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J M Metzger

Publications and source records attributed to J M Metzger.

At least 37 records · Page 2Linked to original sources
PubMed1999

A nemaline myopathy mutation in alpha-tropomyosin causes defective regulation of striated muscle force production.

Nemaline myopathy (NM) is a rare autosomal dominant skeletal muscle myopathy characterized by severe muscle weakness and the subsequent appearance of nemaline rods within the muscle fibers. Recently, a missense mutation inTPM3, which encodes the slow skeletal alpha-tropomyosin (alphaTm), was linked to NM in a large kindred with an autosomal-dominant, childhood-onset form of the disease. We used adenoviral gene transfer to fully differentiated rat adult myocytes in vitro to determine the effects of NM mutant human alphaTm expression on striated muscle sarcomeric structure and contractile function. The mutant alphaTm was expressed and incorporated correctly into sarcomeres of adult muscle cells. The primary defect caused by expression of the mutant alphaTm was a decrease in the sensitivity of contraction to activating Ca(2+), which could help explain the hypotonia seen in NM. Interestingly, NM mutant alphaTm expression did not directly result in nemaline rod formation, which suggests that rod formation is secondary to contractile dysfunction and that load-dependent processes are likely involved in nemaline rod formation in vivo.

Adenoviridae↗
PubMed1998

4,4-Disubstituted piperidine high-affinity NK1 antagonists: structure-activity relationships and in vivo activity.

Previously reported studies from these laboratories described the design of a novel series of high-affinity NK1 antagonists based on the 4,4-disubstituted piperidine ring system. Further structure-activity studies have now established that for high NK1 affinity the benzyl ether side chain must be 3,5-disubstituted and highly lipophilic, the optimal side chain being the 3, 5-bis(trifluoromethyl)benzyl ether, 12 (hNK1 IC50 = 0.95 nM). Additional studies have shown that this class of NK1 antagonist tolerates a wider range of substituents on the piperidine nitrogen, including acyl (38) (hNK1 IC50 = 5.3 nM) and sulfonyl (39) (hNK1 IC50 = 5.7 nM) derivatives. Following preliminary pharmacokinetic analysis, two compounds (32 and 43) were selected for in vivo study in the resiniferotoxin-induced vascular leakage model, both showing excellent profiles (ID50 = 0.22 and 0.28 mg/kg, respectively).

Animals↗
PubMed1998

An NK1 receptor antagonist affects the circadian regulation of locomotor activity in golden hamsters.

Substance P (SP) is a neuromodulator which may participate in the photic regulation of the circadian timing system in mammals. The biological effects of SP are mediated by interaction with specific receptors, designated as NK1, NK2, and NK3. The NK1 subtype receptor is expressed in the circadian system. Experiment 1 was designed to test whether an NK1 antagonist mimics the effects of dark pulses. Hamsters were housed in constant lighting conditions, either constant darkness or constant light (around 250 lx), and they received an i.p. injection of either the specific NK1 receptor antagonist, L-760,735 (5 mg/kg), or saline during the mid-subjective day, a time when dark pulses cause a phase-advance in circadian rhythm of locomotor activity. After treatment with the NK1 antagonist, significant phase-advances of wheel-running activity rhythm were found in constant light, but not in constant darkness. Experiment 2 was designed to test the ability of the NK1 antagonist to block the phase-delaying and/or the phase-advancing effects of light in animals kept in constant darkness. Phase-advances of locomotor activity rhythm that can normally be induced by light pulses given during the late subjective night were markedly reduced by pre-treatment with the NK1 antagonist. By contrast, phase-delays that can be induced by lights pulses given during the early subjective night were unaffected by the NK1 antagonist. These data support the hypothesis that SP within the circadian system may, by interacting with NK1 receptors, modulate photic responses of the SCN pacemaker.

Animals↗
PubMed1998

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.

Adipose Tissue, Brown↗
PubMed1998

Stability of the contractile assembly and Ca2+-activated tension in adenovirus infected adult cardiac myocytes.

Adenovirus-mediated gene transfer into adult cardiac myocytes in primary culture is a potentially useful method to study the structure and function of the contractile apparatus. However, the consequences of adenovirus infection on the highly differentiated state of the cultured myocyte have not been determined. We report here a detailed analysis of myofilament structure and function over time in primary culture and after adenovirus infection. Adult rat ventricular myocytes in primary culture were infected with a recombinant adenovirus vector expressing either the LacZ or alkaline phosphatase reporter gene. Control and infected myocytes were collected at days 0-7 post-isolation/infection, and myofilament isoform expression was determined by SDS-PAGE and Western blot. Laser scanning densitometry showed that the alpha- to beta-myosin heavy chain ratio, the stoichiometry of the myosin light chains and the expression of the adult troponin T isoform did not change over time in culture or with adenovirus treatment. Importantly, examination of Ca2+-activated tension in single myocytes showed no change in the shape or position of the tension-pCa relationship in the control and adenovirus infected myocytes during primary culture. These results indicate that the structure and function of adult cardiac myocytes are stable in short term primary culture and are not affected by adenovirus infection per se, and therefore provide the foundation for the use of adenovirus-mediated myofilament gene transfer to study contractile apparatus structure and function in adult cardiac myocytes.

Adenoviruses, Human↗
PubMed1998

Peak power output is maintained in rabbit psoas and rat soleus single muscle fibers when CTP replaces ATP.

The chemomechanical coupling mechanism in striated muscle contraction was examined by changing the nucleotide substrate from ATP to CTP. Maximum shortening velocity [extrapolation to zero force from force-velocity relation (Vmax) and slope of slack test plots (V0)], maximum isometric force (Po), power, and the curvature of the force-velocity curve [a/Po (dimensionless parameter inversely related to the curvature)] were determined during maximum Ca2+-activated isotonic contractions of fibers from fast rabbit psoas and slow rat soleus muscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTP as the nucleotide substrate. In addition to a decrease in the maximum Ca2+-activated force in both fiber types, a change from 4 mM ATP to 10 mM CTP resulted in a decrease in Vmax in psoas fibers from 3.26 to 1.87 muscle length/s. In soleus fibers, Vmax was reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by the change in nucleotide as the result of a three- to fourfold decrease in the curvature of the force-velocity relationship. The results are interpreted in terms of the Huxley model of muscle contraction as an increase in f1 and g1 coupled to a decrease in g2 (where f1 is the rate of cross-bridge attachment and g1 and g2 are rates of detachment) when CTP replaces ATP. This adequately accounts for the observed changes in Po, a/Po, and Vmax. However, the two-state Huxley model does not explicitly reveal the cross-bridge transitions that determine curvature of the force-velocity relationship. We hypothesize that a nucleotide-sensitive transition among strong-binding cross-bridge states following Pi release, but before the release of the nucleotide diphosphate, underlies the alterations in a/Po reported here.

Adenosine Triphosphate↗
PubMed1997

Growth and muscle defects in mice lacking adult myosin heavy chain genes.

The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying null mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both null strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb null mutants are generally milder than in the MyHC-IId/x null strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for null expression of the two genes. Most striking is that while both null strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb null mice has significantly reduced ability to generate force while IId null mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.

Age Factors↗
PubMed1997

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function.

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension-Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.

Actin Cytoskeleton↗
PubMed1997

Ultrastructure and cell-cell coupling of cardiac myocytes differentiating in embryonic stem cell cultures.

Differentiation cultures of embryonic stem (ES) cells can be a useful in vitro system for understanding cardiac myocyte development. However, cell morphometry, sarcomere development, and functional cell-cell junction formation have not been examined in detail to determine whether ES cell-derived cardiac myocytes exhibit structural and functional characteristics similar to cardiac myocytes within the developing heart. Therefore, we examined cellular dimensions, sarcomere formation, and cell-cell contacts in differentiating cardiac myocytes derived from mouse D3-ES cell cultures. Cells exhibited rod-shaped morphology and had single centrally located nuclei, typical of maturing cardiac myocytes. The cellular dimensions of 59 individual cardiac myocytes within contracting foci of ES cell cultures were analyzed (length = 42.2 +/- 2.1 microns, area = 197 +/- 19 microns2, and diameter = 5.5 +/- 0.3 microns) and found to be similar to myocytes in vivo. Transmission electron micrographs of ES cell-derived cardiac myocytes indicated myofibrillar architecture ranged from sparse and disorganized to densely packed, parallel arrays of myofibrils organized into mature sarcomeres. This pattern of myofibrillar assembly in maturing sarcomeres was similar to that observed during in vivo myocyte differentiation. Another hallmark of cardiac development is the formation of intercalated discs, which functionally couple adjacent cardiac myocytes. Electron micrographs indicated nascent intercalated discs were forming in foci of ES cell-derived cardiac myocytes. In addition, indirect immunostaining with anti-connexin 43 antibody (Ab), a monoclonal Ab to the gap junction component of the intercalated disc, indicated that gap junctions were present in contracting ES cell foci. Furthermore, microinjection of single cardiac myocytes with Lucifer yellow (2.5 microM) resulted in the spread of fluorescence to adjacent cells within a contracting focus, an indication of functional cell-cell coupling across these gap junctions. Together, these results indicate ES cell-derived cardiac myocytes exhibit cell morphology, sarcomere formation, and cell-cell junctions similar to those observed in cardiac myocytes developing in vivo.

Animals↗
PubMed1997

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus.

The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for beta-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of beta-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were beta-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 d postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.

Adenoviridae↗
PubMed1997

Nucleotide-dependent contractile properties of Ca(2+)-activated fast and slow skeletal muscle fibers.

The relation between single skinned skeletal fiber contractile mechanics and the myosin mechanoenzyme was examined by perturbing the actomyosin interaction with the ATP analog CTP in fibers from both rabbit psoas and rat soleus. Tension, instantaneous stiffness, and the rate of tension redevelopment (ktr), under software-based sarcomere length control, were examined at 15 degrees C for a range of Ca2+ concentrations in both fiber types. CTP produced 94% of the maximum ATP-generated tension in psoas fibers and 77% in soleus fibers. In psoas, CTP also increased stiffness to 106% of the ATP stiffness, whereas in soleus stiffness decreased to 92%. Thus, part of the greater difference between maximum ATP- and CTP-generated tension in soleus fibers appears to be due to a decrease in strongly bound cross-bridge number. Interestingly, although the nucleotide exchange produced substantial increases in the steepness (nH) of the tension- and stiffness-pCa relationships in soleus fibers, only minor changes were seen in psoas fibers. At maximum Ca2+ and nominal P(i) levels, ktr in psoas fibers increased from 11.7 s-1 with ATP to 16.6 s-1 with CTP and in soleus fibers from 4.9 to 8.4 s-1. Increased P(i) levels decreased the maximum Ca(2+)-activated tension in both fiber types and increased the ktr of psoas fibers, but the ktr of soleus fibers was not significantly altered. Thus, although the nucleotide exchange generally produced similar changes in the mechanics, there were significant muscle lineage differences in the tension- and stiffness-pCa relations and in the effects of P(i) on ktr, such that differences in contractile mechanics were lessened in the presence of CTP.

Actomyosin↗
PubMed1996

N-heteroaryl-2-phenyl-3-(benzyloxy)piperidines: a novel class of potent orally active human NK1 antagonists.

The preparation of a series of N-heteroarylpiperidine ether-based human NK1 antagonists is described. Two of the compounds 3-[-(2S,3S)-3-(((3,5-bis(trifluoromethyl)phenyl)methyl)oxy)- 2-phenylpiperidino}methyl]-1,2,4-triazole (11) and 5-[¿(2S,3S)-3-(((3,5-bis(trifluoromethyl)-phenyl)methyl)oxy)-2- phenylpiperidino}methyl]-3-oxo-1,2,4-triazolone (12)), in particular, are orally bioavailable and exhibited significant improvements in potency, both in vitro and in vivo, over the lead (carboxamidomethyl)piperidine ether 1. Rat liver microsome studies on a selected number of compounds from this series show the triazolone heterocycle to be considerably more stable than the others. Furthermore, both 11 and 12 have been profiled in a number of assays that may be predictive of the clinical utility of substance P antagonists.

Animals↗
PubMed1996

Effects of troponin C isoforms on pH sensitivity of contraction in mammalian fast and slow skeletal muscle fibres.

1. The effects of troponin C (TnC) isoforms on the acidic pH-induced rightward shift in the tension-pCa (-log[Ca2+]) relationship were examined in slow soleus and fast psoas skeletal muscle fibers. Endogenous TnC was partially extracted from skinned single fibres and the extracted fibres were subsequently reconstituted with purified TnC. The pCa producing one-half maximal tension (pCa50) was determined at pH 7.00 and 6.20 in each fibre and then the pH-induced shift in pCa50 (delta pCa50) was calculated. 2. In control fast fibres which express fast skeletal TnC (sTnC), the delta pCa50 was 0.64 +/- 0.02 pCa units (n = 10), and this increased significantly to 0.78 +/- 0.04 pCa units (n = 8) following extraction and reconstitution with cardiac TnC (cTnC). In each fibre, the reconstituted delta pCa50 was subtracted from the control delta pCa50 which yielded a significant shift of -0.13 +/- 0.05 pCa units (n = 8; P < 0.05). Thus, the pH sensitivity of contraction was increased in the cTnC-reconstituted psoas fibres. 3. In extracted psoas fibres that were reconstituted with fast sTnC the pH sensitivity of contraction was unchanged, indicating that the above effects were related to the TnC isoform and not a non-specific effect of the extraction procedure. 4. In a second series of experiments cTnC was specifically extracted from slow soleus fibres which were subsequently reconstituted with purified fast sTnC. Skeletal TnC reconstituted soleus fibres demonstrated a significant decrease in pH sensitivity. In each fibre, the reconstituted delta pCa50 (mean, 0.58 +/- 0.02 pCa units) was subtracted from the control delta pCa50 (mean, 0.63 +/- 0.02 pCa units) which yielded a significant shift of 0.05 +/- 0.01 pCa units (n = 4; P < 0.05). The pH sensitivity was not altered in cTnC-reconstituted soleus fibres (-0.01 +/- 0.01 pCa units, n = 4). 5. These findings indicate that TnC isoforms alter the pH sensitivities of contraction in slow and fast skeletal muscle fibres. However, the magnitude of the change in pH sensitivity is muscle lineage dependent, indicating that differential expression of other myofilament protein isoforms, together with TnC, is necessary to confer full pH sensitivity of contraction in striated muscles.

Animals↗
PubMed1996

Troponin I isoform expression is developmentally regulated in differentiating embryonic stem cell-derived cardiac myocytes.

We studied troponin I (TnI) isoform expression in the mouse embryonic stem (ES) cell model of cardiogenesis as an essential first step to understanding the relationship between TnI isoform transitions and myofibrillar function. Cultures of differentiating ES cells were grown on coverslips to permit microscopic inspection of foci of spontaneously contracting cardiac myocytes developing in culture. TnI expression was followed over time to test whether the cardiac myocytes undergo the developmental pattern of expression characteristic of vertebrate cardiogenesis, in which slow skeletal TnI (ssTnI) is expressed initially, followed by induction of cardiac (cTnI) isoform expression. Cardiac TnI expression was examined using the cardiac-specific, monoclonal TI-1 antibody (Ab) while all striated muscle ThI isoforms were detected using the monoclonal TI-4 Ab. Cardiac-specific TnI expression was detected in only 8% (8/96) of foci contracting less than 5 days while TI-4 positive staining was present in 95% (71/73) of foci. These results indicate that other striated muscle TnI isoforms were being expressed in most of the TI-4 positive staining foci. The proportion of contracting foci expressing the cardiac isoform increased steadily over time, such that 100% of foci contracting more than 20 days (13/13) stained positive with the TI-1 Ab. Dual labeling experiments with both TI-1 and TI-4 anti-TnI Abs in the same culture confirmed that within each foci, the area expressing cTnI increased with the days of spontaneous contraction. Western blot analysis of micro-dissected ES cell derived cardiac myocytes confirmed that TI-4 immunostaining at early developmental time points represented ssTnI, and not the fast skeletal TnI isoform. We conclude that ES cell-derived cardiac myocytes display the developmental induction of cardiac TnI expression characteristic of vertebrate cardiac development. Thus, this model should be useful for studying the regulation and functional significance of TnI isoform expression during in vitro cardiogenesis.

Aging↗
PubMed1996

Effects of phosphate and ADP on shortening velocity during maximal and submaximal calcium activation of the thin filament in skeletal muscle fibers.

The effects of added phosphate and MgADP on unloaded shortening velocity during maximal and submaximal Ca2+ activation of the thin filament were examined in skinned single skeletal fibers from rabbit psoas muscle. During maximal Ca2+ activation, added phosphate (10-30 mM) had no effect on unloaded shortening velocity as determined by the slack-test technique. In fibers activated at submaximal concentrations of Ca2+ in the absence of added phosphate, plots of slack length versus duration of unloaded shortening were biphasic, consisting of an initial high velocity phase of shortening and a subsequent low velocity phase of shortening. Interestingly, in the presence of added phosphate, biphasic slack-test plots were no longer apparent. This result was obtained in control fibers over a range of submaximal Ca2+ concentrations and in maximally Ca2+ activated fibers, which were first treated to partially extract troponin C. Thus, under conditions that favor the appearance of biphasic shortening (i.e., low [Ca2+], troponin C extraction), added phosphate eliminated the low velocity component. In contrast, in fibers activated in the presence of 5 mM added MgADP, biphasic slack-test plots were apparent even during maximal Ca2+ activation. The basis of biphasic shortening is not known but it may be due to the formation of axially compressed cross-bridges that become strained to bear a tension that opposes the relative sliding of the myofilaments. The present findings could be explained if added phosphate and MgADP bind to cross-bridges in a strain-dependent manner. In this case, the results suggest that phosphate inhibits the formation of cross-bridges that bear a compressive strain. Added MgADP, on the other hand, may be expected to detain cross-bridges in strong binding states, thus promoting an increase in the population of cross-bridges bearing a compressive strain. Alterations in the population of strained cross-bridges by added phosphate and MgADP would alter the internal load within the fiber and thus affect the speed of fiber shortening.

Actomyosin↗
PubMed1996

pH dependence of myosin binding-induced activation of the thin filament in cardiac myocytes and skeletal fibers.

The pH dependence of myosin binding-induced thin filament activation was determined in permeabilized cardiac myocytes and slow- and fast-twitch single skeletal muscle fibers by experimental lowering of [MgATP] in the Ca(2+)-free solutions bathing the permeabilized preparations. As the pS (where S is [MgATP] and pS is -log[MgATP]) was increased from 3.0 to 8.0, isometric tension increased to a peak value in the pS range of 4.9-5.3. At pH 7.00, the transition from the relaxed to the activated rigor state was steep in cardiac myocytes [Hill value (nH) = 21.2 +/- 3.1 (SE)] and due to the apparent effect of strongly bound cross bridges to cooperatively activate the thin filament in the absence of added Ca2+. At pH 6.20, the steepness of the tension-pS relationship was markedly reduced (nH = 6.1 +/- 1.0) and the midpoint of the relationship (pS50) was shifted to higher pS values in cardiac myocytes. In comparison, reduced pH had no effect on the steepness or position of the tension-pS relationship in single slow- or fast-twitch skeletal muscle fibers. These findings suggest that myosin binding-induced activation of the thin filament is pH dependent in cardiac myocytes but not in skeletal muscle fibers under these experimental conditions in which Ca2+ is absent.

Animals↗