Edward P. Bornet

Biochemical and morphologic correlates of cardiac ischemia. I. Membrane systems.

Functions of membrane-linked myocardial systems and morphology of the myocardial cell were examined in normal and acutely and chronically ischemic myocardium. Hemodynamic measurements of ischemic tissue showed depressed force development as well as decreased (and variable) ventricular peak pressures. Mitochondrial respiratory function was reduced, with state 3 (phosphorylating) respiration showing the most marked impairment. Losses in cytochromes c and a 3 were observed. Diminished mitochondrial calcium uptake with subsequent release of the calcium taken up during continued respiratory activity was characteristic of severely ischemic tissue, that is, 7 days after ligation. Defects in the carnitine-mediated oxidation of palmitic acid in isolated mitochondria were severe between 1 and 8 days after ligation. Losses in tissue carnitine also occurred simultaneously with decreased mitochondrial carnitine palmityltransferase activity. During this period the oxidation of hexanoic acid was unaffected. The binding and release cycle of calcium was studied in sarcoplasmic reticulum (cardiac relaxing system) isolated from control tissues and from acutely and chronically ischemic heart. An early impairment in the release phase of calcium from an acutely ischemic preparation occurred at times (12 to 60 minutes after ligation) when the other membrane-associated functions maintained normal integrity. In the chronically ischemic dog, there was marked impairment of calcium-binding variables in the first 2 weeks after ligation. This impairment occurred at times when both mitochondrial and sodium, potassium adenosine triphosphatase (Na + ,K + -ATPase) activity levels were severely impaired. The Na + ,K + -ATPase activity level was consistently lower than the control level by 7 days after ligation. However, there was no change in kinetic indexes or in the ouabainbinding characteristics in the functional enzyme remaining. Morphologic studies of tissue taken from left posterior papillary muscle 3 to 8 days after ligation revealed significant and specific changes in ultrastructure (disrupted Z and I bands, appearance of pseudo-N band and appearance of dense bodies in mitochondria); the greatest occurrence of damaged cells occurred 7 to 8 days after ligation.

Mechanisms of calcium accumulation and transport in cardiac relaxing system (sarcoplasmic reticulum membranes): Effects of Verapamil, D-600, X537A and A23187

Verapamil and D-600, two potent (I50 = 10−8 to 10−7m) calcium-dependent negative inotropic agents, are thought to act by decreasing calcium conductance (transmembrane calcium influx) across sarcolemmal membranes. These agents, although inhibiting cardiac relaxing system (sarcoplasmic reticulum fragments) calcium binding and uptake, did so at very high concentrations (I50 ∼ 1 mm) suggesting that their negative inotropic effect occurs by an action on a site other than the sarcoplasmic reticulum. The agents did not influence the rate of spontaneous release of calcium from cardiac relaxing system, nor did they affect the rapid release induced by a sudden increase in pH or by the addition of X537A or A23187, potent ionophoric agents that release calcium from sarcoplasmic reticulum fragments. The lack of effect on release suggests that “calcium channels” are not an important component of calcium release from cardiac relaxing system.

Association of glycogenolysis with cardiac sarcoplasmic recticulum: II. Effect of glycogen depletion, deoxycholate solubilization and cardiac ischemia: Evidence for a phosphorylase kinase membrane complex

The glycogenolytic enzymes associated with cardiac sarcoplasmic reticulum have been shown to have two major features which make the munique. The conversion of phosphorylase b to phosphorylase a can take place in the presence of 20 m m eGTA which ordinarily inhibits this reaction by chelating the calcium which is required for the enzyme phosphorylase b kinase of muscle. In addition, inhibitors of “debrancher” enzyme such as Tris also inhibit phosphorylase b activity in the sarcoplasmic reticulum-glycogen complex, but do not inhibit purified phosphorylase. The phosphorylase b to a conversion which occurs through the addition of ATP alone is very rapid, being complete within 5–10 min in normal cardiac sarcoplasmic reticulum and converting approximately 30% of the total phosphorylase activity. DOC solubilization irreversibly perturbed the sarcoplasmic reticulum membrane so that EGTA resistance could not be reconstituted although phosphorylase b to a conversion continued to some extent. Thus, EGTA sensitivity depended, at least in part, on some specific property of the native membrane system. Examination of skeletal muscle sarcoplasmic reticulum from fast, slow and mixed skeletal muscle revealed that phosphorylase b to a conversion occurred, but was EGTA sensitive in a manner similar to that of the solubilized cardiac preparation. Glycogen depletion solubilized phosphorylase and uncoupled it from its activating enzymes. This uncoupling could not be reversed by the addition of glycogen at any concentration. A relationship was demonstrated between the phosphorylase activity in sarcoplasmic reticulum fractions and the glycogen concentration in both normal sarcoplasmic reticulum that was amylase treated, and from sarcoplasmic reticulum which was isolated from ischemic tissue. A correlation between density observed on a sucrose density gradient and glycogen concentration was described. The relationship between phosphorylase and debrancher activity depended only on the presence of glycogen and therefore was not as specific a structural feature. The results suggest that the sarcoplasmic reticulum glycogen complex is highly specific and that specific functional couplings between glycogen and sarcoplasmic reticulum membranes exist. Thus, modulation of the complex could occur through control of glycogenolysis or alterations in membrane conformation, and this complex might represent a link between energy metabolism and excitation-contraction coupling.

Phasic components of calcium binding and release by canine cardiac relaxing system (sarcoplasmic reticulum fragments).

Abstract Cardiac relaxing system (sarcoplasmic reticulum fragments) isolated from canine myocardium exhibit cyclic changes initiated by ATP. The addition of ATP results in rapid calcium binding followed by a slower spontaneous release. After release has occurred, restimulation of calcium binding can be effected by additional ATP provided the ATP which initiated the first binding is hydrolyzed to a low level. The evidence suggests that calcium binding and calcium release represent two independent phasic changes in a calcium binding site with different responses to temperature ( Q 10 1.5 and 2.6, respectively), pH, Mg 2+ and ATP. The ability of ATP to restimulate binding may represent still a third phasic change. Evidence is also presented that each site must go through each phase independently in order to proceed to the next. The first phase is initiated by ATP and results in calcium binding; the second phase requires calcium binding and results in calcium release; the third phase requires calcium release. The implications of these changes and the source of energy transduction are discussed.

The effect of taurine on cardiac sarcoplasmic reticulum

Abstract A recent communication reported that the rate of calcium uptake by sarcoplasmic reticulum (SR) isolated from rat skeletal muscle could be increased by the isolation of the SR in 15 mM taurine, and that exposure of the SR to taurine throughout the isolation procedure resulted in an increased yield of SR. Because of these results in rat skeletal muscle SR studies were carried out on dog myocardial SR. Sarcoplasmic reticulum isolated from adult dog cardiac muscle was not affected by taurine in concentrations as high as 15 millimolar. The addition of taurine to isolation media did not affect calcium transport, ATPase, binding, or release. Sarcoplasmic reticulum fragments were stored and re-examined over a period of a week without appreciable difference in stability of activity between those isolated in the presence of taurine and the control group. This lack of effect suggests that the role of taurine in cardiac muscle metabolism is not likely to be found in regulation of the sarcoplasmic reticulum.

Spontaneous calcium release from sarcoplasmic reticulum. A re-examination.

A recent study by Blayney and co-workers (Blayney, L., Thomas, H., Muir, J. and Henderson, A. (1977) Biochim. Biophys. Acta 470, 128--133) purported to demonstrate that apparent spontaneous calcium release in sarcoplasmic reticulum is an artifact of the uptake of murexide dye. This report demonstrates that spontaneous calcium release (1) takes place despite equilibration of murexide sarcoplasmic reticulum to a stable baseline; (2) may be reversed by addition of ATP or oxalate after release has begun. The identical phenomenon can be demonstrated utilizing the indicator arsenazo III or Millipore filtration methods. The results suggest that equilibration of the murexide with sarcoplasmic reticulum vesicles must occur prior to ATP addition in order to achieve a stable baseline but that spontaneous calcium release is not an artifact.

146 COMPARISON OF METHODS OF CARDIOPLEGIA

The purpose of our study was to compare the effectiveness of three techniques of coronary artery perfusion for protection of the canine myocardium during ischemic cardiac arrest (IA).In each of three different methods, the perfusate, chilled to 4°C., consisted of 5% dextrose in water, 15 meq KCl, 5 meq NaHC03 and 10,000 units heparin/L. In method 1, during aortic crossclamping, the solution was infused continuously at a rate sufficient to maintain myocardial temperature at 18°C. In method 2 the solution was given as a single bolus of 10 cc/kg. In method 3 the solution was administered also by a single bolus and to the solution was added 2 mM CaCl2 and 25 mM MgCl2. In a control preparation (method 4), no perfusate was administered during IA. In the table are listed the number of immediate survivors of the experiment, the indices of subcellular and hemodynamic myocardial function (the latter expressed as % of control).HR=heart rate; MAR=mean arterial pressure; C0=cardiac output; LVdF/dt=LV force of contraction; SR=sarcoplasmic reticulum.Continuous perfusion appears to provide better protection of the myocardium than the single bolus technique.