ryanodine and Cardiomyopathies

The present experiments were performed in order to study abnormal action potential configuration and ion channel activity in ventricular myocytes obtained from 23 male myopathic Syrian hamsters (Biobreeders strain 14.6, 32-52 weeks old) compared with 10 age-matched healthy control hamsters (Biobreeders F1B) by means of whole-cell patch-clamp techniques. The results show that the myopathic myocytes had a longer action potential duration, a reduced transient outward K(+) current on depolarization and a smaller transient inward current on repolarization after prolonged depolarizing pulses (> 500 msec). However, the L-type Ca(2+) current and the inwardly rectifing K(+) current were not significantly different from those of healthy myocytes. The oscillatory transient inward currents could be diminished by treatment with ryanodine (0.01-1 micromol/L), a sarcoplasmic reticulum (SR) Ca(2+) release channel blocker, or with Na(+)-free superfusate. We conclude that the hereditary myopathic hamsters are less likely to develop delayed after depolarization-related transient inward currents and triggered arrhythmias owing to a smaller SR Ca(2+) content.

Topics: Action Potentials; Algorithms; Animals; Calcium; Calcium Channels, L-Type; Cardiomyopathies; Cell Separation; Cricetinae; Heart Failure; In Vitro Techniques; Ion Channels; Male; Mesocricetus; Myocytes, Cardiac; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Ryanodine; Sodium

The recently devised domain peptide probe technique was used to identify and characterize critical domains of the cardiac ryanodine receptor (RyR2). A synthetic peptide corresponding to the Gly(2460)-Pro(2495) domain of the RyR2, designated DPc10, enhanced the ryanodine binding activity and increased the sensitivity of the RyR2 to activating Ca(2+): the effects that resemble the typical phenotypes of cardiac diseases. A single Arg-to-Ser mutation made in DPc10, mimicking the recently reported Arg(2474)-to-Ser(2474) human mutation, abolished all of these effects that would have been produced by DPc10. On the basis of the principle of the domain peptide probe approach (see Model 1), these results indicate that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. This domain peptide approach opens the new possibility in the studies of the regulatory and pathogenic mechanisms of the cardiac Ca(2+) channel.

Topics: Amino Acid Sequence; Animals; Calcium; Cardiomyopathies; Dogs; Dose-Response Relationship, Drug; Microsomes; Models, Biological; Molecular Sequence Data; Myocardium; Peptide Fragments; Peptides; Polylysine; Polymorphism, Genetic; Protein Structure, Tertiary; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

Decreased cardiac contractility has been observed in cirrhosis, but the cause remains unclear. Because cardiomyocyte contraction depends on Ca2+ influx entering via L-type Ca2+ channels (I(Ca,L)s) to activate Ca2+ release from the sarcoplasmic reticulum, we postulated that the Ca2+ transients may be abnormal in cirrhotic cardiomyocytes. We aimed to investigate the status of the cellular Ca2+-regulatory system in a rat model of cirrhotic cardiomyopathy.. Cirrhosis was induced by bile duct ligation. The I(Ca,L) protein expression was detected by Western blotting. Ca2+ currents were measured electrophysiologically. The intracellular Ca2+ system, which includes the ryanodine receptor 2 (RYR2), sarcoplasmic reticulum Ca2+-pump adenosine triphosphatase (SERCA2), and Ca2+-binding protein were quantitatively assayed by reverse-transcription polymerase chain reaction and Western blots and functionally by 3H-ryanodine binding and radiolabeled Ca2+ uptake.. I(Ca,L) protein expression was reduced in cirrhotic rats compared with controls, and the peak inward Ca2+ current was significantly less. At all membrane potentials examined, I(Ca,L)s current densities from cirrhotic animals were consistently lower, and the response to maximal isoproterenol stimulation was also significantly lower. Protein expression and messenger RNA transcription for RYR2, SERCA2, and calsequestrin were quantitatively unchanged, and 3H-ryanodine binding characteristics and Ca2+ uptake were also unaltered.. We conclude that the decreased cardiac contractility in cirrhotic cardiomyocytes is caused by dysfunction of the Ca2+-regulatory system. Plasma membrane I(Ca,L)s are quantitatively reduced and functionally depressed, whereas intracellular systems are intact.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cardiomyopathies; Colforsin; Isoproterenol; Liver Cirrhosis, Experimental; Male; Myocardial Contraction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases

In human ventricular myocardium, contractile force increases at higher stimulation frequencies (positive force-frequency relation). In failing hearts, the force-frequency relation (FFR) is negative. Data on the effect of volatile anesthetics on FFR are very limited.. The authors obtained left ventricular tissue from 18 explanted hearts from patients undergoing cardiac transplantation and tissue of 8 organ donors. The negative inotropic effect of halothane, isoflurane, and sevoflurane on isometric force of contraction of isolated muscle preparations at a stimulation frequency of 1 and 3 Hz and the effect of each anesthetic on the FFR were studied. Ryanodine and verapamil were studied for comparison. In addition, the effect of the anesthetics on Ca(2+)-dependent (3)H-ryanodine binding was investigated.. In nonfailing myocardium, halothane was the strongest negative inotropic compound, and the positive FFR was not affected by either drug. In failing myocardium, halothane also showed the strongest negative inotropic effect, but the positive shape of FFR was restored by halothane and ryanodine. In contrast, isoflurane, sevoflurane, and verapamil did not change FFR. Only halothane shifted the Ca(2+)-dependent (3)H-ryanodine binding curve toward lower Ca(2+) concentrations.. In nonfailing human myocardium, none of the anesthetics affect FFR, but halothane is the strongest negative inotropic compound. In failing myocardium, halothane, but not isoflurane or sevoflurane, restores the positive shape of FFR. Both the more pronounced negative inotropic effect of halothane and the restoration of the positive shape of FFR in failing myocardium in the presence of halothane can be explained by its interaction with the myocardial sarcoplasmic reticulum calcium-release channel.

Topics: Anesthetics, Inhalation; Calcium Channel Blockers; Calcium Channels; Cardiomyopathies; Culture Techniques; Drug Interactions; Electric Stimulation; Halothane; Humans; Isoflurane; Methyl Ethers; Myocardial Contraction; Myocardium; Ryanodine; Sarcoplasmic Reticulum; Sevoflurane; Vasodilator Agents; Verapamil

Altered Ca2+ metabolism of the sarcoplasmic reticulum results in changes of the contractile behavior in failing human myocardium. The ryanodine-sensitive Ca2+ release channel of the sarcoplasmic reticulum plays a key role in the intracellular Ca2+ handling in cardiac myocytes. Recently, we showed that the density of 3H-ryanodine binding sites which correspond to the SR Ca2+ release channel in human myocardial homogenates is unchanged in failing human myocardium. However, the sensitivity of the channel towards Ca2+, which acts as the trigger signal of channel activation and thereby initiates contraction, has not yet been investigated in failing and nonfailing myocardium.. Homogenates (100 micrograms protein) from hearts with dilated (DCM, n = 10) or ischemic (ICM, n = 9) cardiomyopathy were incubated with a saturating concentration of 3H-ryanodine (12 nM) in the presence of different Ca2+ concentrations ranging from 1 nM to 10 mM. For comparison, myocardium of 8 nonfailing hearts which could not be transplanted for technical reasons was investigated. Non-specific binding was determined in the presence of a high concentration (10 microM) of unlabeled ryanodine.. 3H-ryanodine binding to the Ca2+ release channel showed a bell-shaped pattern with an increase in specific binding at submicromolar Ca2+ concentrations and a decrease at higher Ca2+ concentrations than 0.5 mM, whereas nonspecific binding was not influenced by different Ca2+ concentrations. In nonfailing myocardium, maximal 3H-ryanodine binding (Bmax) was 85.2 +/- 3.1 fmol/mg protein and half-maximal binding was reached at a free Ca2+ concentration of 0.25 (0.22-0.30) microM (EC50). Neither EC50 values nor maximal specific 3H-ryanodine binding differed between nonfailing and failing myocardium of both etiologies. EC50 values were 0.24 (0.23-0.26) microM (DCM, n = 10) or 0.28 (0.25-0.31) microM (ICM, n = 9), respectively. Caffeine (2 mM) and the ATP-analogon AMP-PCP (1 mM) led to a shift towards lower Ca2+ concentrations consistent with an activation of the channel by these compounds, whereas Mg2+ (0.7 mM) shifted the Ca(2+)-dependence of 3H-ryanodine binding towards higher Ca2+ concentrations indicating inhibition of channel opening. After activation of the Ca2+ release channel by caffeine or AMP-PCP as well as after the inhibition with Mg2+ EC50 values were the same in failing and nonfailing myocardium.. Caffeine and AMP-PCP sensitize, whereas Mg2+ desensitizes the myocardial Ca2+ release channel to Ca2+. The determination of Ca(2+)-dependent 3H-ryanodine binding to the human myocardial Ca2+ release channel is a useful tool to investigate its open probability. Furthermore, the Ca(2+)-sensitivity and the pharmacological behavior of the human SR Ca2+ release channel are similar in failing and nonfailing myocardium.

Topics: Calcium; Calcium Channels; Cardiomyopathies; Heart; Humans; Ion Channel Gating; Myocardial Contraction; Myocardium; Ryanodine; Sarcoplasmic Reticulum

The cardiomyopathic Syrian hamster develops a progressive cardiomyopathy characterized by cellular necrosis, hypertrophy, cardiac dilatation, and congestive heart failure. This study aimed to identify alterations in cardiac mechanical function and in the cellular content of sarcoplasmic reticulum (SR) Ca(2+)-release channels (ryanodine receptors, RyR) in the heart of the UM-X7.1 cardiomyopathic hamster during the development of heart failure. Experimental and healthy control hamsters were examined at 8, 18, and 28 wk of age. The UM-X7.1 hamsters had developed left ventricular (LV) hypertrophy at 8 wk and a marked LV dilatation at 18-28 wk. During the latter stage, the UM-X7.1 hamster hearts showed global hypokinesis. Equilibrium binding assays of high-affinity sites for [3H]ryanodine were performed in ventricular homogenate preparations. There was no significant difference between the two groups in the maximum number of [3H]ryanodine binding sites (Bmax) at either 8 or 18 wk of age, although the cardiac pump function was impaired in UM-X7.1 hamsters at 18 wk of age. By 28 wk, Bmax was significantly lower in the UM-X7.1 hamsters. Quantitative immunoblot assay revealed that the content of RyR protein in cardiomyopathic hearts, which was increased at the early stage, declined to below normal as heart failure advanced. These results suggest that the number of RyR in the UM-X7.1 cardiomyopathic hamsters was preserved at both the hypertrophic and early stages of heart failure with a possibly compensatory increase in the level of protein expression, although the cardiac function already showed a tendency to be impaired.

Topics: Aging; Animals; Cardiomyopathies; Cricetinae; Echocardiography; Female; Heart; Heart Failure; Male; Mesocricetus; Myocardium; Reference Values; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

We compared the contractile responses to vasoconstrictors in aortas from 20- to 22-week old cardiomyopathic hamsters, BIO 53.58 strain, and age-matched F1b strain controls. Aortas from cardiomyopathic hamsters exhibited greater contractions in response to phenylephrine, angiotensin II, and high K+ than did the controls. Neither endothelium removal nor the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA) affected the enhanced contractile responses to these vasoconstrictors, indicating no involvement of endogenous prostanoids and nitric oxide from the endothelium. The contractile response to phorbol-12,13-dibutyrate (PDB) was also more markedly increased in cardiomyopathic aortas regardless of whether extracellular Ca2+ was present. The contractile response of cardiomyopathic aorta to phenylephrine was more sensitive to the inhibitory actions of the protein kinase C inhibitors staurosporine and calphostin C than was that of control aorta. These results suggest that activation of protein kinase C is partly involved in the enhanced phenylephrine response of cardiomyopathic aorta. None of nifedipine, ryanodine, and cyclopiazonic acid modified the maximum contractions induced by phenylephrine in either cardiomyopathic aortas or controls. The Ca2+ sensitivity of tension was significantly increased in beta-escin-skinned smooth muscle of mesenteric artery from cardiomyopathic hamsters compared to that of controls. PDB induced Ca2+ sensitization, but significantly only in cardiomyopathic hamsters. We propose that the enhanced vascular reactivity in cardiomyopathic hamsters may primarily result from increased Ca2+ sensitivity of contractile proteins. In addition, protein kinase C-mediated Ca2+ sensitization may further contribute to the enhanced vascular response to agonists.

Topics: Actin Cytoskeleton; Angiotensin II; Animals; Aorta; Calcium; Calcium Channel Blockers; Cardiomyopathies; Cricetinae; Indoles; Male; Mesocricetus; Muscle Contraction; Muscle, Smooth, Vascular; Naphthalenes; Nifedipine; Phenylephrine; Phorbol 12,13-Dibutyrate; Potassium; Ryanodine; Staurosporine; Vasoconstrictor Agents

Abnormal intracellular calcium handling in cardiomyopathic human hearts has been associated with an impaired function of the sarcoplasmic reticulum, but previous reports on the gene expression of the ryanodine receptors (Ry2) are contradictory. We measured the mRNA levels, the protein levels and the number of high affinity [3H]ryanodine binding sites in the left ventricle of non-failing (n = 9) and failing human hearts [idiopathic dilated (IDCM n = 16), ischemic (ICM n = 7) or mixed (MCM n = 8) cardiomyopathies]. Ry2 mRNA levels were significantly reduced in IDCM (-30%) and unchanged in MCM and ICM and Ry2 protein levels were similar. In contrast, we observed a two-fold increase in the number of high affinity Ry2 (B(max) = 0.43 +/- 0.11 v 0.22 +/- 0.13 pmol/mg protein, respectively; P<0.01) and an unchanged K(d). Furthermore, levels of myosin heavy chain mRNA and protein per g of tissue were similar in failing and non-failing hearts, suggesting that the observed differences in Ry2 are not caused by the increase in fibrosis in failing heart. Therefore, the dissociation between the two-fold increase in the number of high affinity ryanodine receptors observed in all failing hearts and the slightly decreased mRNA level or unchanged protein level suggests that the ryanodine binding properties are affected in failing myocardium and that such modifications rather than a change in gene expression alter the channel activity and could contribute to abnormalities in intracellular Ca2+ handling.

Topics: Adult; Binding Sites; Blotting, Western; Calcium Channels; Cardiomyopathies; Cardiomyopathy, Dilated; Female; Humans; Male; Middle Aged; Muscle Proteins; Myocardial Ischemia; Myocardium; RNA Processing, Post-Transcriptional; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tritium

The mechanisms for the progression of the anthracycline-induced cardiomyopathy to contractile failure has not been defined. In vitro, doxorubicin (DOX) appears to modify calcium-mediated excitation-contraction coupling, which depresses cardiac contractility. This study characterizes the onset of contractile failure associated with the development of DOX-induced cardiomyopathy. Rabbits were treated with DOX (1 mg/kg i.v. twice weekly, 12-18 doses; DOX-treated group) and compared with a pair-fed Control group infuse with saline vehicle. The severity of the cardiomyopathy was determined by numerically-scored histopathology. Myocardial contractility was determined in thin fiber bundles from right ventricular (RV) papillary muscles and left atria that were removed and mounted on a force transducer in oxygenated Krebs-bicarbonate buffer (pH=7.4 at 30 degrees C) to record the amplitude (DT) and maximum rate (+dT/dt ) of isometric tension. Myofibrillar and calcium loading properties were determined by the calcium and caffeine-activated tension responses respectively in chemically-permeabilized fibers. With the onset of the cardiomyopathy (score <2) DT at low frequency (0.5 Hz) was depressed (0.61+/-0.01 mN/mg; n=14) compared to Control (0.93+/-0.09 mN/mg; n=15). Contractility at higher rates (1 Hz) was not different in this DOX-treated and Control groups. Maximum calcium and caffeine-activated force and the pCa to half-maximum force of permeabilized fibers were comparable in DOX-treated and Control groups. The loss of contractility of the DOX-treated group was related to reduction in sarcoplasmic reticulum calcium release channel density, as determined by Bmax for 3H-ryanodine binding in cardiac microsomal membrane fraction. Post-rest potentiation of contractility, as well as frequency-dependent (0.25-1.5 Hz) and post-extrasystolic potentiation of contractility were preserved in the DOX-treated group. In vitro, DOX depressed post-rest potentiation of contractility. Thus, the onset of contractile failure of the DOX-induced cardiomyopathy is characterized by effects consistent with disordered calcium-mediated excitation-contraction coupling and these effects are qualitatively different than in vitro effects of DOX.

Topics: Animals; Calcium Channels; Cardiomyopathies; Doxorubicin; Male; Myocardial Contraction; Rabbits; Ryanodine

The molecular basis of human heart failure is unknown. Alterations in calcium homeostasis have been observed in failing human heart muscles. Intracellular calcium-release channels regulate the calcium flux required for muscle contraction. Two forms of intracellular calcium-release channels are expressed in the heart: the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we showed that these two cardiac intracellular calcium release channels were regulated in opposite directions in failing human hearts. In the left ventricle, RyR mRNA levels were decreased by 31% (P < 0.025) whereas IP3R mRNA levels were increased by 123% (P < 0.005). In situ hybridization localized both RyR and IP3R mRNAs to human cardiac myocytes. The relative amounts of IP3 binding sites increased approximately 40% compared with ryanodine binding sites in the failing heart. RyR down-regulation could contribute to impaired contractility; IP3R up regulation may be a compensatory response providing an alternative pathway for mobilizing intracellular calcium release, possibly contributing to the increased diastolic tone associated with heart failure and the hypertrophic response of failing myocardium.

Topics: Adolescent; Adult; Blotting, Northern; Calcium Channels; Cardiomyopathies; Cells, Cultured; DNA Probes; Female; Gene Expression; Heart Failure; Heart Transplantation; Homeostasis; Humans; In Situ Hybridization; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Middle Aged; Muscle Proteins; Myocardium; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel

The objective of this study was to evaluate the tension-frequency relationship in normal and cardiomyopathic myocardium from one species with a negative or biphasic relationship, the hamster, and one with a positive relationship, the dog. Left ventricular papillary muscles from 100-day-old normal Syrian and cardiomyopathic (CHF-147) hamsters and right ventricular papillary muscles or trabeculae from normal mongrel dogs and dog with pacing-induced heart failure were used for the study. Stimulation frequency was varied from 1 to 90/min and isometric contractions recorded at each frequency prior to and after the addition of phenylephrine 10 microM. A tension-frequency relationship at varying extracellular calcium concentrations (1.25, 2.5 and 5.0 mM) was also constructed in normal hamster myocardium. Ryanodine 1.2 microM was added to a bath with normal hamster muscles and a force-frequency relationship constructed prior to and after adding phenylephrine 10 microM. A calcium dose-response curve in normal and cardiomyopathic dog myocardium was also constructed. Normal and cardiomyopathic hamster myocardium had a biphasic tension-frequency relationship with the increase in tension during the second phase being greater in normal v cardiomyopathic hamster myocardium (0.66 +/- 0.19 v 0.12 +/- 0.03 g/mm2, P < 0.05). The initial decrease in tension in response to increasing stimulation frequency was markedly attenuated in normal hamster myocardium by increasing extracellular calcium concentration. Developed tension was eliminated at lower stimulation rates by ryanodine such that when developed tension did occur, it increased with increasing stimulation rates. The addition of phenylephrine to hamster myocardium modified the tension-frequency relationship of both normal and cardiomyopathic dog myocardium and their response to phenylephrine were similar. In each case, tension increased progressively with increasing stimulation rate. Although the absolute increase in tension caused by increasing extracellular calcium was less in cardiomyopathic dog myocardium, the percent increase in tension and shortening was greater. We conclude that the tension-frequency relationship of normal and cardiomyopathic hamster myocardium are biphasic, with the initial negative phase being the result of limitations of sarcoplasmic reticulum calcium handling. Phenylephrine modifies this relationship to a uniphasic positive one, likely by its effects on both the sarcolemma and the sarcoplasmi

Topics: Animals; Calcium; Cardiomyopathies; Cricetinae; Dogs; Dose-Response Relationship, Drug; Electric Stimulation; Heart Failure; Heart Rate; In Vitro Techniques; Mesocricetus; Myocardial Contraction; Papillary Muscles; Phenylephrine; Ryanodine; Species Specificity

We have determined the densities of sarcolemmal voltage-dependent Ca2+ channels (VDCC) and Ca(2+)-induced Ca2+ release channels (CICR) of sarcoplasmic reticulum (SR) in the cardiomyopathic hamster heart using [3H]PN-200 and [3H]ryanodine, respectively. Partially purified cardiac membrane preparations from myopathic animals exhibit a twofold higher capacity to bind both [3H]PN-200 and [3H]ryanodine. Crude particulate membrane fractions from normal and cardiomyopathic animals reveal no significant difference in receptor densities for [3H]PN-200, whereas densities for [3H]ryanodine binding sites and mRNA levels are significantly (P < 0.05) diminished in cardiomyopathic animals. Inhibition of [3H]ryanodine binding by either Ca2+ or Mg2+ (in mM) as well as temperature dependence for receptor activation for [3H]ryanodine (Q10) is not significantly different, whereas membranes isolated from cardiomyopathic hearts are 1.4-fold and threefold more sensitive to activation by doxorubicin and Ca2+ (in microM), respectively. Vesicles isolated from myopathic hearts are more sensitive to inhibition of Ca2+ uptake by doxorubicin. The higher densities of binding sites for [3H]PN-200 and [3H]ryanodine observed in partially purified membrane fractions from cardiomyopathic hearts are more likely the result of altered patterns with which T-tubule and CICR channels fractionate in preparations from cardiomyopathic hamster heart rather than transcriptional upregulation and may be a consequence of the deficiency in a dystrophin-associated glycoprotein recently identified. Downregulation and functional changes in CICR channels may alter SR Ca2+ transport and contribute to the progression of cardiomyopathy in the hamster.

Topics: Animals; Calcium; Calcium Channels; Cardiomyopathies; Cricetinae; Dihydropyridines; Doxorubicin; Electrophysiology; Isradipine; Membrane Proteins; Muscle Proteins; Myocardium; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel

Several laboratories have provided indirect evidence that the myocardium of the cardiomyopathic Syrian hamster (CMH) is chronically ischemic on the basis of microvascular spasm. We previously reported evidence supporting a defect in the ryanodine-sensitive sarcoplasmic reticulum calcium release channel (SRCRC) in CMH. A relation between alterations in SRCRC and chronic ischemia has not yet been explored. A potential mechanism could be the effects of changes in redox state on thiol groups. Thiol reagents have previously been shown to regulate calcium release from SRCRC. Accordingly, we studied the inotropic effects of the sulfhydryl donors, acetylcysteine (AC), cysteine, and cystine in CMH. AC was a positive inotrope in isolated papillary muscles prepared from CMH, but not F1B controls (F1B) (p < 0.01). No significant differences were noted in inotropic responses to cysteine or cystine. AC blunted the response of CMH > F1B control papillary muscle preparations to stimulation frequency (p < 0.01). The actual tension generated (in mg/mm2) by CMH was no longer different than F1B with addition of AC (10(-3) M), ryanodine (10(-8) M), or verapamil (5 x 10(-7) M). These findings are consistent with a defect in SRCRC in CMH. This defect may be primary or may provide a novel mechanism for hibernating myocardium owing to chronic ischemia.

Topics: Acetylcysteine; Analysis of Variance; Animals; Cardiomyopathies; Cricetinae; Cysteine; Cystine; Male; Mesocricetus; Myocardial Contraction; Papillary Muscles; Ryanodine; Stimulation, Chemical; Verapamil

Doxorubicin is a highly effective cancer chemotherapeutic agent that produces a dose-dependent cardiomyopathy that limits its clinical usefulness. Clinical and animal studies of morphological changes during the early stages of doxorubicin-induced cardiomyopathy have suggested that the sarcoplasmic reticulum, the intracellular membrane system responsible for myoplasmic calcium regulation in adult mammalian heart, may be the early target of doxorubicin. To detect changes in the calcium pump protein or the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum during chronic doxorubicin treatment, rabbits were treated with intravenous doxorubicin (1 mg/kg) twice weekly for 12 to 18 doses. Pair-fed controls received intravenous normal saline. The severity of cardiomyopathy was scored by light and electron microscopy of left ventricular papillary muscles. Developed tension was measured in isolated atrial strips. In subcellular fractions from heart, [3H]ryanodine binding was decreased in doxorubicin-treated rabbits (0.33 +/- 0.03 pmol/mg) compared with control rabbits (0.66 +/- 0.02 pmol/mg; P < 0.0001). The magnitude of the decrease in [3H]ryanodine binding correlated with both the severity of the cardiomyopathy graded by pathology score (light and electron microscopy) and the decrease in developed tension in isolated atrial strips. Bmax for [3H]ryanodine binding and the amount of immunoreactive ryanodine receptor by Western blot analysis using sequence-specific antibody were both decreased, consistent with a decrease in the amount of calcium release channel of sarcoplasmic reticulum in doxorubicin-treated rabbits. In contrast, there was no decrease in the amount or the activity of the calcium pump protein of the sarcoplasmic reticulum in doxorubicin-treated rabbits. Doxorubicin treatment did not decrease [3H]ryanodine binding or the amount of immunoreactive calcium release channel of sarcoplasmic reticulum in skeletal muscle. Since the sarcoplasmic reticulum regulates muscle contraction by the cyclic uptake and release of a large internal calcium pool, altered function of the calcium release channel could lead to the abnormalities of contraction and relaxation observed in the doxorubicin cardiomyopathy.

Topics: Animals; Atrial Function; Calcium Channels; Calcium-Transporting ATPases; Cardiomyopathies; Doxorubicin; Heart Atria; Muscle, Smooth; Myocardial Contraction; Rabbits; Ryanodine; Sarcoplasmic Reticulum

Alterations in the native function of the ryanodine-sensitive Ca2+ release channel complex of sarcoplasmic reticulum (SR) isolated from rat cardiac ventricles during acute and chronic exposure to doxorubicin are examined. Compared to control SR, actively loaded SR from animals exposed to a single intravenous dose of doxorubicin exhibit faster rates of doxorubicin-induced Ca2+ release and the occupancy of [3H]ryanodine is significantly enhanced with subsequent exposure of SR membranes to doxorubicin in vitro. One week after acute exposure to doxorubicin in vitro, the EC50 for activation of the binding of [3H]ryanodine by Ca2+ is not significantly different from control SR. However, the persistence of doxorubicin-sensitized SR channels appears to be latent since repeated exposure to doxorubicin in vitro significantly enhances receptor occupancy in SR obtained from the treated rats compared to control SR. Ryanodine receptors from rats chronically exposed to doxorubicin consistently exhibit a higher sensitivity to activation Ca2+ which persists at least 4 weeks following the last injection of drug. Chronic exposure produces a concomitant reduction in the capacity of [3H]ryanodine binding sites. The marked decrease in receptor density observed with SR from doxorubicin-treated rats coincides with significant reduction in body weight, suggesting a possible influence of nutrition. However, sodium dodecyl sulfate polyacrylamide electrophoresis indicates no significant loss of the high molecular weight subunit of the ryanodine receptor, suggesting that loss of [3H]ryanodine-binding capacity may be the result of progressive and permanent channel desensitization. Consistent with desensitized receptors, membrane vesicles prepared from rats chronically exposed to doxorubicin take up significantly more Ca2+ and exhibit significantly reduced rates of doxorubicin or Ca2+/ryanodine induced Ca2+ release. The data demonstrates (i) doxorubicin inflicts cumulative SR channel lesions in vivo, (ii) a persistent sensitization of the SR channel to activation by Ca2+ and (iii) a significant and apparently irreversible reduction in the number of functional channel complexes.

Topics: Animals; Calcium; Calcium Channels; Cardiomyopathies; Doxorubicin; Injections, Intravenous; Male; Microsomes; Rats; Rats, Inbred Strains; Ryanodine; Sarcoplasmic Reticulum

We compared the radioligand-binding and inotropic effects of ryanodine [known specific regulator of sarcoplasmic reticulum (SR) calcium release channel] on BIO 14.6 cardiomyopathic Syrian hamsters with those on age-matched F1B controls. Scatchard analyses of [3H]ryanodine binding to cardiac membranes prepared from 1-2-month-old BIO 14.6 and F1B Syrian hamsters revealed the presence of a significant increase in binding capacity (Bmax = 942 +/- 49 vs. 567 +/- 33 fmol/mg protein) with no difference in affinity (KD = 3.9 +/- 0.4 vs. 3.5 +/- 0.5 nM, p less than 0.01). Ryanodine is a significantly less potent negative inotrope in isolated papillary muscles prepared from 1-2-month-old BIO 14.6 hamsters than in muscles from F1B controls (IC50 = 4.3 +/- 1 vs. 0.5 +/- 0.4 microM, p less than 0.05). Ryanodine was 200-fold less potent in the 4-6-month-old myopathic muscles than in controls (IC50 = 20 +/- 5 vs. 0.1 +/- 0.01 microM, p less than 0.01). A paradoxic positive inotropic effect of ryanodine observed in 4-6-month-old myopathic muscles at low concentrations was not seen in controls (ECmax = 126 +/- 5% at 1 +/- 0.1 x 10(-8) M). These data support the presence of a defect both biochemical and physiologic in the ryanodine-sensitive SR calcium release channel in the BIO 14.6 cardiomyopathic Syrian hamster. Purification, cloning, sequencing, and expression studies will be required to distinguish among primary, secondary, intrinsic, and regulatory defects in the ryanodine-sensitive SR calcium release channel in the cardiomyopathic Syrian hamster.

Topics: Animals; Calcium Channels; Cardiomyopathies; Cricetinae; Depression, Chemical; Mesocricetus; Myocardial Contraction; Nitrendipine; Papillary Muscles; Radioligand Assay; Ryanodine; Sarcoplasmic Reticulum

Rat cardiac membrane vesicles enriched in biochemical markers of the junctional region of sarcoplasmic reticulum (SR) and exhibiting ruthenium red-sensitive rapid Ca2+ release have been prepared. Doxorubicin and seven congeners are shown to enhance the binding of [3H]ryanodine to the ryanodine receptor with a strong structural requirement. Doxorubicin enhances the binding of [3H]ryanodine to SR membranes and soluble receptor preparations and induces Ca2+ release from SR vesicles in a highly Ca2(+)-dependent manner, suggesting that anthraquinones promote the open state of the junctional Ca2+ release channel by increasing the affinity of the Ca2+ activator site for Ca2+. Doxorubicin reduces the Kd of [3H]ryanodine binding solely by enhancing the rat of association. Caffeine competes for the same site with anthraquinones, because the caffeine-activated binding of [3H]ryanodine is inhibited by doxorubicin and vice versa. The acute effect of doxorubicin on the cardiac Ca2+ release channel is fully reversible; however, long term treatment (up to 24 hr) with doxorubicin increases the sensitivity of the preparation to subsequent acute challenge with doxorubicin. The thiol-reductive agent dithiothreitol enhances, whereas the reactive disulfide 4,4'-dithiodipyridine reduces, the doxorubicin-enhanced binding of [3H]ryanodine. These results demonstrate that the acute and chronic cardiotoxicity of anthraquinones may be accounted for by a receptor-mediated mechanism. Our findings suggest that the chronic effects observed with the clinical use of anthraquinones may be the result of a receptor-mediated shift in the redox equilibrium of allosteric thiols at the ryanodine receptor complex, which in turn leads to long term sensitization of the Ca2+ release channel.

Topics: Alkaloids; Animals; Anthraquinones; Binding, Competitive; Caffeine; Calcium; Calcium Channels; Cardiomyopathies; Daunorubicin; Doxorubicin; Drug Interactions; Electrophoresis, Polyacrylamide Gel; Female; Heart; Kinetics; Male; Rats; Rats, Inbred Strains; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Structure-Activity Relationship

Intracellular Ca2+ release and reuptake are essential for contraction and relaxation of normal heart muscle. Intracellular Ca2+ transients were recorded with aequorin during isometric contraction of myocardium from patients with end-stage heart failure. In contrast to controls, contractions and Ca2+ transients of muscles from failing hearts were markedly prolonged, and the Ca2+ transients exhibited 2 distinct components. Muscles from failing hearts showed a diminished capacity to restore low resting Ca2+ levels during diastole. These experiments provide the first direct evidence from actively contracting human myocardium that intracellular Ca2+ handling is abnormal and may cause systolic and diastolic dysfunction in heart failure.

Topics: Adolescent; Adult; Calcium; Cardiomyopathies; Diastole; Female; Heart; Hemodynamics; Humans; Intracellular Fluid; Male; Middle Aged; Myocardial Contraction; Myocardium; Ryanodine; Sarcolemma; Sarcoplasmic Reticulum; Systole; Temperature; Verapamil