PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin.

In myocardium, protein kinase A (PKA) is known to phosphorylate troponin I (TnI) and myosin-binding protein-C (MyBP-C). Here, we used skinned myocardial preparations from nontransgenic (NTG) mouse hearts expressing 100% alpha-tropomyosin (alpha-Tm) to examine the effects of phosphorylated TnI and MyBP-C on Ca2+ sensitivity of force and the rate constant of force redevelopment (k(tr)). Experiments were also done using transgenic (TG) myocardium expressing approximately 60% beta-Tm to test the idea that the alpha-Tm isoform is required to observe the mechanical effects of PKA phosphorylation. Compared with NTG myocardium, TG myocardium exhibited greater Ca2+ sensitivity of force and developed submaximal forces at faster rates. Treatment with PKA reduced Ca2+ sensitivity of force in NTG and TG myocardium, had no effect on maximum k(tr) in either NTG or TG myocardium, and increased the rates of submaximal force development in both kinds of myocardium. These results show that PKA-mediated phosphorylation of myofibrillar proteins significantly alters the static and dynamic mechanical properties of myocardium, and these effects occur regardless of the type of Tm expressed.

Cardiac myosin heavy chains lacking the light chain binding domain cause hypertrophic cardiomyopathy in mice.

Myosin is a chemomechanical motor that converts chemical energy into the mechanical work of muscle contraction. More than 40 missense mutations in the cardiac myosin heavy chain (MHC) gene and several mutations in the two myosin light chains cause a dominantly inherited heart disease called familial hypertrophic cardiomyopathy. Very little is known about the biochemical defects in these alleles and how the mutations lead to disease. Because removal of the light chain binding domain in the lever arm of MHC should alter myosin's force transmission but not its catalytic function, we tested the hypothesis that such a mutant MHC would act as a dominant mutation in cardiac muscle. Hearts from transgenic mice expressing this mutant myosin are asymmetrically hypertrophied, with increases in mass primarily restricted to the cardiac anterior wall. Histological examination demonstrates marked cellular hypertrophy, myocyte disorganization, small vessel coronary disease, and severe valvular pathology that included thickening and plaque formation. Skinned myocytes and multicellular preparations from transgenic hearts exhibited decreased Ca2+ sensitivity of tension and decreased relaxation rates after flash photolysis of diazo 2. These experiments demonstrate that alterations in myosin force transmission are sufficient to trigger the development of hypertrophic cardiomyopathy.

Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart.

1. To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene-encoded ssTnI. 2. The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. 3. Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP-dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild-type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non-transgenic cardiomyocytes at baseline and after treatment with isoprenaline. 4. The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild-type myocytes. However, the half-time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re-lengthening time of the transgenic cells. 5. These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. 6. Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to beta-adrenergic receptor stimulation.

Altered kinetics of contraction of mouse atrial myocytes expressing ventricular myosin regulatory light chain.

To investigate the role of myosin regulatory light chain isoforms as a determinant of the kinetics of cardiac contraction, unloaded shortening velocity was determined by the slack-test method in skinned wild-type murine atrial cells and transgenic cells expressing ventricular regulatory light chain (MLC2v). Transgenic mice were generated using a 4.5-kb fragment of the murine alpha-myosin heavy chain promoter to drive high levels of MLC2v expression in the atrium. Velocity of unloaded shortening was determined at 15 degrees C in maximally activating Ca2+ solution (pCa 4.5) containing (in mmol/l) 7 EGTA, 1 free Mg2+, 4 MgATP, 14.5 creatine phosphate, and 20 imidazole (ionic strength 180 mmol/l, pH 7.0). Compared with the wild type (n = 10), the unloaded shortening velocity of MLC2v-expressing transgenic murine atrial cells (n = 10) was significantly greater (3.88 +/- 1.19 vs. 2.51 +/- 1.08 muscle lengths/s, P < 0.05). These results provide evidence that myosin light chain 2 regulates cross-bridge cycling rate. The faster rate of cycling in the presence of MLC2v suggests that the MLC2v isoform may contribute to the greater power-generating capabilities of the ventricle compared with the atrium.

Results of three to 10 year follow up of balloon dilatation of the pulmonary valve.

BACKGROUND: The results of immediate and short term follow up of balloon dilatation of the pulmonary valve have been well documented, but there is limited information on long term follow up. OBJECTIVE: To evaluate the results of three to 10 year follow up of balloon dilatation of the pulmonary valve in children and adolescents. SETTING: Tertiary care centre/university hospital. DESIGN: Retrospective study. METHODS AND RESULTS: 85 patients (aged between 1 day and 20 years, mean (SD) 7.0 (6.4) years) underwent balloon dilatation of the pulmonary valve during an 11 year period ending August 1994. There was a resultant reduction in the peak to peak gradient from 87 (38) to 26 (22) mm Hg. Immediate surgical intervention was not required. Residual gradients of 29 (17) mm Hg were measured by catheterisation (n = 47) and echo Doppler (n = 82) at intermediate term follow up (two years). When individual results were scrutinised, nine of 82 patients had restenosis, defined as a peak gradient of 50 mm Hg or more. Seven of these patients underwent repeat balloon dilatation of the pulmonary valve: peak gradients were reduced from 89 (40) to 38 (20) mm Hg. Clinical evaluation and echo Doppler data of 80 patients showed that residual peak instantaneous Doppler gradients were 17 (15) mm Hg at long term follow up (three to 10 years, median seven), with evidence for late restenosis in one patient (1.3%). Surgical intervention was necessary to relieve fixed infundibular stenosis in three patients and supravalvar pulmonary stenosis in one. Repeat balloon dilatation was performed to relieve restenosis in two patients. Actuarial reintervention free rates at one, two, five, and 10 years were 94%, 89%, 88%, and 84%, respectively. Pulmonary valve regurgitation was noted in 70 of 80 patients at late follow up, but neither right ventricular dilatation nor paradoxical interventricular septal motion developed. CONCLUSIONS: The results of late follow up of balloon dilatation of the pulmonary valve are excellent. Repeat balloon dilatation was performed in 11% of patients and surgical intervention for subvalvlar or supravalvar stenosis in 5%. Most patients had mild residual pulmonary regurgitation but right ventricular volume overload was not required. Balloon dilatation is the treatment of choice in the management of moderate to severe stenosis of the pulmonary valve. Further follow up studies should be undertaken to evaluate the significance of residual pulmonary regurgitation.

Transgenic remodeling of the regulatory myosin light chains in the mammalian heart.

The regulatory myosin light chain (MLC) regulates contraction in smooth muscle. However, its function in striated muscle remains obscure, and the different functional activities of the various isoforms that are expressed in the mammalian heart (ventricle- and atrium-specific MLC2) remain undefined. To begin to explore these issues, we used transgenesis to determine the feasibility of effecting a complete or partial replacement of the cardiac regulatory light chains with the isoform that is normally expressed in fast skeletal muscle fibers (fast muscle-specific MLC2). Multiple lines of transgenic mice were generated that expressed the transgene at varying levels in the heart in a copy number-dependent fashion. There is a major discordance in the manner in which the different cardiac compartments respond to high levels of overexpression of the transgene. In atria, isoform replacement with the skeletal protein was quite efficient, even at low copy number. The ventricle is much more refractory to replacement, and despite high levels of transgenic transcript, protein replacement was incomplete. Replacement could be further increased by breeding the transgenic lines with one another. Despite very high levels of transgenic transcript in these mice, the overall level of the regulatory light chain in both compartments remained essentially constant; only the protein isoform ratios were altered. The partial replacement of the ventricular with the skeletal isoform reduced both left ventricular contractility and relaxation, although the unloaded shortening velocity of isolated ventricular cardiomyocytes was not significantly different.

Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation.

To examine the role of alterations in myofibrillar function in human dilated cardiomyopathies, we determined isometric tension-calcium relations in permeabilized myocytesized myofibrillar preparations (n = 16) obtained from left ventricular biopsies from nine patients with dilated cardiomyopathy (DCM) during cardiac transplantation or left ventricular assist device implantation. Similar preparations (n = 10) were obtained from six normal hearts used for cardiac transplantation. Passive and maximal Ca2+-activated tensions were similar for the two groups. However, the calcium sensitivity of isometric tension was increased in DCM compared to nonfailing preparations ([Ca2+]50=2.46+/-0.49 microM vs 3.24+/-0.51 microM, P < 0.001). In vitro treatment with the catalytic subunit of protein kinase A (PKA) decreased calcium sensitivity of tension to a greater degree in failing than in normal preparations. Further, isometric tension-calcium relations in failing and normal myofibrillar preparations were similar after PKA treatment. These findings suggest that the increased calcium sensitivity of isometric tension in DCM may be due at least in part to a reduction of the beta-adrenergically mediated (PKA-dependent) phosphorylation of myofibrillar regulatory proteins such as troponin I and/or C-protein.

Correlates of cochlear implantation, 1986-1992.

There is a paucity of information regarding the use of cochlear implants within large populations. This article describes correlates of cochlear implantation procedures using a statewide hospital discharge database. Among the 146 implant procedures, 55% involved female patients, whereas the largest groups of patients were represented by younger and older persons (27% between ages 2 and 9 years and 24% 60 years and older). A bimodal distribution was apparent for average annual age-specific rates of cochlear implantation, with the highest rates among persons aged 2 to 9 years (5.4 implants per 1 million) and persons aged 60 to 69 years (3.7 implants per 1 million). Total hospital charges, excluding professional fees, exceeded $12,000 per implant and were found to vary significantly when examined by length of stay and by calendar year of procedure. The data presented are unique with regard to the total number of cochlear implant procedures included and the large, diverse population used. This study expands knowledge concerning the epidemiology and utilization of cochlear implantation.

Anthracycline-induced tension in permeabilized cardiac fibers: evidence for the activation of the calcium release channel of sarcoplasmic reticulum.

Anthracyclines, such as doxorubicin (DOX), are important cancer chemotherapeutic agents that are cardiotoxic. The mechanism for the cardiotoxicity is not well-defined. Recent studies have concluded that anthracyclines release calcium (Ca2+) from membrane fractions containing sarcoplasmic reticulum (SR). To determine whether anthracyclines release Ca2+ in situ from cardiac SR, the effects of DOX on Ca(2+)-activated contractions were analyzed in membrane-permeabilized and membrane-intact fibers from rabbit heart. DOX (10-120 microM) induced tension development in calcium-preloaded permeabilized fibers. DOX-induced tension required submicromolar Ca2+, and was blocked by ruthenium red (20 microM) and Triton X-100 treatment, characteristics shared by caffeine-induced tension referable to SR Ca(2+)-release. DOX (50 microM) did not alter the maximum Ca(2+)-activated tension or shift the Ca2+ concentration-tension relationship of permeabilized fibers, indicating no effect of DOX on the myofilaments. DOX (44-350 microM) depressed post-rest isometric contractility of membrane-intact fibers but did not inhibit steady-state contractility (at 1 Hz; 2.5 Mm Ca2+), similar to effects of caffeine and submicromolar ryanodine. The specific effects of DOX on post-rest contractility of membrane-intact fibers are consistent with DOX-induced Ca2+ release from the SR of membrane-permeabilized fibers. Thus, DOX alters SR Ca2+ release in situ which may contribute to the inotropic and lusitropic dysfunction observed with anthracyclines.

A new class of high affinity ligands for the neurokinin A NK2 receptor: psi (CH2NR) reduced peptide bond analogues of neurokinin A4-10.

Analogues of [Leu10]NKA4-10 were synthesized in which each of the amide bonds was sequentially replaced with the reduced amide psi (CH2NH) bond to determine the effect of this structural modification on the antagonism of NKA binding to the HUB NK2 receptor. [psi (CH2-NH)9,Leu10]NKA4-10 (6) retained significant affinity for the NK2 receptor (IC50 = 115 nM) and showed weak partial stimulation of PI turnover (approximately 10-15% of NKA maximum). 6 behaves as a competitive antagonist of NKA-stimulated PI turnover with a pA2 = 6.7. The secondary amine of the psi (CH2NH) moiety of 6 was converted to a tertiary amine by alkylation. This modification was found to have a small effect upon receptor affinity but did result in attenuation of partial agonist activity. A combination of amino acid substitutions and psi (CH2NH) alkylation yielded [beta Ala8,psi (CH2N(CH2)2CH3)9,Phe10]NKA4-10 (21) which has very high affinity for the HUB NK2 receptor. This compound inhibited [125I]NKA binding with an IC50 = 1 nM which is equal to the receptor affinity of NKA. Compound 21 also shows very weak partial agonism of PI turnover (< or = 5% of NKA maximum) which makes this the most potent member of a new class of NKA ligands: psi(CH2NR)9-NKA4-10 analogues which potently antagonize NKA binding and possess minimal partial agonist activity.

Demonstration of a neurokinin A receptor subtype in transfected fibroblasts.

In competitive radioligand binding assays, the NK2 receptor antagonists [Tyr5,D-Trp6,8,9,Arg10]NKA(4-10) (MEN 10207) and [Tyr5,D-Trp6,8,9,Arg10]NKA(3-10) (MEN 10208) had high and low affinity, respectively, in bovine stomach membranes and SKLKB82#3 cells, a murine fibroblast cell line transfected with a cDNA encoding for the bovine NK2 receptor. These antagonists also had different affinities when inhibiting neurokinin A-induced polyphosphoinositide hydrolysis in SKLKB82#3 murine fibroblasts. Thus, the de novo protein expressed by the SKLKB82#3 murine fibroblasts may represent a distinct NK2 receptor subtype.

Comparison of the effects of neuropeptide K and neuropeptide gamma with neurokinin A at NK2 receptors in the hamster urinary bladder.

Neuropeptide K (NPK) and neuropeptide gamma (NP gamma) are two endogenous N-terminally extended forms of neurokinin A (NKA). Here, we compared their effects with those of NKA on 125I-NKA binding, phosphatidylinositol (PI) turnover and smooth muscle contraction in the hamster urinary bladder. NPK, NP gamma and NKA were equipotent in competing 125I-NKA from NK2 receptors in crude hamster bladder membranes. All three peptides stimulated PI turnover by approximately 750% with similar potency. In a third series of experiments, these peptides had similar efficacy in inducing a dose-dependent contraction of bladder smooth muscle. The NK2 receptor selective antagonist L-659,877 (cyclo[Leu-Met-Gln-Trp-Phe-Gly]) inhibited the stimulation of PI turnover and bladder contractions induced by all three tachykinins. The present results show that NKA, NPK and NP gamma display a similar biological profile. The N-terminal extensions of NPK and NP gamma appear not to influence binding of these peptides to NK2 receptors, NK2 receptor mediated stimulation of PI turnover, or smooth muscle contraction in hamster urinary bladder.

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula.

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK1 receptor. The pharmacology of the other site, which specifically bound [125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II approximately scyliorhinin I greater than eledoisin approximately substance P approximately neurokinin A greater than phyllomedusin approximately physalaemin greater than [Sar9Met(O2)11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK1 receptor and of a nonmammalian tachykinin receptor in the dogfish.

Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells.

Membranes isolated from a murine fibroblast B82 cell line (SKLKB82#3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of [His(125I)1]neurokinin A (125I-NKA) to a single population of sites with a Bmax of 147 fmol/mg of protein and a Kd of 0.59 nM. Control cell lines had little or no specific binding. The ligand binding in SKLKB82#3 cells was reversible and was inhibited by peptides in the potency rank of neuropeptide gamma greater than neuropeptide K greater than neurokinin A greater than [10-norleucine]neurokinin A-(4-10) greater than substance P much greater than senktide (succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2). Specific binding was enhanced by Mn2+, Mg2+, and Ca2+ and was inhibited by guanine nucleotide analogues. Thus, SKLKB82#3 cells have been transfected with NK2 receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK2 receptors, NK2 receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of 125I-NKA binding by nucleotide analogues was markedly different in SKLKB82#3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK2 receptors.

C-terminal modifications of neuropeptide Y and its analogs leading to selectivity for the mouse brain receptor over the porcine spleen receptor.

Neuropeptide Y (NPY) is known to bind to at least two types of receptors (Y1 & Y2). One type (Y2) is able to bind and undergo activation by both NPY and its C-terminal fragments with good potency while the other (Y1) requires the full length of NPY for good potency. For most NPY analogs that have been examined, potency for the Y2 system (porcine spleen) is greater than or equal to that for the Y1 system (mouse brain), since the Y2 system is generally less selective. However, modifications of NPY and its analogs at position 34 can lead to materials with some Y1 selectivity. For example, [Pro34]-pNPY binds to mouse brain with an affinity of 0.14 nM. Its affinity for porcine spleen is 140 nM. [His34]-pNPY was also found to be Y1 selective (19-fold), but not to the degree of the [Pro34] analog (1000-fold). The Pro34 modification in the Y2 selective C-terminal fragment NPY (20-36) converted it into an essentially non-selective analog. The selectivity from the Pro34 substitution results from a loss of Y2 binding potency along with little effect on the Y1-receptor binding. Therefore, Y1 and Y2 receptors have differing requirements for the C-terminal region of NPY in addition to their different requirements for NPY's N-terminus.