Isolation of highly active preparations of sarcoplasmic reticulum and Ca2-dependent ATPase from cardiac muscle]

A microsomal preparation with a high ability for Ca2+ uptake has been isolated from pigeon heart. A method of further purification of Ca2+-accumulating system of heart, based on the ability of sarcoplasmic reticulum for the energy-dependent Ca2+ accumulation in the presence of oxalate, has been developed. Upon centrifugation in the gradient of sucrose and KCl concentration the fragments of sarcoplasmic reticulum, rendered "heavy" by calcium oxalate, can be separated from foreign cell membranes. The main component of heart "calcium pump" is Ca2+-dependent ATPase (making up to about 50% of all proteins of the purified reticulum), having a molecular weight of 100.000--105.000. Specific activity of heart Ca2+-ATPase as well as the ability of purified heart sarcoplasmic reticulum for Ca2+ uptake are only slightly less than those of the skeletal muscle reticulum. The data obtained suggest that heart sarcoplasmic reticulum may be efficient for providing heart muscle relaxation.     

Phosphorylation of titin modulates passive stiffness of cardiac muscle in a titin isoform-dependent manner

We investigated the effect of protein kinase A (PKA) on passive force in skinned cardiac tissues that express different isoforms of titin, i.e., stiff (N2B) and more compliant (N2BA) titins, at different levels. We used rat ventricular (RV), bovine left ventricular (BLV), and bovine left atrial (BLA) muscles (passive force: RV > BLV > BLA, with the ratio of N2B to N2BA titin, approximately 90:10, approximately 40:60, and approximately 10:90%, respectively) and found that N2B and N2BA isoforms can both be phosphorylated by PKA. Under the relaxed condition, sarcomere length was increased and then held constant for 30 min and the peak passive force, stress-relaxation, and steady-state passive force were determined. Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV. Fitting the stress-relaxation data to the sum of three exponential decay functions revealed that PKA blunts the magnitude of stress-relaxation and accelerates its time constants. To investigate whether or not PKA-induced decreases in passive force result from possible alteration of titin-thin filament interaction (e.g., via troponin I phosphorylation), we conducted the same experiments using RV preparations that had been treated with gelsolin to extract thin filaments. PKA decreased passive force in gelsolin-treated RV preparations with a magnitude similar to that observed in control preparations. PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length. Finally, we investigated the effect of the beta-adrenergic receptor agonist isoprenaline on diastolic force in intact rat ventricular trabeculae. We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations. Taken together, these results suggest that during beta-adrenergic stimulation, PKA increases ventricular compliance in a titin isoform-dependent manner.     

Phosphorylation of isolated components of the troponin complex of skeletal and cardiac muscle phosphorylase kinase from bird skeletal muscles

Pigeon and chicken skeletal muscle phosphorylase kinase purified to a nearly homogeneous state is able to phosphorylate both cardiac and skeletal troponin I and T. After 1-hr incubation, the enzyme transfers up to 0.35 mole of phosphorus per mole of skeletal troponin I, up to 0.5 mole of cardiac troponin I and up to 0.1 mole of cardiac and skeletal troponin T. Avian muscle phosphorylase kinase does not phosphorylate the first serine residue of cardiac and skeletal troponin T, but catalyzes the phosphate incorporation into the site(s) of troponin T located in the central or C-terminal parts of the protein molecule. The rate of troponin phosphorylation by pigeon muscle phosphorylase kinase is pH-dependent: the 6.8/8.2 ratio for troponin I is close to 0,2, whereas that with troponin T varies in the range of 0.5-0.7. Troponin phosphorylation by avian phosphorylase kinase depends on the presence of Ca2+ in the incubation mixture. In the presence of 3 mM EGTA troponin I phosphorylation is inhibited by 70-90%, whereas that of troponin T--by 50%. The experimental results indicate that the phosphorylation of troponin I and T is catalyzed either by two different active centers or by different conformations of the single center of avian phosphorylase kinase.     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Cytosolic enzyme leakage from isolated smooth muscle preparations

Several isolated smooth muscle preparations were shown to release cytosolic enzymes including adenosine deaminase, lactate dehydrogenase, and nucleoside phosphorylase under experimental conditions resembling those used in contractility or drug uptake studies. This enzyme leakage indicates significant general cell damage and suggests that experiments dealing with drug metabolism, binding, and uptake in isolated tissues must be interpreted with caution.     

Phosphorylation of phosphofructokinase from skeletal muscle: correlations between phosphorylation and muscle function.

The specific radioactivity of [³²P]-phosphate incorporated into muscle phosphofructokinase was in equilibrium with the specific radioactivity of the γ-phosphate group of ATP. The incorporation was independent of the presence of cycloheximide. The total content of covalently bound phosphate in phosphofructokinase was correlated with the functional state of the muscle from which the enzyme was purified. Muscle dissected post mortem led to phosphofructokinase containing less than 2 phosphate groups per tetramer. Muscle dissected in vivo gave phosphofructokinase with 4 phosphates per tetramer when kept at rest and 8 phosphates per tetramer when stimulated to contract.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of C-protein in intact amphibian cardiac muscle. Correlation between 32P incorporation and twitch relaxation

The molecular mechanisms by which neurotransmitters modulate the force of contraction of cardiac muscle are incompletely understood. Hartzell and Titus (1982. J. Biol. Chem. 257:2111-2120) have recently reported that C-protein, an integral component of the thick filament, is reversibly phosphorylated in response to ionotropic agents. In this communication, C-protein phosphorylation (as measured by isotopic labeling with 32P) is correlated with changes in the rate of relaxation of twitch tension. On the average, isoproterenol simultaneously increases peak systolic tension twofold, decreases twitch relaxation time from a control value of approximately 450 to approximately 300 ms, and increases C-protein phosphorylation two- to threefold, with a maximum effect occurring less than 60 s after addition of 1 microM isoproterenol. Carbamylcholine, in contrast, decreases peak systolic tension more rapidly than it affects relaxation or C-protein phosphorylation. The maximum decrease in peak tension (60%) occurs within 1 min of addition of 0.5 microM carbamylcholine, but relaxation time increases slowly to 800 ms over approximately 6 min. The increase in relaxation time correlates well with the decrease in 32P incorporation into C-protein (r = 0.94). Changing beat frequency between 0.2 and 1/s has no effect on C-protein phosphorylation but does alter relaxation time (relaxation time decreases approximately 100 ms when beat frequency is changed from 0.5 to 1/s) and thus alters the quantitative relationship between C-protein phosphorylation and relaxation rate. These results suggest that two separate processes affect relaxation. It is proposed that the level of C-protein phosphorylation sets the boundaries over which relaxation is regulated by a second process that is dependent upon beat frequency and probably involves changes in intracellular Ca.     

Pyruvate potentiates inotropic effects of isoproterenol and Ca(2+) in rabbit cardiac muscle preparations

Catecholamines and elevated extracellular Ca(2+) concentration ([Ca(2+)](o)) augment contractile force by increased Ca(2+) influx and subsequent increased sarcoplasmic reticulum (SR) Ca(2+) release. We tested the hypothesis that pyruvate potentiates Ca(2+) release and inotropic response to isoproterenol and elevated [Ca(2+)](o), since this might be of potential importance in a clinical setting to circumvent deleterious effects on energy demand during application of catecholamines. Therefore, we investigated isometrically contracting myocardial preparations from rabbit hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At a [Ca(2+)](o) of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 1.89 +/- 0.42 to 3.62 +/- 0.62 (SE) mN/mm(2) (n = 8, P < 0.05) and isoproterenol (10(-6) M) alone increased F(dev) from 2.06 +/- 0. 55 to 25.11 +/- 2.1 mN/mm(2) (P < 0.05), whereas the combination of isoproterenol and pyruvate increased F(dev) overproportionally from 1.89 +/- 0.42 to 33.31 +/- 3.18 mN/mm(2) (P < 0.05). In a separate series of experiments, we assessed SR Ca(2+) content by means of rapid cooling contractures and observed that, despite no further increase in F(dev) by increasing [Ca(2+)](o) from 8 to 16 mM, 10 mM pyruvate could still increase F(dev) from 26.4 +/- 6.8 to 29.7 +/- 7. 1 mN/mm(2) (P < 0.05, n = 9) as well as the Ca(2+) load of the SR. The results show that the positive inotropic effects of pyruvate potentiate the inotropic effects of isoproterenol or Ca(2+), because in the presence of pyruvate, Ca(2+) and isoproterenol induced larger increases in inotropy than can be calculated by mere addition of the individual effects.     

Native connectin from porcine cardiac muscle

Native connectin was isolated from porcine cardiac muscle using the method developed for the preparation of native connectin from chicken breast muscle (Kimura et al. (1984) J. Biochem. 96, 1947-1950). It was not necessary to keep cardiac muscle at 0 degrees C before preparation: the proteolysis of alpha-connectin to beta-connectin proceeded during the preparation of myofibrils. Cardiac connectin showed almost the same properties as those of skeletal muscle connectin: mobility in SDS gel electrophoresis, filamentous structure under an electron microscope, circular dichroism spectra, UV absorption spectra, and amino acid composition. Porcine cardiac connectin cross-reacted with antiserum against chicken breast muscle connectin as revealed by an immunoblot method. Immunoelectron microscopical observations revealed an abundance of connectin antigenic sites around the A-I junction area of cardiac myofibrils. Cardiac connectin also interacted with myosin and actin filaments at low ionic strengths to form aggregates. The extent of interaction was somewhat weaker in the case of cardiac connectin than skeletal muscle connectin, regardless of the origin of myosin and actin (porcine cardiac and rabbit skeletal muscles). In conclusion, cardiac connectin is very similar, but not identical to skeletal muscle connectin.