Role of protein phosphatases in regulation of cardiac inotropy and relaxation

We studied the effects of the protein phosphatase (PP) inhibitor cantharidin (Cant) on time parameters and force of contraction (FOC) in isometrically contracting electrically driven guinea pig papillary muscles. We correlated the mechanical parameters of contractility with phosphorylation of the inhibitory subunit of troponin (TnI-P) and with the site-specific phosphorylation of phospholamban (PLB) at serine-16 (PLB-Ser-16) and threonine-17 (PLB-Thr-17). Cant (after 30 min) started to increase FOC (112 ± 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 ± 5 and 128 ± 7% of control) without any alteration of relaxation time. Cant (10 μM) started to increase PLB-Ser-16, but the relaxation was shortened at only 100 μM (from 140 ± 9 to 116 ± 12 ms, n = 9). Moreover, 100 μM Cant, 3 min after application, started to increase PLB-Thr-17, TnI-P, and FOC. Cant (100 μM) began to increase PLB-Ser-16 after 20 min. This was accompanied by shortening of relaxation time. Differences in protein kinase activation or different substrate specificities of PP may explain the difference in Cant-induced site-specific phosphorylation of PLB in isometrically contracting papillary muscles. Moreover, PLB-Thr-17 may be important for inotropy, whereas PLB-Ser-16 could be a major determinant of relaxation time.

Biochemical mechanism(s) of stunning in conscious dogs

The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca2+ homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning. Conscious dogs were chronically instrumented, and the left anterior descending artery (LAD) was occluded for 10 min. Thereafter, reperfusion of the LAD was initiated. Tissues from reperfused LAD (stunned) and Ramus circumflexus (control) areas were obtained when left ventricular regional wall thickening fraction had recovered by 50%. Northern and Western blotting revealed no differences in the expression of the following genes: phospholamban, calsequestrin, sarco(endo)plasmic reticulum Ca2+-ATPase 2a, and the inhibitory subunit of troponin I (TnI). However, the phosphorylation state of TnI and phospholamban were reduced in the LAD area. Fittingly, cAMP levels were reduced by 28% ( P < 0.05). It is concluded that the contractile dysfunction in cardiac stunning might be mediated in part by decreased levels of cAMP and subsequently a reduced phosphorylation state of phospholamban and TnI.

Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells.

Bundles of cells from rat right ventricular myocardium were made "hyperpermeable" by an overnight soak in 10 mM EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764). In this preparation the cytoplasmic concentration of Ca++ and ATP could be controlled while sarcolemmal receptors and enzymes were retained. The Ca sensitivity of the tissues (as indicated by the pCa for 50% maximum activation) was altered to different extents in the presence of [32Pgamma]ATP by treatment with cyclic nucleotides, catecholamines, or a low concentration of nonionic detergent. The proteins of the tissue were then isolated by SDS-polyacrylamide gel electrophoresis, and the identity of 32P-labeled proteins was determined. The Ca sensitivity is inversely related to the relative amount of 32P incorporated into the inhibitory subunit of troponin (TNI). Extrapolation of the relation to the lowest Ca sensitivity observed gives a stoichiometry of about 0.8 mol PO4 per mol TNI. These results support the hypothesis that Ca sensitivity of cardiac myofibrils is regulated by a phosphrylation of TNI that is stimulated by cyclic AMP (cAMP) and inhibited by cGMP.

Cyclic nucleotide regulation of the contractile proteins in mammalian cardiac muscle.

The contractile system of rat cardiac muscle that has been made hyperpermeable by soaking the tissue in EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764) can be probed directly with Ca buffer from the bathing solution without significant interference from either sarcoplasmic reticulum or mitochondria on the Ca concentration. Changes in Ca-activated force are due therefore to changes in the properties of the contractile system itself and not to regulation of Ca concentration. The addition of cAMP, cGMP, and GTP, guanylyl imidodiphosphate (GMP-PNP), or epinephrine to the bath does not alter maximum Ca-activated force, but when these drugs are added with 1% nonionic detergent to the bath, contractility increases by as much as 180%. An inhibitor of phosphodiesterase must be present for the inotropic effect of cAMP but not cGMP, GTP, GMP-PNP, or epinephrine. The inotropic response to cAMP is independent of the Ca sensitivity of the contractile system, but guanine nucleotides enhance contractility only when Ca sensitivity is not high. The inotropic effect of epinephrine is inhibited to a large extent by cGMP but not by GMP-PNP. These data can be explained by a model in which contractility is enhanced by a cAMP-regulated phosphorylation that can be controlled through the beta-receptor adenylate cyclase complex in the sarcolemma. The regulation involves two reactions, one a phosphorylation and a second that occurs in the presence of detergent. Phosphorylation of neither the myosin light chain nor the inhibitory subunit of troponin appears to be involved in this mechanism for regulating contractility.

Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart

1. Rat hearts were perfused with 32Pi, and contractile force was increased by positive inotropic agents (agents that increase contractility). The inhibitory subunit of troponin (troponin I) was then isolated by affinity chromatography in 8M-urea, and its 32P content measured. Incorporation of phosphate into the subunit was calculated on the basis of the [gamma-32P]ATP specific radioactivity in the hearts. 2. When hearts were perfused with 30 nM-DL-isoprenaline (N-isopropylnoradrenaline), there was an increase in contractile force over 30s which was paralleled by an increase in troponin I phosphorylation. When hearts were perfused for 25s with increasing concentrations of isoprenaline from 1 NM to 0.6 muM, there was again a parallel increase in contractile force and troponin I phosphorylation. The maximum phosphorylation observed was 1.5 mol of phosphate/mol of troponin I, which was reached after 25s with 0.1 muM-isoprenaline. 3. Hearts were stimulated with a 15s pulse perfusion of 30nM-DL-isoprenaline. There was an increase in contractile force which was followed by a return to the control value within 50s. Troponin I phosphorylation increased to a plateau value which was reached within 30s, and remained constant for 60s after the isoprenaline pulse. Phosphorylase a and 3′:5′-cyclic AMP concentration showed changes similar to that of the contractile force. There was no change in 3′:5′-cyclic GMP concentration. 4. When hearts stimulated with a 15S pulse of isoprenaline were subsequently perfused with 0.6 muM-acetylcholine, the changes in contractile force, phosphorylase a and 3′:5′-cyclic AMP were very similar to those seen with the 15s pulse of isoprenaline alone. Troponin I phosphorylation increased to a maximum 30s after the end of the isoprenaline pulse, but then rapidly decreased during the subsequent 30s. This decrease was preceded by a 60% increase in the concentration of 3′:5′-cyclic GMP. 5. Hearts were perfused with 0.2 muM-glucagon for periods up to 60s. Contractile force showed little change for the first 30s, but then increased rapidly. This was paralleled by changes in 3′:5′-cyclic AMP concentration. Troponin I phosphorylation increased slowly, but the increase in contractile force had reached a maximum before significant phosphorylation had occurred. 6. It is concluded that under certain conditions, e.g. immediately after β-adrenergic stimulation, there is a good correlation between contractile force and troponin I phosphorylation. However, under other conditions, e.g. when contractile force is decreasing after removal of β-adrenergic stimulation or in the presence of glucagon, contractile force and troponin I phosphorylation are not well correlated. These results suggest that mechanisms for modifying cardiac contractility, other than troponin I phosphorylation, must be present in rat heart.

The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases

1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.