Effect of exogenous phosphocreatine on cardiomycytic apoptosis and expression of Bcl-2 and Bax after cardiopulmonary resuscitation in rats

BACKGROUND： Ischemia-reperfusion injury in the myocardium after cardiac arrest （CA）and cardiopulmonary resuscitation （CPR） is an important pathologic basis of post-cardiac arrestof syndrome （PCAS）, and apoptosis is one of the major mechanisms in myocardial ischemiareperfusioninjury. To lessen myocardial ischemia-reperfusion injury after cardiac arrest and CPR,it is important to reduce energy consumption and to increase energy supply in the myocardium.This study aimed to observe changes of cell apoptosis and expression of Bcl-2 and Bax proteinon the myocardium after CPR in rats, and the protective effects of different doses of exogenousphosphocreatine （creatine phosphate, CP） on them.METHODS： A total of 32 male adult Sprague-Dawley rats were randomly divided into 4 groups：control group （group A）, CPR group （group B）, low-dose CP group （group C, CP 0.5 g/kg at beginning ofCPR and 1.0 g/kg at 2 hours after CPR） and high-dose CP group （group D, CP 1.0 g/kg at beginning ofCPR and 2.0 g/kg at 2 hours after CPR）. Cardiac arrest was induced by asphyxiation and CPR startedat 7 minutes after asphyxiation in groups B, C and D. Myocardium samples were taken at 24 hoursafter CPR. Cardiomycytic apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling（TUNEL） method. The expression of Bcl-2 and Bax protein was measured by immunohistochemistry.RESULTS： Cardiomyocytic apoptosis index （AI） and expression of Bcl-2 and Bax proteinincreased more significantly in groups B, C and D than in group A （P〈0.01）, but Bcl-2/Bax ratiosignificantly decreased （P〈0.01）. Cardiomyocytic AI and expression of Bcl-2 and Bax proteindecreased more significantly in groups C and D than in group B （P〈0.01）, but Bcl-2/Bax ratioincreased more significantly （P〈0.01）. Cardiomyocytic AI and expression of Bcl-2 and Bax proteindecreased more signifi cantly in group D than in group C （P〈0.05）, but Bcl-2/Bax ratio increased moresignifi cantly （P〈0.05）.CONCLUSION： Exogenous phosphocreatine, especially at a large dose, could inhibitcardiomyocytic apoptosis and alleviate myocardial injury after CPR in rats.

Efficacy of Danshen Injection Combined with Phosphocreatine Disodium in Treating Frequent Premature Contractions during Pregnancy and Its Effects on Maternal and Infant Outcomes

[Objectives]To explore the efficacy of Danshen Injection combined with phosphocreatine disodium in treating frequent premature contractions during pregnancy and its effect on maternal and infant outcomes.[Methods]A total of 200 pregnant women with normal pregnancy and frequent premature contractions who were treated in the outpatient department of internal medicine in Women and Children's Hospital of Hubei Province and Xinzhou District People's Hospital of Wuhan Central Hospital during September 2015 and October 2018 were selected and randomly divided into the control group and observation group,100 cases for each group.The observation group was treated with Danshen Injection combined with phosphocreatine disodium,and the control group was treated with phosphocreatine disodium alone.The course of treatment in both groups was one week.During the treatment,the changes of heart rate,heart rhythm,electrocardiogram and 24-h dynamic electrocardiogram(DCG)of both groups were observed.[Results]After treatment,the clinical efficacy of the observation group was better than that of the control group,and the difference was statistically significant(P<0.01).There were no adverse drug reactions in both groups.However,in the control group,non-sustained ventricular tachycardia(NSVT)was found in 24-h dynamic electrocardiogram(DCG)of 2 pregnant women with premature ventricular contraction.The pregnancy process was smooth,with full-term natural delivery and no fetal malformation.There was no significant difference in gestational age and neonatal weight between the two groups(P>0.05).However,there was a statistically significant difference in Apgar score between the two groups(P<0.05).During the 6-month postpartum follow-up,the mother and child were unharmed,and examination of repeated electrocardiograms showed that the conditions were normal.[Conclusions]Danshen Injection combined with phosphocreatine disodium has better efficacy in the treatment of frequent premature contractions during pregnancy and the maternal and infant outcomes than the treatment with phosphocreatine disodium alone,and it has good safety and can prevent premature contractions from progressing to tachyarrhythmias.

An Ion-Pair HPLC Method for Simultaneous Determination of Exogenous Phosphocreatine and Its Metabolite Creatine and Related ATP in Rabbit Plasma and RBC: Application to a Pharmacokinetic Study

A specific, precise and accurate ion-pair HPLC-UV method has been developed and validated for simultaneous determination of phosphocreatine (PCr), and its metabolite creatine (Cr) as well as related ATP in plasma and red blood cell (RBC) of rabbits. After addition of TMP as IS, the samples were deproteinized with 6% PCA. The analytes were separated on a Kromasil C18 column using a tertiary gradient mobile phase composed of buffer A (0.2% KH2PO4 + 0.08% tetrabutyl ammonium hydrogen sulphate, pH 3.0), buffer B (buffer A adjusted to pH 7.5 with 1 mol/L NaOH) and MeOH. Detection wavelengths were set at 210 nm for PCr and Cr and 260 nm for ATP and TMP. Some blank samples were initially run for baseline subtraction. The linear detection responses were obtained for PCr concentration over a range of 10 - 7500 mg/ml (plasma) and 5 - 2500 mg/ml (RBC) and for both Cr and ATP concentrations of 10 - 1500 mg/ml (plasma) and 5 - 750 mg/ml (RBC) (r > 0.99). The QC samples of 3 analytes showed intra-day and inter-day precisions (RSD) of - 107%. The method was successfully used to simultaneously determine plasma and RBC concentrations of the 3 analytes and to study pharmacokinetics after iv administration of PCr to rabbits.

The Effect of Extrogenous Phosphocreatine on L-type Calcium Current in Ischemic Guinea Pig Ventricular Myocytes

Objectives Heart failure （HF） is one of the most common outcome for all kinds of heart diseases, the effects of energetic therapy on HF remains controversial, especially to ischemic HF. The aim of this study was to explore the effect of exogenous phosphocreatine with different concentration on L-type calcium（I Cc-L） current in ischemic ventricular myocytes of guinea pig and to investigate its underlying electrophysiological mechanism for the treatment of ischemic HF. Methods Single ventricular myocytes were isolated enzymatically from left ventricle of guinea pig. Peak I Ca-L current were recorded using patch clamp techniques in the whole-cell configuration when myocytes had been superfused with normal Tyrode solution, simple ischemic solution, ischemic solution containing phosphocreatine with different concentration for 10 minutes respectively. Results Peak I Ca-L current density of myocytes superfused with simple simulated ischemic solution was remarkably inhibited by 80.6 ± 5.2% compared with myocytes superfused with normal Tyrode solution（P〈0.05）. Ischemic solution containing phosphocreatine of 5, 10, 20, 30mmol/L inhibited Peak I Ca-L current density by （53.8±6.7）%, （41.8 ± 8.2）%, （38.1±7.4）%, （36.6±9.7）% respectively. There was no statistical significance among phosphocreation of 10, 20, 30 mmol / L. Conclusions Extrogenous phosphocreatine could reverse the inhibition of I Ca-L current under ischemic condition, which could be the ionic basis for the treatment of ischemic heart failure. 0-10 mmol/L phosphocreatine exerted significant dose-effect relationship which no longer existed as concentration more than 10 mmol/L. It is supposed that phosphocreatine increased I Ca-L current by many pathways rather than simple substrate for ATP synthesis.

Pyruvate-fortified resuscitation stabilizes cardiac electrical activity and energy metabolism during hypovolemia

AIM: To test the hypothesis that fluid resuscitation with Ringer's solution enriched with pyruvate(PR), a physiological antioxidant and energy substrate, affords protection of myocardial metabolism and electrophysiological performance superior to lactated Ringer's(LR) during hypovolemia and hindlimb ischemia-reperfusion.METHODS: Male domestic goats(25-30 kg) were exsanguinated to a mean arterial pressure of 48 ± 1 mm Hg. Right hindlimb ischemia was imposed for 90 min by applying a tourniquet and femoral crossclamp. LR or PR, infused iv, delivered 0.05 mmol/kg per minute L-lactate or pyruvate, respectively, from 30 min hindlimb ischemia until 30 min post-ischemia. Time controls(TC) underwent neither hemorrhage, hindlimb ischemia nor resuscitation. Goats were sacrificed and left ventricular myocardium biopsied at 90 min fluid resuscitation(n = 6 per group) or 3.5 h later(n = 9 LR, 10 PR, 8 TC).RESULTS: Myocardial 8-isoprostane content, phosphocreatine phosphorylation potential, creatine kinase activity, and heart rate-adjusted QT interval(QTc) vari- ability were evaluated at 90 min resuscitation and 3.5 h post-resuscitation. PR sharply lowered pro-arrhythmic QTc variability vs LR(P < 0.05); this effect persisted 3.5 h post-resuscitation. PR lowered myocardial 8-isoprostane content, a product of oxidative stress, by 39 and 37% during and 3.5 h after resuscitation, respectively, vs LR. Creatine kinase activity fell 42% post-LR vs TC(P < 0.05), but was stable post-PR(P < 0.02 vs post-LR). PR doubled phosphocreatine phosphorylation potential, a measure of ATP free energy state, vs TC and LR(P < 0.05); this energetic enhancement persisted 3.5 h post-resuscitation.CONCLUSION: By augmenting myocardial energy state and protecting creatine kinase activity, pyruvateenriched resuscitation stabilized cardiac electrical function during central hypovolemia and hindlimb ischemiareperfusion.