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Molecular and Cellular Mechanisms of Action for the Cardioprotective and Therapeutic Role of Creatine Phosphate
Abstract
Studies on the biochemical basis for a therapeutic effect of creatine phosphate (PCr) demonstrate a role in preserving contractile function, maintaining intracellular adenosine triphosphate (ATP), PCr, and reducing creatine kinase (CK) loss. The anti-ischaemic effect is Ca2+ -dependent and an antioxidant property has been confirmed. The mechanism of action is thought to relate to the preservation of sarcolemmal membranes. While reservations about membrane penetration of the molecule are expressed, pharmacokinetic data have shown the uptake of exogenous PCr by heart, skeletal muscle, brain and to some extent by kidney, but not by lung or liver tissues. Thus, the protective effect of PCr is directly related to the tissue uptake due to some specific phospholipid composition of the cell membranes, and in this respect, it would be interesting to analyze the available data for all tissues studied. A mechanism of protection related to stabilization of the sarcolemma without significant penetration into the cells can also be envisaged. The results of the various studies on PCr present some important points for consideration.
... It is possible that increased muscle phosphocreatine levels resulting from creatine supplementation could reduce muscle dysfunction, reducing muscle soreness or enhancing recovery. Exogenous phosphocreatine reduces muscle damage in cardiac tissue by stabilising the membrane phospholipid bilayer, decreasing membrane fluidity, and turning the membrane into a more ordered state 47,48 . In cardiac tissue, this decreases the loss of cardiac muscle proteins, which indicates less muscle tissue damage 47 . ...
... Official Journal of FIMS (International Federation of Sports Medicine)48 ...
Creatine supplementation is a widely used and heavily studied ergogenic aid. Athletes use creatine to increase muscle mass, strength, and muscle endurance. While the performance and muscle- building effects of creatine supplementation have been well documented, the mechanisms responsible for these muscular adaptations have been less studied. Objective: The purpose of this review is to examine studies of the mechanisms underlying muscular adaptations to creatine supplementation. Data sources: PubMed and SPORTDiscus databases were searched from 1992 to 2007 using the terms creatine, creatine supplementation, creatine monohydrate, and phosphocreatine. Study selection: Studies of creatine supplementation in healthy adults were included. Data extraction: Due to the small number of studies identified, a meta-analysis was not performed. Data synthesis: Several potential mechanisms underlying muscular adaptations to creatine supplementation were identified, including: metabolic adaptations, changes in protein turnover, hormonal alterations, stabilization of lipid membranes, molecular modifications, or as a general training aid. The mechanisms with the greatest amount of support (metabolic adaptations, molecular modifications, and general training aid) may work in concert rather than independently. Conclusions: Creatine supplementation may alter skeletal muscle directly, by increased muscle glycogen and phosphocreatine, faster phosphocreatine resynthesis, increased expression of endocrine and growth factor mRNA, or indirectly, through increased training volume. Keywords: dietary supplement, creatine monohydrate, phosphocreatine, muscle, sport nutrition
... For example, transgenic mice expressing mtCK in their liver acquire, after Cr supplementation, a remarkable tolerance against hypoxia (Miller et al. 1993) and liver toxins (Hatano et al. 1996), as well as against tumour necrosis factor-induced apoptosis (Hatano et al. 2004). Since PCr has been shown to bind to and protect biological membranes (Saks et al. 1996; Tokarska-Schlattner et al. 2003), it is conceivable that the PCr generated by mtCK in the mitochondrial intermembrane space would also bind to mitochondrial membranes and stabilize them against swelling, as this was shown for plasma membranes of erythrocytes (TokarskaSchlattner et al. 2003). Thus, mtCK plus Cr seem to exert cell protection not only by improving cellular energetics , but also by more or less energy-independent actions that also affect apoptosis (O'Gorman et al. 1997a; Brdiczka et al. 2006) (seeTable 1). ...
... In addition, the glyocogen content in these muscles is elevated indicating that instead of PCr, glycogen/glucose is taken as a more or less immediate source of energy for muscle contraction. SuchTable 1 Pleiotropic effects of creatine for cell function and cell protection Energy-related effects of creatine Cr improves cellular energy state (PCr/ATP ratio) and muscle performance (Harris et al. 1992; Greenhaff et al. 1993) Cr facilitates intracellular energy transport (PCr circuit or shuttle) (Wallimann 1975; Saks et al. 1978 Saks et al. , 2006b Bessman and Geiger 1981; Wallimann and Eppenberger 1985; Bessman 1986; Wallimann et al. 1992 Wallimann et al. , 1998 Kaasik et al. 2003; Wallimann et al. 2007) Cr improves the efficiency of cellular energy utilization (e.g. for Ca 2? -handling) (Rossi et al. 1990; Steeghs et al. 1997; Pulido et al. 1998; van Leemputte et al. 1999) Cr stimulates mitochondrial respiration (improved energy provision) (Kay et al. 2000 et al. 2007, 2008) Cr lowers homocysteine levels and lipid peroxidation (heart risk factors) (Deminice et al. 2009) Cr acts as an osmolyte, protecting cells against hypertonic stress (Alfieri et al. 2006) PCr binds to cell membranes and stabilizes and protects erythrocyte cell membranes (Saks et al. 1996; Tokarska-Schlattner et al. 2003) The creatine kinase system and pleiotropic effects of creatine 1281 interesting compensatory alterations give new insight into the kinds of problems that may have been generated in a given tissue by knocking out of either cytosolic and/or mitochondrial CK. A further interesting observation relates to the fact that CK exists as isoforms and that in a given cell usually a cytosolic CK isoform is co-expressed with a mitochondrial mtCK isoform, although the relative proportion may vary depending on cell type and organ (Wallimann and Hemmer 1994). ...
The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure-function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans.
... In theory, increased muscle phosphocreatine levels resulting from creatine supplementation may reduce muscle dysfunction because it is known that exogenous phosphocreatine reduces muscle damage in cardiac tissue (28,29). Phosphocreatine binds to the polar phospholipid heads of the cardiac muscle membrane, stabilizes the membrane phospholipid bilayer, decreases membrane fluidity, and turns the membrane into a more ordered state (29). ...
... This subsequently decreases the loss of cardiac muscle proteins, indicating less cytoplasmic leakage and potentially less muscle tissue damage (29). In fact, phosphocreatine is used as a cardio-protective agent during heart surgery and to reduce infarct size after myocardial infarction (28,29). ...
Previous studies have shown that creatine supplementation reduces muscle damage and inflammation following running but not following high-force, eccentric exercise. Although the mechanical strain placed on muscle fibers during high-force, eccentric exercise may be too overwhelming for creatine to exert any protective effect, creatine supplementation may protect skeletal muscle stressed by a resistance training challenge that is more hypoxic in nature. The purpose of this study was to examine the effects of short-term creatine supplementation on markers of muscle damage (i.e., strength, range of motion, muscle soreness, muscle serum protein activity, C-reactive protein) to determine whether creatine supplementation offers protective effects on skeletal muscle following a hypoxic resistance exercise test. Twenty-two healthy, weight-trained men (19-27 years) ingested either creatine or a placebo for 10 days. Following 5 days of supplementation, subjects performed a squat exercise protocol (5 sets of 15-20 repetitions at 50% of 1 repetition maximum [1RM]). Assessments of creatine kinase (CK) and lactate dehydrogenase activity, high-sensitivity C-reactive protein, maximal strength, range of motion (ROM), and muscle soreness (SOR) with movement and palpation were conducted pre-exercise and during a 5-day follow up. Following the exercise test, maximal strength and ROM decreased, whereas SOR and CK increased. Creatine and placebo-supplemented subjects experienced significant decreases in maximal strength (creatine: 13.4 kg, placebo: 17.5 kg) and ROM (creatine: 2.4 degrees , placebo: 3.0 degrees ) immediately postexercise, with no difference between groups. Following the exercise test, there were significant increases in SOR with movement and palpation (p < 0.05 at 24, 48, and 72 hours postexercise), and CK activity (p < 0.05 at 24 and 48 hours postexercise), with no differences between groups at any time. These data suggest that oral creatine supplementation does not reduce skeletal muscle damage or enhance recovery following a hypoxic resistance exercise challenge.
... 5 The CK/PCr system is both a buffer and a shuttle for energy-rich phosphates between the sites of ATP use and production, and the molecular and cellular basis for the cardioprotective action of PCr is well established. 7 Not surprisingly, there is also wide ranging evidence for the beneficial effects of creatine supplementation, especially in the elderly, 8 and in patients with muscle wasting disease. 9 But even more importantly, the PCr system has long been regarded essential for energy transfer in muscle. ...
... Cytokines and chemokines released by epithelial cells are also involved in eosinophil and Th 2 lymphocyte recruitment [19,23,24] . Creatine (Cr) is a nitrogenous amino acid derivative found in beef and fi sh and is produced endogenously from arginine, methionine and glycine [4,10,25,30,31] . It is widely used as a nutritional supplement by athletes to increase muscle mass and strength [4] . ...
Airway epithelium plays important roles in the pathophysiology of asthma. Creatine supplementation (Cr) was shown to increase asthma features in a murine model of allergic asthma; however, the role of the airway epithelium in this inflammatory response is not known. BALB/c mice were divided into control, creatine supplementation, ovalbumin-sensitized (OVA) and OVA plus creatine supplementation groups. OVA sensitization occurred on days 0, 14, 28 and 42, and ovalbumin challenge from days 21-53. Cr was also given on days 21-53. Total and differential cells counts in BALF were evaluated. Quantitative epithelial expression of interleukin (IL)-4, IL-5, IL-13, CCL11, CCL5, CCL2, iNOS, VCAM-1, ICAM-1, NF-κB, VEGF, TGF-β, IGF-1, EGFR, TIMP-1, TIMP-2, MMP-9, MMP-12 and arginase II were performed. Cr increased the number of total cells and eosinophils in BALF, the epithelial content of goblet cells and the epithelial expression of IL-5, CCL2, iNOS, ICAM-1, NF-κB, TGF-β, TIMP-1 and MMP-9 when compared to the control group (p<0.05). Creatine supplementation also exacerbated goblet cell proliferation, and IL-5 and iNOS expression by epithelial cells compared to the OVA group (p<0.01). Creatine up-regulates the pro-inflammatory cascade and remodelling process in this asthma model by modulating the expression of inflammatory mediators by epithelial cells.
The literature on creatine supplementation supporting its efficacy has grown rapidly and has included studies in both healthy volunteers and patient populations. However, the first rule in the development of therapeutic agents is safety. Creatine is well-tolerated in most individuals in short-term studies. However, isolated reports suggest creatine may be associated with various side effects affecting several organ systems including skeletal muscle, the kidney and the gastrointestinal tract. The majority of clinical studies fail to find an increased incidence of side effects with creatine supplementation. To date, studies have not found clinically significant deviations from normal values in renal, hepatic, cardiac or muscle function. Few data are available on the long-term consequences of creatine supplementation.
This investigation evaluated the effects of oral creatine (Cr) supplementation on markers of exercise-induced muscle damage following high-force eccentric exercise in subjects randomly administered Cr or placebo (P) in a double-blind fashion. When injected, exogenous phosphocreatine has been shown to stabilize the muscle membrane in cardiac tissue and enhance recovery of strength and power following injury. Twenty-three men aged 18-36 years ingested either 20 g of Cr or P for 5 days. Criterion measures were maximal isometric force of the elbow flexors (MIF), range of motion (ROM) about the elbow, mid and distal arm circumference (CIR; to assess swelling), soreness with movement and palpation (SOR), and blood levels of creatine kinase (CK) and lactate dehydrogenase (LDH). Following the supplementation period, subjects performed 50 maximal eccentric contractions of the elbow flexors. Criterion measures were assessed pre-exercise, immediately postexercise, and for 5 days after exercise. Both groups experienced a significant loss of MIF and ROM (time effect, p < 0.05). There was a significant increase in CIR of the mid and distal biceps, SOR with movement and palpation, CK, and LDH (time effect, p < 0.05), indicating that there was significant muscle damage. However, there were no significant differences in any of the criterion measures between groups (group x time interaction term, p > 0.05). The pattern of change over the 6 days, in response to the eccentric exercise, was nearly identical between groups. These data suggest that 5 days of Cr supplementation does not reduce indirect markers of muscle damage or enhance recovery from high-force eccentric exercise.
Creatine supplement is the most popular nutritional supplement, and has various metabolic functions and sports medicine applications. Creatine supplementation increases muscle mass and can decrease muscular inflammation. Some studies have also suggested a beneficial role of creatine supplementation on chronic pulmonary diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Among athletes, the prevalence of asthma is high, and many of these individuals may be taking creatine. However, the effects of creatine supplementation on chronic pulmonary diseases of allergic origin have not been investigated. In the present study, we analyzed the effects of creatine supplementation on a model of chronic allergic lung inflammation. Thirty-one Balb/c mice were divided into four groups: control, creatine (Cr), ovalbumin (OVA), and OVA+Cr. OVA and OVA+Cr groups were sensitized with intraperitoneal injections of OVA on Days 0, 14, 28, and 42. OVA challenge (OVA 1%) and Cr treatment (0.5 g/kg/d) were initiated on Day 21 and lasted until Day 53. We determined the index of hyperresponsiveness, the serum levels of OVA-specific immunoglobulin (Ig)E and IgG(1), and the total and differential cell counts in bronchoalveolar lavage fluid. We also quantified airway inflammation, and the airway density of IL-4+, IL-5+, IL-2+, IFN-gamma+, and insulin-like growth factor (IGF)-1+ cells, collagen and elastic fibers, and airway smooth muscle thickness. Our results showed that creatine in OVA-sensitized mice increased hyperresponsiveness; eosinophilic inflammation; airway density of IL-4+, IL-5+, and IGF-1 inflammatory cells; airway collagen and elastin content; and smooth muscle thickness. The results show that creatine supplementation exacerbates the lung allergic response to OVA through a T helper cell type 2 pathway and increased IGF-1 expression.
To probe adriamycin-phospholipid interactions, the effects of this cytotoxin on the hydrolysis of a pyrene-labeled acidic alkyl-acyl phospholipid analog 1-octa-cosanyl-2-(6-pyren-1-yl)hexanoyl-sn-glycero-3-phos p hatidylmethanol (C28-O-PHPM) by porcine pancreatic phospholipase A2 (PLA2) were studied. In the absence of added Ca2+ adriamycin caused a 3-4-fold activation of hydrolysis of this pyrenelipid whereas an inhibition of action of PLA2 on the corresponding phosphatidylcholine derivative C28-O-PHPC was observed. Under similar conditions adriamycin also enhanced the rate of hydrolysis of the pyrene-labeled diacyl lipid 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylgly cer ol and inhibited the hydrolysis of PLA2 on the phosphatidylcholine derivative. Increasing calcium concentrations abolished the activating and most of the inhibitory effects of adriamycin with the above phospholipid substrates. Quenching of pyrene excimer fluorescence by adriamycin revealed efficient binding of the drug to acidic lipids. Addition of 1 mM calcium reduced fluorescence quenching by adriamycin maximally by approximately 90%. In comparison, quenching by adriamycin of pyrene-labeled phosphatidylcholine was much weaker and calcium had only an insignificant effect. Monolayer experiments at an air/water interface showed a rapid and surface pressure-dependent penetration of the drug into a film of C28-O-PHPM. Increase in surface pressure was reversed by 80% by the inclusion of 1 mM Ca2+ into the subphase. Penetration of adriamycin into a monolayer of C28-O-PHPC was much weaker. In agreement with earlier studies two types of binding of adriamycin to C28-O-PHPM are proposed.
A phosphorus 31-nuclear magnetic resonance method was used to study the effect of exogenous phosphocreatine on the isolated perfused rat heart. The hearts were chemically arrested by St. Thomas' Hospital solution and made totally ischemic for 35 minutes at 37 degrees C. In the presence of phosphocreatine, 10 mmol/L, during ischemia, almost complete recovery of heart function and phosphocreatine content and 61% recovery of adenosine triphosphate content were observed after 30 minutes of postischemic reperfusion; in the control experiments without phosphocreatine, contractile function, intracellular phosphocreatine, and adenosine triphosphate contents were restored to 33%, 43%, and 26% of their normal values, respectively. Ultrastructural studies with a lanthanum tracer method showed remarkable protection of sarcolemma against ischemic injury by exogenous phosphocreatine at the level of the glycocalyx.
A model of immune (cytotoxic) injury of the heart in the dog was used to evaluate the effects of exogenous phosphocreatine on the structure and function of coronary vessels. Immune damage was caused by administration of cytotoxic anticardiac serum (produced by immunization of rabbits by a supernatant obtained after centrifugation of dog heart homogenate) into one of the branches of the left coronary artery. In immune-damaged myocardium, phosphocreatine (500 mg/kg injected 10 to 15 minutes before immune damage) exerted a protective effect in all phases of the pathologic process, preventing development of hypotension, decreasing cardiac output, and increasing coronary vessel resistance.
Given the fact that creatine phosphate (CP) has a protective action on the myocardium, the question posed is whether this polar molecule may cross the cell membrane. Uptake of 14C-Creatine 32P-Phosphate by rabbit myocardium was examined in vitro and in vivo. There was significant myocardial uptake of 14C and 32P-labels. The ratio 14C:32P (40:60) was unchanged on cellular uptake. The 14C and 32P-labels were predominantly (85% in vitro and 50% in vivo) associated within the mitochondria and homogenization of the mitochondrial fraction resulted in solubilization of the radioactivity. TLC analysis confirmed that the 14C and 32P labels comigrated predominantly with CP. The results imply that CP may cross the cell (and mitochondrial ?) membrane. This uptake may be energy-dependent or may depend on membrane integrity in that mitochondrial 'in vitro' loading decreased in anoxia.
(1) The effects of the anti-tumor drug adriamycin on lipid polymorphism in cardiolipin-containing model membranes and in isolated inner mitochondrial membranes has been examined by 31P-NMR. (2) Adriamycin binding does not affect the macroscopic structure or local order in the phosphate region of cardiolipin liposomes. (3) In cardiolipin liposomes and in cardiolipin-phosphatidylcholine (1:1) liposomes, the drug inhibits the ability of Ca2+ to induce the hexagonal HII phase. (4) Adriamycin interaction with both dioleoylphosphatidylethanolamine-cardiolipin (2:1) and dioleoylphosphatidylethanolamine-phosphatidylserine (1:1) liposomes results in structural phase separation into a liquid-crystalline hexagonal HII phase for the phosphatidylethanolamine and a liquid-crystalline lamellar phase for the negatively charged phospholipid. (5) Combined high-resolution 31P-NMR, electron microscopy and light scattering studies reveal the prominent fusion capacity of adriamycin towards cardiolipin-phosphatidylcholine small unilamellar vesicles. (6) Addition of Ca2+ to total rat liver inner mitochondrial membrane lipids, dispersed in excess buffer, results in hexagonal HII formation for part of the phospholipids. By contrast, the original bilayer structure is completely conserved when the above experiment is performed in the presence of adriamycin. (7) 31P-NMR spectra of isolated inner mitochondrial membranes are indicative of a bilayer organization for the majority of the phospholipids. Approximately 15% of the signal intensity originates from phospholipids which experience isotropic motion. Adriamycin addition almost completely eliminates the latter spectral component. In the absence of adriamycin, Ca2+ addition greatly increases the percentage of the phospholipids giving rise to an isotropic signal possibly indicating the formation of non-lamellar lipid structures. Adriamycin which specifically binds to cardiolipin (K. Nicolay et al. (1984) Biochim. Biophys. Acta 778, 359–371) completely blocks the Ca2+-induced structural reorganization of the lipids in this membrane.
Radiotracer measurements of adsorption showed that the binding of Ca2+ with the phosphate groups of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine monolayers depended largely on the molecular conformation of the lipid in the monolayer. The monolayers of phosphatidylethanolamine and phosphatidylcholine bind small amounts of Ca2+ when the lipid molecules are packed closer than 150 Å2 per molecule. The distribution of bound Ca2+ on both phosphate and carboxyl groups of phosphatidylserine was analyzed from the differences in accessibility of Ca2+ to each group. The amount of Ca2+ binding is larger for the carboxyl than the phosphate groups of phosphatidylserine by a factor of about 3 at molecular areas larger than 150 Å2/molecule at pH 7.5 and Ca2+ 0.05 mM. The apparent reactivities of these phospholipids to Ca2+ varied in the order phosphatidylserine > phosphatidylethanolamine ⪢ phosphatidylcholine. The low reactivity of phosphate groups of phosphatidylethanolamine and phosphatidylcholine may reasonably be understood as a lowered ionic nature of the phospholipid owing to the intra- or intermolecular neutralization of the charge between ammonium cation and phosphate anion groups of the lipids. The Ca2+ binding is explained by taking into account those ionic equilibria of the acid and base dissociations, the intra- or intermolecular neutralization among phosphate, carboxyl and ammonium groups, and the calcium soap formation at each anionic site. The equilibrium constants, which determine the ionic properties of these phospholipids, are calculated from the adsorption data of Ca2+ at various pH values, and at various degrees of molecular packing of the phospholipid monolayers.
Adriamycin and several derivatives were found to inhibit the last oxidation site of the respiratory chain (cytochrome c oxidase EC.1.9.3.1.) both on mitochondria and on purified reconstituted systems. A new mechanism of membrane enzyme inactivation is proposed to explain the experimental results: adriamycin does not interact directly with cytochrome c oxidase but inactivates it by changing the cardiolipin environment essential for its activity. In presence of adriamycin, cardiolipin is extracted from the lipid surrounding environment of cytochrome c oxidase and segregates in a separate phase inaccessible for the enzyme. We suggest that other cardiolipin dependent enzymes could be inactivated by adriamycin.
We have characterized the interaction of the antitumor drug doxorubicin woth model membranes of the anionic phospholipids dioleoylphospatidic acid (DOPA), dioleoylphosphatidylserine (DOPS), cardiolipin and dioleoylphosphatidylglycerol (DOPG) as compared to the zwitterionic dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). The saturating binding levels were: 2.4 (DOPA), 1.3 (cardiolipin), 1.5 (DOPS, DOPG) and 0.02 (DOPC) doxorubicin per lipid phosphorus (mol/mol). The half-saturating free drug concentrations were comparable for DOPA, cardiolipin, DOPS and DOPG: 20, 16, 35 and 18 μM, respectively. Doxorubicin fluorescence revealed the simultaneous existence of at least two populations of bound drug in the various anionic phospholipids: (1) fluorescent molecules with chromophores that reside between the lipid molecules and (2) above 0.01–0.02 doxorubicin bound per lipid phosphorus: non-fluorescent drug-stacks that are closer to the aqueous phase than the fluorescent molecules. Small-angle X-ray scattering indicated that doxorubicin can reorganize anionic phospholipid dispersions into closely-packed multilamellar structures. Addition of the drug caused leakage of entrapped 6-carboxyfluorescein. Neither 2H-NMR on [2-2H]serine-labelled DOPS nor 31P-NMR revealed any significant effect of doxorubicin on headgroup conformation, but 2H-NMR on di[11,11-2H2]oleoyl-labelled phospholipids showed that the drug had a strong acyl chain-disordering effect on anionic phospholipids. 2H-NMR relaxation measurements indicated that the drug immobilized the headgroups and acyl chains of anionic phospholipids. The implications of these observations for the cellular activity of the drug are indicated.
The investigation focuses on the phospholipid composition of the sarcolemma of cultured neonatal rat heart cells and on the distribution of the phospholipid classes between the two monolayers of the sarcolemma. The plasma membranes are isolated by ‘gas-dissection’ technique and 38% of total cellular phospholipid is present in the sarcolemma with the composition: phosphatidylethanolamine (PE) 24.9%, phosphatidylcholine (PC) 52.0%, phosphatidylserine/phosphatidylinositol (PS/PI) 7.2%, sphingomyelin 13.5%. The cholesterol /phospholipid ratio of the sarcolemma is 0.5. The distribution of the phospholipids between inner and outer monolayer is defined with the use of two phospholipases A2, sphingomyelinase C or trinitrobenzene sulfonic acid as lipid membrane probes in whole cells. The probes have access to the entire sarcolemmal surface and do not produce detectable cell lysis. The phospholipid classes are asymmetrically distributed: (1) the negatively charged phospholipids, PS/PI are located exclusively in the inner or cytoplasmic leaflet; (2) 75% of PE is in the inner leaflet; (3) 93% of sphingomyelin is in the outer leaflet; (4) 43% of PC is in the outer leaflet. The predominance of PS/PI and PE at the cytoplasmic sarcolemmal surface is discussed with respect to phospholipid-ionic binding relations between phospholipids and exchange and transport of ions, and the response of the cardiac cell on ischemia-reperfusion.
The effects of various doses of creatine phosphate have been examined in a rat model of acute myocardial ischaemia. When given directly into the lumen of the left ventricle in pentobarbitone-anaesthetised male rats, creatine phosphate (50 and 100 mg/kg) markedly reduced the incidence of ventricular ectopic beats, and especially the incidence and duration of ventricular tachycardia and fibrillation which normally resulted from acute coronary artery ligation in this model. This protection was observed even if 1 of 2 h elapsed between creatine phosphate administration and coronary artery ligation. Electrophysiological studies on papillary muscles removed from rats 1 h after administration showed that creatine phosphate both decreased the maximum rate of depolarisation and prolonged the duration of the action potential. These results confirm our previous work in dogs that creatine phosphate is effective against early postinfarction ventricular arrhythmias, at least if given locally. It is suggested that these effects are due, at least in part, to a prolongation of the cardiac muscle action potential. Whether this is the result of maintaining energy production early in myocardial ischaemia is unclear.
This paper summarizes the data concerning the role of the creatine phosphokinase system in muscle cells with main attention to the cardiac muscle. Creatine phosphokinase isoenzymes play a key role in the intracellular energy transport from mitochondria to myofibrils and other sites of energy utilization. Due to the existence of the creatine phosphate pathway for energy transport, intracellular creatine phosphate concentration is apparently an important regulatory factor for muscle contraction which influences the contractile force by determining the rate of regeneration of ATP directly available for myosin ATPase, and at the same time controls the activator calcium entry into the myoplasm across the surface membrane of the cells.
The effects of creatine phosphate on frog heart muscle contraction have been studied further. It has been shown that after inhibition of mitochondrial oxidative phosphorylation by sodium cyanide the frog heart ventricle contraction can be maintained at a high level by addition of creatine phosphate. The effect of creatine phosphate on the contractile force and action potential is similar for frog heart ventricle and atrium. It has been directly demonstrated by using the voltage-clamp technique that creatine phosphate controls the slow inward calcium current through the surface membrane of frog atrium cells.
The anticancer drug doxorubicin penetrates into Langmuir monolayers containing phosphoinositides. Upon binding of doxorubicin to phosphoinositide-containing SUV, its fluorescence is self-quenched due to self-association. As compared to other anionic phospholipids, as much as 2- to 3-fold larger effects were obtained with PIP and PIP2, in mixtures of these lipids with DOPC. Doxorubicin competes efficiently with the non-penetrating antibiotic neomycin for binding to PIP2. According to its penetration, specific binding of doxorubicin was half-maximal at 5-15 microM. It is likely that also in biological membranes doxorubicin binds specifically to PIP and PIP2.
The purpose of this study was to find out whether Ca2+ is necessary for the protective effect of phosphocreatine (PCr) on ischemic myocardium. Isolated Langendorff-perfused rat hearts were used in the study. When ischemic arrest was induced in Ca2+-free buffer, PCr did not exert a protective effect on ischemic myocardium. PCr improved postischemic contractile recovery of hearts subjected to ischemia in perfusion media containing 0.5 and 1.2 mmol/l Ca2+.
Phosphoarginine, a structural analogue of PCr which possesses Ca2+-binding property similar to that of PCr did not exert any protective effect on ischemic myocardium. The effects of PCr and Ca2+ on lipid order of sarcolemmal vesicles from canine heart were studied by using ESR spectroscopy. PCr made membrane phospholipids more tightly packed at mildly acidic and neutral pH, but did not at pH 8.5. Although Ca2+ itself did not influence the membrane structure at pH 5.5, it potentiated the effect of PCr on sarcolemmal phospholipids. Thus, the protective effect of PCr on ischemic myocardium is not attributed to its Ca2+ binding properly, but Ca2+ is a necessary component of the mechanism of protective effect of PCr on ischemic myocardium.
The aim was to attempt to get further insight into the mechanism of the cardioprotective action of phosphocreatine (PCr).
Three experimental protocols were used: (1) The effect was examined of exogenous PCr (10 mmol.litre-1) on myocardial oxidative damage produced by H2O2 perfusion (90 mumol.litre-1) of isolated rat heart. (2) Isolated rat hearts were subjected to 35 min cardioplegic ischaemia followed by reperfusion. A control group was studied along with two PCr groups, one corrected for Ca2+ to compensate its binding with PCr (1.4 mmol.litre-1 CaCl2 in St Thomas's Hospital cardioplegic solution), and the other not (1.2 mmol.litre-1). (3) The effect was studied of PCr alone and in combination with the antioxidant tocopherol phosphate (0.1 mumol.litre-1) on contractile and metabolic recovery of isolated rat heart reperfused after 40 min cardioplegic ischaemia.
Studies were performed on hearts of 84 male Wistar rats, weighing 250-300 g.
(1) Oxidative stress resulted in irreversible contracture and impairment of sarcolemmal integrity revealed by using the transmembrane tracer ionic lanthanum. These effects coincided with the decrease of developed pressure from 116 (SEM 3) to 38(3) mm Hg and rate-pressure product from 498(13) to 165(16) mm Hg.s-1. The Ca2+ binding property of PCr was estimated experimentally and the stability constant of the complex CaPCr was found to be 35.4(0.7) mmol; from this the Ca2+ bound by PCr was calculated to be 14% in the experimental conditions used. Ca2+ concentration in K-H buffer containing PCr was increased to compensate its binding with PCr. PCr prevented H2O2 induced contracture, preserved sarcolemmal integrity, and attenuated H2O2 induced decrease in developed pressure and rate-pressure product [73(6) mm Hg and 340(28) mm H.s-1, respectively, p less than 0.05 compared with control]. (2) PCr reduced the diastolic pressure [29(10) v 68(10) mm Hg in control group at 30 min of reperfusion, p less than 0.05] and enhanced the developed pressure [81(10) v 46(10) mm Hg in controls, p less than 0.05] and rate-pressure product [325(44) v 158(40) mm Hg.s-1 in controls, p less than 0.05]. When CaCl2 was increased to 1.4 mmol.litre-1 the protective effect of PCr was not abolished. (3) PCr resulted in improvement of developed pressure [49(7) v 18(5) mm Hg in controls at 40 min of reperfusion, p less than 0.05] and rate-pressure product [184(27) v 71(20) mm Hg.s-1 in controls, p less than 0.05]. The degree of contractile recovery in the tocopherol group was almost the same as in the PCr group. Combined addition of PCr and tocopherol further increased the developed pressure and rate-pressure product to 72(4) mm Hg and 284(23) mm Hg.s-1, respectively. Similarly, PCr and tocopherol in combination provided substantial inhibition of creatine kinase release into perfusate, at 3.8(0.4) v 10.9(2.5) IU in controls, p less than 0.05.
PCr decreases the vulnerability of myocardium to oxidative stress and ischaemic damage. These effects cannot be explained by PCr induced shifts in Ca2+ concentration. Protective effects of PCr and tocopherol are quantitatively additive, most probably due to their different mechanisms of action, and tocopherol may be effective in extending the ability of PCr to stabilise cell membrane structure.
Doxorubicin (Adriamycin) and the related anthracycline antibiotic daunorubicin play a central part in cancer therapy because of their efficacy in the treatment of hematologic cancers (both acute leukemias and lymphomas), as well as carcinomas of the breast, lung, and thyroid and bone and soft-tissue sarcomas.1 Furthermore, these drugs are widely used in both adults and children in treatment regimens aimed at the cure of neoplasms as well as at palliation. The discovery more than 20 years ago that therapy with anthracyclines could produce irreversible and possibly life-threatening cardiac injury has led to limitations on the use of these drugs in . . .
Much of the damage arising during ischemia and reperfusion can be attributed to the consequences of flow deprivation. However, while reperfusion is a prerequisite for the survival of tissue, it may have an injurious component, which, if counteracted, might enhance postischemic recovery. The complex and dynamic changes that occur during ischemia in the diseased human heart are difficult to model in experimental preparations. As a consequence, much remains to be learned about the identity and manipulability of cellular changes leading to irreversible injury. Although the subject of most studies, injury to the myocyte may not be the primary determinant of tissue injury and changes in the endothelium or vascular smooth muscle may play an important role. Once critical ischemia-induced cellular changes have been identified, interventions can be developed to delay their progression such that at the time of reperfusion more cells are potentially salvable. Suboptimal reperfusion may limit the recovery of the tissue through the induction of "reperfusion injury." Much controversy surrounds the importance and even the existence of this phenomenon. It is proposed that reperfusion injury may express itself in four distinct forms: a) reperfusion-induced arrhythmias, which are potentially lethal (but preventable or reversible) events occurring in otherwise viable tissue; b) myocardial stunning, which is expressed as prolonged (but eventually fully reversible) contractile and metabolic dysfunction; c) the induction of lethal injury in tissue that was potentially viable in the moments before reperfusion; d) accelerated necrosis in tissue that is already irreversibly injured (the "oxygen paradox"). All but the third of these categories has been shown to exist experimentally and clinically, and can be advantageously manipulated. Although it is likely that lethal reperfusion injury also exists, there is as yet no definitive proof. Clarification of this issue is of considerable importance to those undergoing angioplasty or thrombolytic procedures.
Acute adriamycin cardiotoxicity was studied in the isolated, perfused rat heart by 31P and 13C NMR spectroscopy at flow rates of 15 and 5 ml/min. Treated hearts received a total dose of 13.5 mg of adriamycin. 31P NMR spectra were collected at the beginning and end of each experiment, and cardiac function was recorded throughout. Hearts were perfused with [1-13C]glucose, and 13C NMR spectra were recorded in the presence and absence of the drug. At normal flow (15 ml/min), adriamycin caused a decline in cardiac function which was reversible when the drug was removed. There were no changes in high energy phosphate levels. The labeling of glutamate was unchanged in the presence of adriamycin; however, there was a slight increase in the labeling of lactate and alanine. At reduced flow (5 ml/min), control hearts exhibited a small decrease in ATP and phosphocreatine levels, and cardiac function was depressed. These changes were reversible when normal flow was restored. Nevertheless, adriamycin treatment at low flow caused an irreversible decline in function and in hydrolysis of ATP and phosphocreatine. At reduced flow, the control and drug-treated hearts showed similar labeling of the glutamate pool; however, there was significantly greater labeling of lactate and alanine during adriamycin treatment. These results indicate that adriamycin is more toxic under reduced flow conditions. Impairment of cardiac function by adriamycin without changes in glutamate labeling suggests that this drug alters the relationship between cardiac function and energy production.
The aim was to determine the mechanism of the cardiotoxic effect of adriamycin, particularly at the level of the function of the cardiac myocyte.
After chronic exposure to adriamycin, the contractile responses of single isolated cardiac myocytes to increasing calcium and isoprenaline were measured, as well as oxygen consumption of myocyte suspensions. Creatine kinase and myosin isoforms were investigated in whole ventricle. The degree of fibrosis of the ventricle was quantified using histological methods.
24 white male New Zealand rabbits were treated with adriamycin (1 mg.kg-1) twice a week for eight weeks, and allowed to recover for two weeks. There were 29 untreated controls. Six further rabbits were implanted with mini osmotic pumps delivering a constant infusion of isoprenaline for one week; five controls had pumps containing saline.
Cardiac myocytes were enzymatically isolated, and their contraction amplitude and velocity monitored. Cells isolated from adriamycin treated rabbits had a lower contraction amplitude than those from controls when maximally activated with calcium, at 11.1(0.9)% shortening (n = 11) v 13.6(0.5)% (n = 14), p less than 0.02; or with isoprenaline, at 11.6(0.6)% shortening v 13.1(0.4)%, p less than 0.05. Contraction, but not relaxation, velocity in maximum calcium or isoprenaline was also significantly lower in cells from adriamycin treated animals. Oxygen consumption per 10(6) cells was lower in preparations from treated animals (p less than 0.05), but the relative effects of glucose, acetate, 2,4-DNP, and cyanide were unaffected. There was no significant change in creatine kinase or myosin isozyme composition or in amounts of fibrosis following adriamycin treatment. However, the quantity of myosin per g wet weight of tissue was significantly reduced from 8.04(0.45) mg.g-1 wet tissue, n = 4, in controls to 5.76(1.55) mg.g-1, n = 6, in adriamycin treated animals (p less than 0.001). The EC50 for isoprenaline was unchanged in cells from treated animals. Together with the unaltered maximum isoprenaline/calcium ratio, this implies that there is no change in beta adrenoceptor sensitivity following adriamycin treatment. To confirm that it was possible to desensitise rabbit cardiac beta adrenoceptors, and to detect changes in sensitivity on single cells, rabbits were treated with isoprenaline for one week. Such treatment decreased the maximum isoprenaline/calcium contraction amplitude ratio from 0.97(0.15), n = 5, to 0.47(0.12), n = 6 (p less than 0.05), and increased the EC50 from 7.9 to 224 nM (p less than 0.05).
Single cardiac myocytes isolated from the hearts of adriamycin treated rabbits show a decrease in contraction amplitude, velocity, and oxygen consumption compared to controls. The decreased contractility of individual myocytes may relate to their low myosin content, and could contribute to the reduced cardiac output produced by adriamycin treatment. Heart failure induced by adriamycin in the rabbit is not accompanied by beta adrenoceptor desensitisation.
Anthracyclines, such as doxorubicin and daunorubicin, are highly effective anticancer agents that produce a well-described but incompletely understood cardiac toxicity. According to a popular hypothesis, anthracyclines injure the heart by generating oxygen-centered free radicals. This free radical hypothesis, however, appears to be inconsistent with many observations, such as the frequent failure of anthracyclines at cardiotoxic doses to produce evidence of increased free radical generation. Other explanations of cardiotoxicity involve platelet-activating factor, prostaglandins, histamine, calcium, and C-13 hydroxy anthracycline metabolites. These C-13 hydroxy metabolites, on the basis of in vitro data, are considerably more potent than parent compounds as myocardial depressants and as inhibitors of ATPases of sarcoplasmic reticulum, mitochondria, and sarcolemma. Further studies will be required to determine whether metabolites or the other putative injurious agents discussed contribute substantially to the cardiomyopathy of anthracycline therapy. The hypotheses presented in this paper should provide a useful framework for subsequent investigations into the mechanisms of anthracycline cardiotoxicity.
Changes in the major parameters of central and intracardiac hemodynamics and body's oxygen supply were examined in 93 patients with massive myocardial infarction in the in-hospital period of the disease. Traditional therapy was given to 71 patients; in addition, phosphocreatine infusions (a course dose being 30 g) were used in 22 patients in acute myocardial infarction. Phosphocreatine therapy failed to substantially affect cardiac pump function, but prevented left ventricular dilation and development of congestive heart failure. The patients receiving phosphocreatine showed an increase in body's oxygen consumption due to its elevated tissue extraction. No adverse effects of phosphocreatine were found.
A consistent pattern of intraventricular regional pressure gradients exists under physiological conditions during the rapid filling phase of diastole in the normal dog left ventricle. We hypothesized that this pressure gradient pattern is caused, in part, by early diastolic recoil of the left ventricular walls in conjunction with release of elastic potential energy stored during systole, generating suction and thus contributing to diastolic filling. If so, any condition that interferes with normal regional systolic function might be expected to modify the pattern of the normal early diastolic intraventricular pressure gradients. Accordingly, the present study was designed to determine whether acutely induced regional systolic left ventricular mechanical dysfunction is accompanied by changes in the pattern of the early diastolic intraventricular pressure gradients. Acute myocardial ischemia was induced by balloon occlusion of the left anterior descending coronary artery (LAD) in nine anesthetized closed-chest dogs. The maximum early diastolic intraventricular pressure gradient (MIVP) was measured between the mid-left ventricle and apex with a dual-sensor micromanometer (3-cm spacing between the sensors) before and 20 minutes after LAD occlusion. Ejection fraction (EF) and number of dyskinetic chords (DChords) were measured from left ventricular contrast ventriculograms. Twenty minutes after LAD occlusion, the nine dogs evidenced significant changes in EF (56 +/- 10% to 37 +/- 8%), DChords (0 +/- 0 to 17 +/- 16 chords), left ventricular minimum pressure (-1.7 +/- 0.5 to 0.0 +/- 1.5 mm Hg), left ventricular end-diastolic pressure (4.2 +/- 1.2 to 5.9 +/- 2.2 mm Hg), and heart rate (90 +/- 17 to 103 +/- 18 beats/min).(ABSTRACT TRUNCATED AT 250 WORDS)
Adriamycin (doxorubicin, AdM) is a potent antineoplastic agent which binds specifically and with high affinity to the acidic phospholipid cardiolipin (CL) [Goormaghtigh et al. (1980) Biochim. Biophys. Acta 597, 1]. Duramycin (DM), a polypeptide antibiotic, has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol [Navarro et al. (1985) Biochemistry 24, 4645]. The selectivity of DM-PE interaction was confirmed. AdM and DM were then used to explore the roles of CL and PE in Ca2+ translocation in a phosphatidylcholine (PC)/PE/CL liposome system modeled on the inner mitochondrial membrane with the following results: (i) AdM (100-400 microM) altered Ca2+ uptake by PC/PE/CL (4/4/1, mol/mol) liposomes in a concentration-dependent fashion which varied with temperature, external Ca2+ concentration, and liposome PE content. (ii) Addition of AdM was qualitatively equivalent to increasing temperature, Ca2+ concentration, or liposome PE content, and cooperative interactions among these parameters were observed. An increase in any one factor generally enhanced Ca2+ uptake; simultaneous increases in several factors inhibited uptake. (iii) Inhibition of Ca2+ uptake was correlated with efflux of Arsenazo III. (iv) Ca2+ uptake by PC/PE/CL liposomes is biphasic [Kester and Sokolove (1989) Biochim. Biophys. Acta 980, 127]. DM suppressed the PE-dependent slow phase and stimulated the PE-independent initial phase. Ca2+ uptake by PC/PE/CL liposomes in the presence of DM resembled uptake by PC/CL liposomes. These data confirm the ability of PE to enhance the slow, highly temperature-dependent component of CL-mediated Ca2+ translocation and suggest that this process is sensitive to lipid phase behavior.
Sudden induction of ischemia by occlusion of a major branch of a coronary artery in mammalian heart sets into motion a series of events that culminates in the death of markedly ischemic myocytes. The changes begin within 8-10 seconds of occlusion and include 1) cessation of aerobic metabolism, 2) depletion of creatine phosphate, 3) onset of anaerobic glycolysis (AG), and 4) accumulation of products of anoxic metabolism in the ischemic tissue. Functional defects appear simultaneously, including depressed contractile activity and electrocardiographic changes. The demand of the ischemic myocytes for energy exceeds the supply of high-energy phosphate (approximately P) possible from AG; as a consequence, myocyte adenosine diphosphate increases, and adenylate kinase is activated to capture the approximately P bond of adenosine diphosphate. Adenosine monophosphate is a product of this reaction; it accumulates and is progressively degraded to nucleosides and bases that are lost from the myocyte. The pace of development of the short-term metabolic changes slows after 40-60 minutes of ischemia, at which time most of the severely ischemic myocytes are irreversibly injured. Early in the irreversible phase of injury tissue is characterized as follows by: 1) very low approximately P content (creatine phosphate less than 1-2% and adenosine triphosphate less than 10% of control), 2) a depressed adenine nucleotide pool that consists principally of adenosine monophosphate, 3) virtual cessation of AG, 4) low pH and low glycogen content, 5) high inosine and hypoxanthine contents, 6) a markedly increased osmolar load consisting chiefly of lactate, and 7) characteristic ultrastructural changes including cell swelling and evidence of generalized mitochondrial and marked sarcolemmal damage. Sarcolemmal disruption is the feature that we hypothesize causes irreversibility; however, its pathogenesis is unknown.
The acute effects of doxorubicin on coronary perfusion and left ventricular pressures and intracellular phosphate metabolite levels, the latter obtained by 31P nuclear magnetic resonance, were measured simultaneously in isolated, isovolumic rat hearts (Langendorf preparation) perfused at constant flow. Nineteen experimental hearts were perfused for 70 min with oxygenated HEPES-buffered solution containing 6 mg/L doxorubicin. These were compared with 18 control hearts (C), perfused under identical conditions but without doxorubicin, by repeated measures analysis of variance. In the experimental group, coronary perfusion pressure steadily increased to 226.3 +/- 13.8% (mean +/- SEM) of initial levels (p less than 0.0001 vs. C). Because flow was constant, the increase in coronary perfusion pressure in experimental hearts indicates a greater than twofold increase in coronary resistance. Intracellular phosphocreatine and ATP decreased to 80.3 +/- 3.9% (p less than 0.005 vs. C) and 82.1 +/- 6.4% (p less than 0.05 vs. C), whereas inorganic phosphate increased to 149.7 +/- 19.1% (p less than 0.05 vs. C) of initial levels, respectively. Accompanying these changes, diastolic pressure steadily increased to 521.7 +/- 91.4% of initial levels (p less than 0.0001 vs. C). Developed pressure initially increased to 107.1 +/- 4.5% at 30 min, and thereafter decreased to 76.2 +/- 6.3% at 70 min (p less than 0.05 vs. C). Typical structural alterations in myocyte nuclei were noted. Cellular calcium was not increased in doxorubicin-exposed hearts. Thus, acute doxorubicin cardiotoxicity is characterized by an increase in coronary resistance and is closely correlated with alterations in ventricular function and a decrease in intracellular high-energy phosphate content.(ABSTRACT TRUNCATED AT 250 WORDS)
On the basis of the positive results of recent experimental research, a clinical trial of phosphocreatine (Neoton) was carried out in 60 randomized patients with acute myocardial infarction (30 patients in the Neoton group and 30 patients in the control group). Neoton was given intravenously not later than 6 hours after the onset of symptoms, in a dose of 2 gm as a bolus injection, followed by a 2-hour infusion at the rate of 4 gm/hr. Holter monitoring for 24 hours showed a significant decrease in the frequency of ventricular premature beats: in the Neoton-treated group the total number of ventricular premature beats for 24 hours was 690 +/- 179 vs 2468 +/- 737 in the control group (p less than 0.02). During this period of time the number of ventricular tachycardia paroxysms was 6 +/- 2 in the treated group and 97 +/- 35 in the control group (p less than 0.01). No side effects or complications were found after administration of phosphocreatine. It is concluded that phosphocreatine may be a potentially important antiarrhythmic drug for treatment of patients with acute myocardial infarction.
The effect of regional myocardial ischemia complicated by ventricular fibrillation (VF) on the ultrastructure of subendocardial (SE) and false tendon (FT) Purkinje cells (PC) was studied in anesthetized dogs. In all cases of early ischemia with spontaneous VF, many PC exhibited ultrastructural damage as early as 2 min after the onset of ischemia. The changes noted were: intercalated disk dissociation, sarcoplasmic reticulum vacuolization (SRV), supercontraction, mitochondrial swelling, and sarcolemmal defects (rigor cells). The appearance of at least some rigor PC seemed to precede spontaneous VF, since these cells were absent from the conduction systems in control hearts in which VF was induced by electric shock or reperfusion, from hearts from sham-operated dogs, or from hearts subjected to longer periods of uncomplicated myocardial infarction. These observations indicate that alterations in SE and FTPC may play a role in the pathogenesis of sudden death due to early myocardial ischemia. The mechanism of this rapid damage of PC remains obscure.
The influence of exogenous creatine phosphate (CP) on peroxidative heart injury was investigated in two experimental models: isolated working rat hearts and myocardial membrane preparations. In the first model the addition of 190 microM hydrogen peroxide to the perfusion buffer caused a marked decrease of aortic flow, minute work and peak aortic pressure, and leakage of intracellular enzymes. In the presence of 10 mM CP the hemodynamic damage produced by the same concentration of hydrogen peroxide was significantly lower and enzyme release was also remarkably reduced. The protection was concentration-dependent and the whole structure of the molecule was required since creatine was found to be ineffective. In the absence of hydrogen peroxide, CP and creatine did not affect heart performance. In microsomal membrane preparations CP decreased the formation of thiobarbituric acid-reactive material (malonaldehyde) induced by hydrogen peroxide in the presence of ferrous ions. This protection was concentration-dependent and occurred at physiological concentrations of CP. Also in this experimental model creatine had no effect and creatine plus inorganic phosphate was much less active than CP. The influence of CP on oxidative heart stress could account for the beneficial effect of this substance in different models of ischemic injury.
The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster overy cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2‐APam, 7‐ASte, 12‐ASte and anthracene‐phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Eggyolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems.
Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0–100 μM, the efficiency of energy transfer was 12‐ASte > 7‐ASte > 2‐APam, with 100% energy transfer for 12‐ASte above a drug concentration of 30 μM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7‐ASte > 12‐ASte = 2‐APam was observed. Measurements of fluorescence quenching using the non‐permeant Cu ²⁺ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer.
Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains.
In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of theese cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments.
These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.
In the presence of cardiolipin-containing small unilamellar vesicles, the antitumor compound adriamycin loses its ability to catalyse the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. The data strongly suggest that in the presence of cardiolipin the dihydroanthraquinone moiety is embedded in the phospholipid bilayer and thus inaccessible to the enzyme.
To study the character of the mechanism of protective action of phosphocreatine on ischemic myocardium the effects of phosphocreatine (PCr) and phosphoarginine (PArg) were compared. PCr and PArg were shown to expose identical Ca2+-chelating properties and were used as their Na-salts. Only PCr protected the cardia function during ischemia and simultaneously inhibited the accumulation of lysophosphoglycerides, products of phospholipid degradation. PArg failed to exert both of these effects. By an EPR probe method PCr was shown to increase the order of structural organization of phospholipids in the cardiac sarcolemmal vesicles. The results show that the effect of PCr on ischemic myocardium is not due to nonspecific changes in the ion composition of a solution, but most probably due to highly specific effect of phosphocreatine on the phospholipid membrane of the cardiac cells sarcolemma.
In valve replacement operations on 78 patients with acquired heart disease, the efficiency of phosphocreatine in intraoperative protection of ischemic myocardium was evaluated by clinical, morphologic, and biochemical methods. Phosphocreatine (8 to 10 mmol/L) in a blood cardioplegic solution was used in operations on 41 patients; in the control group (37 patients) standard blood cardioplegia was used. In the group with phosphocreatine treatment we observed more rapid recovery of hemodynamics after release of the aortic cross-clamp, a decreased frequency of fibrillation, and more frequent restoration of sinus rhythm even if there were sinus rhythm disturbances before aortic cross-clamping. Analysis of the biopsy samples taken from the right ventricle showed protection of the sarcolemma against ischemic damage afforded by phosphocreatine and complete preservation of high-energy phosphates. The results obtained confirm the conclusion made by Robinson, Braimbridge, and Hearse (J Thorac Cardiovasc Surg 1984; 87:190-200) that phosphocreatine is an effective additional cardioprotective agent when used in cardioplegic solutions.
The effect of exogenous phosphocreatine on ischemic myocardium was studied in experimental infarction in rabbits and in total ischemia of pig heart tissue (in vitro). It is shown that single dose administration of phosphocreatine is followed by its rapid clearance from blood plasma (with a half lifetime of 4-6 min), but constantly high plasma concentration of phosphocreatine can be maintained by its intravenous infusion. When administered by this method into rabbits during experimental myocardial infarction, phosphocreatine reduces by 40% the size of the necrotic zone. Morphological electron microscopic studies using a lanthanum tracer method showed significant protection of the sarcolemma of cardiomyocytes in the perinecrotic zone by phosphocreatine. In vitro studies on the model of total ischemia also showed significant protection of cardiac sarcolemma from irreversible ischemic injury and reduction in the rate of high-energy phosphate depletion in the presence of phosphocreatine in the extracellular space. Additionally, it is demonstrated that creatine kinase released during myocardial infarction into the blood flow and exogenous phosphocreatine administered intravenously may significantly inhibit platelet aggregation by rapid removal of ADP, and thus potentially improve microcirculation during myocardial infarction.
An isolated perfused working rat heart preparation was used to assess the effect of including creatine phosphate (10 mmol/l) in the perfusion fluid of hearts subjected to aerobic perfusion (20 min), regional ischaemia (15 min) and reperfusion (2 min). Creatine phosphate had no detectable effect upon pre-ischaemic, ischaemic or post-ischaemic contractile function, it also had no statistically significant effect upon myocardial tissue ATP content. However, creatine phosphate was found to afford striking protection against reperfusion-induced arrhythmias. The incidence of ventricular fibrillation was reduced from over 80% (13/16) in the control group to 10% in the creatine phosphate-treated group (P less than 0.001). Possible mechanisms underlying the anti-arrhythmic effects of creatine phosphate were investigated using isolated rat papillary muscles superfused with or without added creatine phosphate (10 mmol/l). During aerobic superfusion at 37 degrees C creatine phosphate did not cause any statistically significant changes in contractile (developed tension) or electrophysiological (dV/dtmax and action potential duration) indices. Creatine phosphate did however influence the extent to which hypoxia (10 min) and reoxygenation (10 min) altered tension and electrophysiological characteristics. It accelerated the hypoxia-induced decline in developed tension and also the reoxygenation-induced recovery of developed tension. Relatively small changes in dV/dtmax and action potential duration were observed during hypoxia and these rapidly normalized during reoxygenation. In general creatine phosphate acted to exacerbate any changes during hypoxia and accelerate the recovery during reoxygenation. While some of the electrophysiological changes observed would indicate an anti-arrhythmic effect, they were relatively small and perhaps insufficient to explain fully the potent anti-arrhythmic properties of creatine phosphate.
This review has attempted to integrate three areas of cellular bioenergetics to present a novel and comprehensive view of heart high-energy phosphate metabolism. The goal has been to provide a rational view for the functions of phosphocreatine, creatine, and creatine kinase in the energy metabolism of muscle. The first point is that mitochondrial respiratory control is influenced by changes in the concentration of ADP, stimulating the adenine nucleotide translocase and oxidative phosphorylation. Secondly, as a consequence of the proximity of mitochondrial creatine kinase to the translocase, there appears to be a kinetic preference for ADP generated by the forward creatine kinase reaction. As a result, in heart, it can be viewed that the end product of oxidative phosphorylation is phosphocreatine. Finally, thermodynamic considerations suggest that phosphocreatine plays a major role to maintain or buffer the ATP content of the myocardium. Under conditions of increased ATP turnover, large-scale increases in the concentration of ADP, along with major decreases in ATP, are minimized by the creatine kinase equilibrium. The system responds to such a demand with substantial changes in phosphocreatine and creatine, which can kinetically increase the rate of mitochondrial creatine kinase and thus oxidative phosphorylation. Theoretical enzymologists have long argued whether enzymes are under kinetic or thermodynamic control. Heart creatine kinase may be a unique example where both types of control simultaneously operate in different microenvironments, with mitochondrial creatine kinase kinetically controlled, while the sarcoplasmic isozyme is influenced by equilibrium thermodynamics. Overall, heart creatine kinase may be a unique example of "kineto-dynamic" metabolic integration.
In this study, the authors examined the effect of the anti-tumor agent Adriamycin, a known cardiotoxin, on mouse heart, diaphragm, and gastrocnemius muscle. Using an established model of Adriamycin cardiac toxicity, they found that 4 days after the intraperitoneal injection of 20 mg/kg of Adriamycin, characteristic heart lesions, including vacuolation of the sarcoplasmic reticulum, interstitial edema, and mitochondrial degeneration, were demonstrated in all treated animals. Furthermore, similar, but much more severe, myocyte damage was demonstrated in the diaphragm; muscle toxicity followed a decreasing gradient of injury from the peritoneal to the thoracic surface of the tissue. On the other hand, treatment with Adriamycin resulted in an increase in the size and number of lipid droplets in the red fibers of the gastrocnemius muscle without any other ultrastructural evidence of drug-induced damage to myocytes. An examination of the pharmacokinetics and metabolism of Adriamycin after intraperitoneal treatment revealed that relative drug levels in muscle (diaphragm much greater than heart much greater than gastrocnemius) paralleled the degree of ultrastructural damage observed. This study indicates that treatment with Adriamycin can produce significant injury to non-cardiac muscle in a fashion that strongly resembles the characteristic pattern of Adriamycin-related damage to the heart, and that the degree of myocyte damage is apparently dependent upon the Adriamycin concentration in the tissue.
The quantitative analysis of the mobile high-energy phosphorus metabolites in isovolumic Langendorff-perfused rabbit hearts has been performed by 31P NMR utilizing rapid pulse repetition to optimize sensitivity. Absolute quantification required reference to an external standard, determination of differential magnetization saturation and resonance peak area integration by Lorentzian lineshape analysis. Traditionally accepted hemodynamic indices (LVDP, dp/dt) and biochemical indices (lactate, pyruvate) of myocardial function were measured concomitantly with all NMR determinations. Hemodynamically and biochemically competent Langendorff-perfused rabbit hearts were found to have intracellular PCr, ATP, GPC, and Pi concentrations of 14.95 +/- 0.25, 8.08 +/- 0.13, 5.20 +/- 0.58 and 2.61 +/- 0.47 mM respectively. Intracellular pH was 7.03 +/- 0.01. Cytosolic ADP concentration was derived from a creatine kinase equilibrium model and determined to be approximately 36 microM. Reduction of perfusate flow from 20 to 2.5 ml/min demonstrated statistically significant decreases in PCr, ATP, and pH as well as an increase in Pi that correlated closely with the independent hemodynamic and biochemical indices of myocardial function. The decrease in ATP and PCr concentrations precisely matched the increase in Pi during reduced flow. These results constitute the first quantitative determination of intracellular metabolite concentrations by 31P NMR in intact rabbit myocardium under physiologic and low flow conditions.