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R J Solaro

Publications and source records attributed to R J Solaro.

At least 73 records · Page 4Linked to original sources
PubMed1995

Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1.

The significance of site-specific phosphorylation of cardiac troponin I (TnI) by protein kinase C and protein kinase A in the regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1 was investigated. The TnI mutants used were T144A, S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase C phosphorylation sites, Thr-144 and Ser-43/ Ser-45, were, respectively, substituted by Ala) and S23A/S24A and N32 (in which the protein kinase A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or deleted). The mutations caused subtle changes in the kinetics of phosphorylation by protein kinase C, and all mutants were maximally phosphorylated to various extents (1.3-2.7 mol of phosphate/mol of protein). Protein kinase C could cross-phosphorylate protein kinase A sites but the reverse essentially could not occur. Compared to wild-type TnI and T144A, un-phosphorylated S43A/S45A, S43A/S45A/T144, S23A/ S24A, and N32 caused a decreased Ca2+ sensitivity of Ca(2+)-stimulated MgATPase of reconstituted actomyosin. S-1. Phosphorylation by protein kinase C of wild-type and all mutants except S43A/S45A and S43A/S45A/T144A caused marked reductions in both the maximal activity of Ca(2+)-stimulated MgATPase and apparent affinity of myosin S-1 for reconstitued (regulated) actin. It was further noted that protein kinase C acted in an additive manner with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a decreased Ca2+ sensitivity of the myofilament. It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca2+ sensitivity of the myofilament, and that phosphorylation of Ser-43/Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca2+ sensitivity, respectively, of actomyosin S-1 MgATPase.

Actomyosin↗
PubMed1995

Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys 84 allowed measurement of the relative distances to the 13C-methyl groups of the 10 methionines of cTnC in the monomer or complex. All 10 1H-13C correlations in the heteronuclear single- and multiple-quantum coherence (HSMQC) spectrum of [13C-methyl] Met cTnC in the complex with cTnI(33-211) were previously assigned [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., & Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735]. In the presence of oxidized spin label, nine of the 10 Met methyl 1H-13C correlations of cTnC were significantly broadened in the cTnC(C35S) monomer. This suggests flexibility within the central helix, or interdomain D/E helical linker, bringing the N- and C-terminal domains in closer proximity than predicted from the crystallographic structure of TnC. In the spin-labeled cTnC(C35S). cTnI(33-211) complex only N-terminal Met methyl 1H-13C correlations of cTnC(C35S) were paramagnetically broadened beyond detection, whereas correlations for Met residues (103, 120, 137, and 157) in the C-terminal domain were not. Thus, complex formation with cTnI decreases interdomain flexibility and maintains cTnC in an extended conformation. This agrees with the recently published study suggesting that sTnC is extended when bound to sTnI [Olah, G. A., & Trewhella, J. (1994) Biochemistry 33, 12800-12806]. The recombinant N-terminal domain of cTnI, cTnI(33-80), gave similar results as observed with cTnI(33-211) when complexed with spin-labeled cTnC(C35S). However, complex formation with the C-terminal fragment, cTnI(86-211), which contains the inhibitory sequence, is insufficient to maintain cTnC extended to the amount observed with either cTnI(33-211) or cTnI(33-80); although compared to that observed in free cTnC, it does cause decreased flexibility in the interdomain linker. In the absence of the N-terminal domain of cTnI, there is a decrease in flexibility within the N-terminal domain of cTnC. Interestingly, the N-terminal domain of cTnC in the reduced spin-labeled complex with cTnI(86-211), in the presence of ascorbate, showed two distinct conformations which were not seen in the complex with cTnI(33-211).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

The unique amino-terminal peptide of cardiac troponin I regulates myofibrillar activity only when it is phosphorylated.

Protein kinase A (PKA) dependent phosphorylation of C-protein and cardiac troponin I (cTnI) is known to be associated with a reduced sensitivity to Ca2+. We have investigated the relative importance of each of these sites of phosphorylation in this effect by use of extraction/reconstitution experiments and mutagenesis of recombinant cTnI. Conditions developed for extraction of troponin (Tn) complex also resulted in extraction of C-protein. A truncated cTnI (cTnI/NH2) lacking the 32 amino acids in the unique amino terminal extension of cTnI was engineered and expressed. In contrast to native cTnI, cTnI/NH2, which lacks Ser23 and Ser24, was not phosphorylated by PKA either in pure form or after incorporation into the myofilament lattice. The relation between pCa (-log molar free Ca2+ concentration) and MgATPase activity of non-phosphorylated native myofibrils or non-phosphorylated myofibrils reconstituted with cTnI, but lacking C-protein, was the same and could not be distinguished from that of control or PKA-treated myofibrils into which we exchanged cTnI with cTnI/NH2. However, PKA-dependent phosphorylation of either native myofibrils or reconstituted myofibrils containing cTnI but lacking C-protein resulted in an identical and significant rightward shift of pCa50 (half-maximally activating pCa) in the pCa-activity relationship. Our results strongly indicate that phosphorylation of cTnI at Ser residues in the unique amino terminal extension of the molecule is both necessary and sufficient for the decrease in myofilament Ca(2+)-sensitivity associated with PKA-dependent phosphorylation.

Animals↗
PubMed1995

Troponin C - troponin I interactions and molecular signalling in cardiac myofilaments.

This chapter describes a current perception of the molecular interactions regulating myofilament activity in heart cells. The focus is on the interaction between troponin-C (TnC), the Ca(2+)-receptor and troponin I (TnI), an inhibitory protein. It is this interaction that appears to form a molecular switch that turns on the thin filament. It will be seen that control of the actin-myosin reaction is not only through Ca(2+)-binding to TnC, but also through steric, cooperative and allosteric processes involving all of the main myofilament proteins-actin, myosin, tropomyosin (Tm), troponin T (TnT), TnC, and TnI. The process is modulated by covalent and non-covalent mechanisms. The process is altered in diverse myopathies and pathologies of the heart and is a target for pharmacological manipulation by a new class of inotropic agents, the "Ca(2+)-sensitizers".

Actin Cytoskeleton↗
PubMed1995

Differential expression of TnI and TnT isoforms in rabbit heart during the perinatal period and during cardiovascular stress.

We have investigated developmental transitions of TnI and TnT isoforms in fetal, neonatal, and adult rabbit hearts by western blot analysis. Our results provide the first evidence for the existence of two developmentally regulated isoforms of TnI in rabbit heart. These isoforms comigrate with adult rabbit cardiac TnI (cTnI) and slow skeletal TnI (ssTnI). At 23 days of gestation, ssTnI was the predominant TnI isoform. At 29 days of gestation, there was a significant increase in the relative amount of cTnI, that continued with maturation. The TnI isoform transition was significantly faster in right than left ventricles at gestation 30 and 32 days. Four TnT isoforms were detected in fetal rabbit ventricles. The relative amount of TnT isoforms did not change from 23 to 29 days of gestation. However, the relative amount of the adult TnT isoform increased significantly around the time of birth with the increase being significantly more prominent in left than in right ventricles. Maternal injection of phenylephrine (PHE), an alpha-1 adrenergic agonist, increased fetal cardiac force and arterial blood pressure, facilitated TnT, but not TnI, isoform transition in fetal heart. Our results indicate that the developmental transition of rabbit cardiac TnI and TnT isoforms is not coordinated and might be regulated by different mechanisms. Our results also provide evidence that the TnT isoform population is influenced by adrenergic stimulation and stress on the cardiovascular system during development.

Aging↗
PubMed1995

Troponin I isoforms and differential effects of acidic pH on soleus and cardiac myofilaments.

Differences in pH sensitivity of tension generation between developing and adult cardiac myofilaments, which contain the same isoform of troponin C (TnC), have been proposed to be due to troponin I (TnI) isoform switching from the slow skeletal (ss) to cardiac (c) TnI isoforms (21). We investigated the effects of acidic pH on Ca(2+)-activation of force in chemically skinned preparations of adult rat trabeculae and single soleus fibers that also share the same TnC isoform. Compared with the soleus fibers, trabeculae demonstrated a greater suppression of tension and a rightward shift in pCa50 (-log half-maximally activating molar Ca2+ concentration) when pH was decreased from 7.0 to 6.2. The pH-induced shift in pCa50 in soleus fibers did not change with sarcomere length. Troponin subunit interactions were also investigated, using cardiac troponin C (cTnCIA) labeled with a fluorescent probe, 2-(4'-iodoacetamidoanilino)-naphthalene-6-sulfonic acid. Under acidic conditions, cTnCIA demonstrated a decrease in Ca(2+)-affinity. This decrease was amplified both in the binary complex cTnCIA-cTnI and in the complex cTnCIA-cTnI-cTnT-tropomyosin to the same extent. In contrast, substitution of ssTnI for cTnI in these complexes produced the same decrease in Ca2+ affinity in response to acidic pH as cTnCIA alone. These results support our hypothesis that differential effects of pH on tension generation and Ca2+ sensitivity between soleus fibers and trabeculae are due to the presence of different isoforms of TnI.

Acids↗
PubMed1995

Caffeine alters cardiac myofilament activity and regulation independently of Ca2+ binding to troponin C.

We investigated the mechanism by which caffeine influences myofilament responsiveness to Ca2+ by measuring isometric force, Ca2+ binding, and ATPase activity of dog cardiac myofilament proteins. Caffeine (20 mM) increased submaximal and depressed maximal force in skinned fiber bundles. Although the Ca2+ sensitivity of myofilament activity was increased by caffeine, there was no effect on Ca2+ binding to troponin C (TnC) in skinned fiber bundles. To determine if caffeine altered actin-myosin interaction or affected myosin directly, myofibrillar, actomyosin, and myosin ATPase activities were measured. Maximal Ca(2+)-activated myofibrillar Mg(2+)-ATPase activity was depressed by 20 mM caffeine, whereas submaximal Mg(2+)-ATPase activities were not changed. Actomyosin Mg(2+)-ATPase activity was significantly depressed by caffeine concentrations > or = 15 mM. Myosin Ca(2+)-ATPase activity was depressed by caffeine, whereas Mg(2+)-ATPase and K(EDTA)-ATPase activities were not affected. These data suggest that caffeine affects myofilament function via a mechanism that is independent of TnC-Ca2+ binding but that may involve direct effects on actin-cross-bridge interaction.

Actomyosin↗
PubMed1995

Effects of Levosimendan, a cardiotonic agent targeted to troponin C, on cardiac function and on phosphorylation and Ca2+ sensitivity of cardiac myofibrils and sarcoplasmic reticulum in guinea pig heart.

A new cardiotonic agent, (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), has been developed and screened for its ability to bind to cardiac troponin C. In perfused hearts, low concentrations of 0.03 or 0.1 mumol/L Levosimendan increased +dP/dt, but did not affect the speed of relaxation and produced only a slight increase in spontaneous heart rate in the hearts perfused with 0.1 mumol/L of the drug. In these same hearts, perfusion with 0.03 mumol/L Levosimendan did not alter the 32P incorporation into troponin I or C protein, whereas a slight but significant increase was noted for phospholamban, with no detectable change in tissue cAMP levels. Administration of 0.1 or 0.3 mumol/L Levosimendan significantly increased myocardial cAMP levels as well as the phosphorylation of phospholamban, troponin I, and C protein. Levosimendan (0.03 to 10 mumol/L) reversibly increased force generated by detergent-extracted fiber bundles over a range of submaximally activating free Ca2+ concentrations with no significant effect on maximum force or on Ca2+ binding to myofilament troponin C. There was no direct effect of Levosimendan on Ca2+ uptake by vesicles of sarcoplasmic reticulum (SR). In contrast, under conditions optimal for cAMP-dependent phosphorylation, Levosimendan slightly but significantly lowered the concentration of Ca2+, yielding half-maximal uptake rates by the SR vesicles. Our results indicate that at low concentrations Levosimendan acts preferably as a Ca2+ sensitizer, whereas at higher concentrations its action as a phosphodiesterase inhibitor contributes to the positive inotropic effect.

Analysis of Variance↗
PubMed1994

NMR studies delineating spatial relationships within the cardiac troponin I-troponin C complex.

NMR spectroscopy and selective isotope labeling of both recombinant cardiac troponin C (cTnC3) and a truncated cardiac troponin I (cTnI/NH2) lacking the N-terminal 32-amino acid cardiac-specific sequence have been used to probe protein-protein interactions central to muscle contraction. Using [methyl-13C]Met-labeled cTnC3, all 10 cTnC Met residues of Ca(2+)-saturated cTnC3 could be resolved in the two-dimensional heteronuclear single- and multiple-quantum coherence spectrum of the cTnI.cTnC complex. Based on the known Met assignments in cTnC3, the largest chemical shift changes were observed for Met81, Met120, Met137, and Met157. Methionines 120, 137, and 157 are all located in the C-terminal domain of cTnC. Methionine 81 is located at the N terminus of the central helix. Minimal chemical shift changes were observed for Met45, Met47, and Met103 of cTnC3 in the cTnI.cTnC complex. All 6 Met residues in [methyl13C]Met-labeled cTnI/NH2 could be resolved in the cTnI.cTnC complex, suggesting that both cTnI and cTnC form a stable homogeneous binary complex under the conditions of the NMR experiment. In the absence of added protease inhibitors in the cTnI.cTnC complex, cTnI/NH2 was found to undergo selective proteolysis to yield a 5.5-kDa N-terminal fragment corresponding to residues 33-80. Judging from the NMR spectra of [methyl13C]Met-labeled cTnC3, cTnI-(33-80) was sufficient for interaction with the C-terminal domain of cTnC in a manner identical to that observed for native cTnI/NH2. However, in the presence of the proteolytic fragment cTnI-(33-80), the chemical shift of Met81 was not perturbed from its position in free cTnC3. Thus, residues located C-terminal to Arg80 in cTnI appear to be responsible for interaction with the N-terminal half of cTnC. Taken together, these results provide strong evidence for an antiparallel arrangement for the two proteins in the troponin complex such that the N-terminal portion of cTnI interacts with the C-terminal domain of cTnC. This interaction likely plays a role in maintaining the stability of the TnI.TnC complex.

Animals↗
PubMed1994

Isoform specific interactions of troponin I and troponin C determine pH sensitivity of myofibrillar Ca2+ activation.

We investigated whether differences in isoforms of troponin I (TnI) and troponin C (TnC) can account for the greater inhibition of Ca(2+)-dependent MgATPase activity by acidic pH in cardiac (c) than in fast skeletal (fs) myofilaments. We studied fast skeletal myofibrils from which whole Tn was extracted by displacement with excess fsTnT (the tropomyosin binding subunit of Tn) followed by reconstitution with TnC-TnI. Exchange of fsTnI with cTnI did not alter the effect of a drop in pH from 7.0 to 6.5 on the relation between pCa (-log[Ca2+]) and MgATPase activity of fast skeletal myofibrils. Exchange of fsTnC with cTnC did, however, induce an increase in the effect of this same pH change on Ca2+ activation. Yet, the pH sensitivity of Ca2+ activation of fast skeletal myofibrils containing cTnC was not as great as that of native cardiac myofibrils. However, when both fsTnC and fsTnI of fast skeletal myofibrils were replaced by cTnC-cTnI, there was a pH-induced shift in Ca2+ sensitivity similar to that of cardiac myofibrils. In studies using fluorescent probes, both pure fsTnC and pure cTnC showed decreased Ca2+ binding as pH was lowered. This decrease was potentiated in the fsTnC-fsTnI and cTnC-cTnI complexes. However, the effect of acidic pH was the same in fsTnC and the hybrid complex, fsTnC-cTnI, and in cTnC and the hybrid complex, cTnC-fsTnI. Thus, isoform specific interactions between TnI and TnC appear important in the differential response of skeletal and cardiac myofilaments to acidosis.

Animals↗
PubMed1994

Mutagenesis of cardiac troponin I. Role of the unique NH2-terminal peptide in myofilament activation.

Phosphorylation of Ser residues in the NH2-terminal extension unique to cardiac troponin I (cTnI) is known to occur through protein kinase A and to alter myofilament Ca2+ activation (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Yet, how the NH2-terminal extension may itself affect thin filament Ca2+ signaling is unknown. To approach this question we have used molecular cloning, mutagenesis, and bacterial synthesis of a full-length cTnI and a truncated mutant (cTnI/NH2) missing the 32 amino acids. Using reconstituted preparations we could show no differences between cTnI and cTnI/NH2 either in inhibition of actomyosin ATPase activity, in Ca(2+)-reversible inhibitory activity, or in the relation between pCa and Ca2+ binding to the regulatory site of cTnC at either pH 7.0 or 6.5. There were also no significant differences at either pH in the pCa-MgATPase activity relation of myofibrils into which the various species of TnI has been exchanged. Our results indicate: 1) that phosphorylation most likely induces a new state of TnI activity rather than altering an intrinsic effect of the NH2-terminal peptide on Ca2+ activation; and 2) that domains outside the NH2-terminal extension are important with regard to differences in effects of acidic pH on Ca2+ activation on cardiac and skeletal myofilaments.

Actin Cytoskeleton↗
PubMed1994

Discoordinate regulation of contractile protein gene expression in the senescent rat myocardium.

The myocardium is a highly adaptive tissue, as evidenced by phenotypic alterations throughout development and under conditions of altered hemodynamic load. With pressure overload, the myocardium displays adult-to-fetal transitions in expression of contractile and non-contractile proteins. Most intriguing is the fact that many of these transitions are also observed in the senescent heart. The purpose of this work was to establish if the thin filament regulatory proteins, troponin I and troponin T, exhibit reexpression of early developmental isoforms, suggestive of coordinate reprogramming of contractile protein isoform expression. As a functional index of reexpression of the early isoform of troponin I, slow skeletal troponin I, myofibrils were isolated from 12 and 24-month-old Fischer 344 rat ventricles and assayed for myofibrillar ATPase activity at pH 7.0 and 6.5. Both preparations displayed rightward shifts in Ca-ATPase relationships with no differences between groups. SDS-PAGE and Western blot analysis showed that whereas myosin heavy chain expression underwent a transition to predominance of the early development isoform, beta-myosin heavy chain, there was no reexpression of the fetal isoforms of either troponin I or troponin T in the rat heart at 24 months of age. Northern blot analysis using cDNA probes specific for cardiac or slow skeletal troponin I also confirmed the lack of slow skeletal reexpression in the 24-month ventricle. These results are significant in that they demonstrate a lack of coordinate expression of contractile protein isoforms under myocardial adaptation to the aging process.

Adenosine Triphosphatases↗
PubMed1994

Differential regulation of slow-skeletal and cardiac troponin I mRNA during development and by thyroid hormone in rat heart.

We have examined mRNA levels for the cardiac troponin I (cTnI) and slow-skeletal (ssTnI) in perinatal rat hearts. Northern blots showed that hypothyroidism was associated with a delay in the expected isoform switching. RNA slot blots showed a six-fold increase in cTnI mRNA from day 3 to day 21 in hearts from postnatal euthyroid rats compared to a three-fold increase in cTnI for the same period in the hypothyroid hearts. On the other hand, ssTnI mRNA levels were higher after 3 days in hearts from the hypothyroid animals and fell to undetectable levels after 21 days. In euthyroid hearts ssTnI was not detectable after 14 days and was not re-expressed in adult hearts. In the ventricles from 28-day-old animals the most significant differences in cTnI mRNA levels were between the euthyroid and T3-treated hypothyroid preparations and in the 87 to 125-day-old group between euthyroid and hypothyroid ventricles. T3 treatment of the 87 to 125-day-old hypothyroid animals did not increase the cTnI mRNA above euthyroid levels despite elevated serum T3. These results show that thyroid hormone influences expression of the cTnI isoform in postnatal and young adult rats, but not to the same extent in animals greater than 28 days of age.

Animals↗
PubMed1994

Tension production and thin-filament protein isoforms in developing rat myocardium.

The calcium sensitivity of tension production and the expression of troponin I (TnI) and troponin T (TnT) isoforms in skinned neonatal (7 days after birth) and adult rat myocardium were determined. Isometric tension was measured after activation at a known resting sarcomere length in ventricular trabeculae at adult and, for the first time, neonatal ages. Analysis of the tension-pCa relationships indicates a greater calcium sensitivity (approximately 0.3 pCa units) in neonatal ventricular trabeculae compared with adult trabeculae. The maximal isometric tension-generating ability (i.e., tension-tissue cross-sectional area) is threefold greater in adult compared with neonatal trabeculae. Developmental transitions in TnI and TnT isoform expression in atrial and ventricular tissue were examined simultaneously and were found to be dissimilar. Shifts in the expression of TnT isoforms precede shifts in TnI isoforms in ventricular tissue. The opposite pattern occurs in atrial tissue, with shifts in TnI preceding those in TnT. The results show that the greater calcium sensitivity of neonatal compared with adult rat ventricular tissue is associated with developmental changes in both TnT and TnI isoform expressions. These isoform expression patterns may facilitate myocardial tension production at the neonatal stage, when the tension-generating ability of individual trabeculae is much lower than that in the adult.

Aging↗
PubMed1993

A highly specific benzimidazole pyridazinone reverses phosphate-induced changes in cardiac myofilament activation.

Both Ca2+ and the actin cross-bridge reaction itself can activate contraction in myofilaments. We are interested in identifying ligands which modify one or both mechanisms of contractile activation with high affinity and specificity. Results presented here suggest that the benzimidazole-substituted pyridazinone, UD-CG 212 Cl, potently modulates myofilament activation by the cross-bridge reaction. Cross-bridge-mediated activation was studied by varying the population of force-generating (strong) cross-bridges with inorganic phosphate (Pi). Addition of Pi to detergent-extracted (skinned) canine ventricular preparations reduces the population of strong cross-bridges, which causes reduced myofilament force and Ca2+ sensitivity. Increased Pi concentration ([Pi]) also favors cross-bridge-mediated myofilament activation, so cooperativity increases. The decreased Ca2+ sensitivity produced by Pi was reversed by a racemic mixture of 10(-10) M UD-CG 212 Cl, but this agent had no effect on maximum force or cooperativity. In experiments with stereoisomers, only (+)-UD-CG 212 Cl stimulated force. At higher doses (10(-6)-10(-4) M), submaximal but not maximal force decreased and (-)-UD-CG 212 Cl was the active stereoisomer. Neither Pi nor UD-CG 212 Cl affected Ca2+ binding to myofilament troponin C (TnC). Thus, UD-CG 212 Cl appears to reverse Pi-induced decreases in submaximal force via high-affinity binding to a myofibrillar domain not directly involved with myofibrillar TnC-Ca2+ binding. The actions of UD-CG 212 Cl were further investigated by reducing [ATP] as another means of varying the cross-bridge population.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1993

RWJ-24517, a positive inotropic agent, has novel effects on action potentials in guinea pig myocardium.

RWJ-24517 is a positive inotropic agent whose mechanism of action is under investigation. We examined the effects of RWJ-24517 on guinea pig papillary muscle action potentials and myofilament response to Ca2+. RWJ-24517 increased the fast action potential duration (APD) in a dose-dependent fashion but had no effect on the myofilament response. Tetraethylammonium (TEA 10 mM), which in itself slightly prolonged the control AP, completely blocked the increase in APD75 (APD at 75% of repolarization) and suppressed the increase in APD25 induced by RWJ-24517 (10 microM). Verapamil (5 microM) had little effect on control APs but did decrease the prolongation of the AP induced by RWJ-24517. The increase in APD25 induced by RWJ-24517 was not completely blocked by TEA even with addition of verapamil or 4-aminopyridine (2 mM). RWJ-24517 enhanced Ca(2+)-dependent slow APs elicited by 0.1 microM isoproterenol in preparations depolarized by high extracellular K+ (25 mM), but had no effects on slow APs elicited by 10 mM TEA. These results suggest that the primary electrophysiologic effect of RWJ-24517 is a substantial AP prolongation, which appears to occur largely through a mechanism which is likely to involve inhibition of Ca(2+)-dependent K+ channels. It also appears to have additional effects on some other channels (possibly Na+ channels), which may contribute to the positive inotropic action of RWJ-24517.

Actin Cytoskeleton↗
PubMed1993

Stereoselective actions of thiadiazinones on canine cardiac myocytes and myofilaments.

Thiadiazinones are cardiotonic agents that have potent, direct, and stereoselective actions on the myofilament response to Ca2+ in intact myocardium. Their mechanism of action is unknown. We studied the effects of racemic thiadiazinone, EMD 53998 (5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-meth yl-3,6- dihydro-2H-1,3,4-thiadiazin-2-one), and its enantiomers on Ca2+ signaling in myocytes, myofilaments, and myofilament proteins. Intact canine ventricular myocytes responded to the positive enantiomer, EMD 57033, with an increase in the extent of shortening during twitch contractions without increasing the peak amplitude of the Ca2+ transient. The negative enantiomer, EMD 57439, also increased the extent of shortening, but in this case there was a concentration-dependent increase in the peak amplitude of the Ca2+ transient. This is predicted from in vitro data showing that this enantiomer is a relatively potent inhibitor of phosphodiesterase activity. There was no effect of EMD 57439 on the relation between pCa and actomyosin Mg-ATPase activity of canine heart myofibrils. In contrast, EMD 57033 shifted the pCa-Mg-ATPase activity relation to the left. There was no effect of either enantiomer on Ca2+ binding to myofilament troponin C. Moreover EMD 57033, but not EMD 57439, stimulated actomyosin ATPase activity of myofilament preparations in which either troponin or troponin-tropomyosin had been extracted. EMD 57033 had no effect on Mg-ATPase activity of pure ventricular myosin. EMD 57033 also stimulated the velocity of actin filament sliding on myosin heads adhered to nitrocellulose-coated glass coverslips. We propose that the action of EMD 57033 is at the actin-myosin interface on a "receptor" that may be on actin or the crossbridge. Drug binding to this domain appears to reverse the inhibition of actin-myosin interactions by troponin-tropomyosin and also to promote transition of crossbridges from weak to strong force-generating states.

Actin Cytoskeleton↗