrhiza (30 mg/kg, 60 mg/kg, n=7) and vehicle control (n=7) in open-chest anaesthetized rats. Data are shown as mean±SD. (A) CI; (B) maximal velocity of flow increase (df/dt_max); (C) MAP; (D) SVR. (▲) vehicle control; (■) depside salts from S miltiorrhiza (30 mg/kg); (●) depside salts from S miltiorrhiza (60 mg/kg); ^bP<0.05, ^cP<0.01 vs baseline; ^eP<0.05, ^fP<0.01 vs vehicle control.

Effects of S miltiorrhiza depside salts on myocardial microperfusion The effect of S miltiorrhiza depside salts (30 mg/kg and 60 mg/kg) on myocardial microperfusion in the LV was measured in 7 open-chest anesthetized rats (Figure 6). Myocardial microperfusion maximally increas-ed by 6.3%±2.9% (P<0.01) and 9.6%±4.0% (P<0.01) for 30 mg/kg and 60 mg/kg S miltiorrhiza depside salts, respectively. The profile of myocardial microperfusion with S miltiorrhiza depside salts intervention returned to the baseline in no more than 15 min.

Figure 6 Responses of myocardial microperfusion to intravenous injection of depside salts from S miltiorrhiza (30 mg/kg, 60 mg/kg, n=7) and vehicle control (n=7) in open-chest anaesthetized rats. MMP change (%) is short for myocardial microperfusion change percentage. Data are shown as mean±SD. (▲) vehicle control; (■) depside salts from S miltiorrhiza (30 mg/kg); (●) depside salts from S miltiorrhiza (60 mg/kg); ^bP<0.05, ^cP<0.01 vs baseline; ^eP<0.05, ^fP<0.01 vs vehicle control.

Discussion

Coronary microcirculation plays a pivotal role in determining the supply of oxygen and nutrients to the myocardium. Analyses of coronary microcirculation are important for understanding the pathophysiology of ischemic hearts and the effect of cardioprotective agents. Although the cardio-protec-tive property of MLB has been studied extensively, as well as those of S miltiorrhiza depside salts, there is still no report concerning their effect on coronary micro-circulation. To our knowledge, we demonstrated here for the first time that S miltiorrhiza depside salts could improve myocardial microperfusion using laser Doppler flowmetry, as well as CO.

A number of strategies have been previously employed to optimize the laser Doppler system to overcome movement artifacts so that it can be used for myocardial microvascular measurement^[13–19]. In the present study, we proposed that the probe was satisfactorily positioned when a distinct and regular waveform pattern was observed during baseline measurements and was altered following intervention, as suggested by Klassen et al^[15]. Kiyooka et al^[20] reported that red blood cell flow in the epimyocardium capillaries was predominant during either systole or diastole, acting as the watershed between diastolic arterial and systolic venous flows. Besides, the diameters of subepicardial arterioles remained basically constant during the entire cardiac cycle^[17]. In our research, data showed that the phasic profile of myocardial microperfusion by laser Doppler flowmetry, which reflected both arterial inflow and venous outflow, was predominately diastole, which was identical to previous studies^[17,20]. Furthermore, the representative vasodilator nifedipine was studied in our system and showed significant increasing effects on myocardial microperfusion as reported^[21,22], so did captopril as predicted, which in turn convinced us that the laser Doppler system was sensitive enough to the responses of coronary microvascular to drugs for measure-ment.

In our study, the improving effect of intravenously administered S miltiorrhiza depside salts on CI was in a dose-related manner. After a bolus injection of S miltiorrhiza depside salts, we observed significant increases in CI and myocardial microperfusion prior to minor changes of MAP, which suggested that S miltiorrhiza depside salts might improve cardiac function directly.

Yokozawa et al^[23] reported that oral administration of MLB produced a significant reduction in blood pressure in rats with sodium-induced hypertension. The studies by Kamata et al^[24,25] also showed that intravenous injection of lithospermic acid B (10–30 mg/kg) decreased the blood pressure in a dose-dependent manner and behaved as an endothelium-dependent vasodilator. In the current study, the effect of 30 mg/kg S miltiorrhiza depside salts on MAP was identical to previous studies, however, 60 mg/kg S miltior-rhiza depside salts resulted in a marked increase in MAP in the initial period of observation when CI was promoted significantly. We presumed that it resulted from the overwhelming CO increasing effect which consequently concealed the vasodilation property of S miltiorrhiza depside salts.

In conclusion, S miltiorrhiza depside salts improve myocardial microperfusion, as well as CO, which provides further evidence for the treatment of S miltiorrhiza depside salts on coronary heart diseases.

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