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Glen F Tibbits

Publications and source records attributed to Glen F Tibbits.

16 recordsLinked to original sources
PubMed2006

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes.

Store-operated Ca2+ entry (SOCE), which is Ca2+ entry triggered by the depletion of intracellular Ca2+ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca2+ loading (load(SR)) that was not altered by inhibition of L-type Ca2+ channels, reverse mode Na+/Ca2+ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca2+] to 0 mM or adding either 0.5 mM Zn2+ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 microM) inhibited load(SR) at rest. Interestingly, inhibition of forward mode NCX with 30 microM KB-R7943 stimulated SOCE significantly and resulted in enhanced load(SR). In addition, manipulation of the extracellular and intracellular Na+ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca2+ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca2+ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca2+ overload and arrhythmogenesis.

Age Factors↗
PubMed2005

L-type Ca2+ channel function and expression in neonatal rabbit ventricular myocytes.

L-type Ca(2+) channel-mediated, Ca(2+)-induced Ca(2+) release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the mature mammalian myocardium but is thought to be absent in the fetal and newborn mammalian myocardium. Furthermore, the characteristics and contributors of E-C coupling at the earliest developmental stages are poorly understood. In this study, we measured [(3)H](+)PN200-110 dihydropyridine binding capacity, functionality and expression of the L-type Ca(2+) channel, and cytosolic [Ca(2+)] ([Ca(2+)](i)) at various developmental stages (3, 6, 10, 20, and 56 days old) to characterize ontogenetic changes in E-C coupling. We found that 1) the whole cell L-type Ca(2+) channel peak current (I(Ca)) density increased slightly in parallel with cell growth, but the current-voltage relationship, the steady-state activation, and the maximum DHP binding and binding affinity did not exhibit significant developmental changes; 2) sarcoplasmic reticulum Ca(2+) dependence of inactivation rates of L-type Ca(2+) channel and peak of I(Ca) density were only observed after 10 days of age, which temporally coincides with transverse (T)-tubule formation; 3) the relationship between [Ca(2+)](i) and voltage changed from a linear relationship at the earliest developmental stages to a "bell-shaped" relationship at the later developmental stages, presumably corresponding to a switch from reverse-mode Na/Ca exchange-dependent to I(Ca)-dependent E-C coupling; and 4) the expression of two different splice variants of Ca(V)1.2, IVS3A and IVS3B, switched from predominantly IVS3A at the earliest stages to IVS3B at the later developmental stages. Our data suggest that whereas the density of functional dihydropyridine receptors (DHPRs) increases only slightly during ontogeny, the enhancement of functional coupling between DHPR and ryanodine receptor is dramatic between the second and third weeks after birth. Furthermore, we found that the differential expression of splice variants during development temporally correlated with the appearance of I(Ca)-dependent E-C coupling and T-tubule formation.

Aging↗
PubMed2005

cDNA cloning and expression of the cardiac Na+/Ca2+ exchanger from Mozambique tilapia (Oreochromis mossambicus) reveal a teleost membrane transporter with mammalian temperature dependence.

The complete cDNA sequence of the tilapia cardiac Na(+)/Ca2+ exchanger (NCX-TL1.0) was determined. The 3.1-kb transcript encodes a protein 957 amino acids in length, with a predicted signal peptide cleaved at residue 31 and two potential N-glycosylation sites in the extracellular N terminus. Hydropathy analysis and sequence comparison predicted a mature protein with nine transmembrane-spanning segments, consistent with the structural topologies of other known mammalian and teleost NCX isoforms. Overall sequence comparison shows high identity to both trout NCX-TR1.0 ( approximately 81%) and mammalian NCX1.1 ( approximately 73%), and phylogenetic analyses confirmed its identity as a member of the NCX1 gene family, expressing exons A, C, D, and F in the alternative splice site. Sequence identity is even higher in the alpha-repeats, the exchanger inhibitory peptide (XIP) site, and Ca(2+)-binding domains, which is reflected in the functional and regulatory properties of tilapia NCX-TL1.0. When NCX-TL1.0 was expressed in Xenopus oocytes and the currents were measured in giant excised patches, they displayed both positive regulation by Ca2+ and Na(+)-dependent inactivation in a manner similar to trout NCX-TR1.0. However, tilapia NCX-TL1.0 exhibited a relatively high sensitivity to temperature compared with trout NCX-TR1.0. Whereas trout NCX-TR1.0 currents displayed activation energies of approximately 7 kJ/mol, tilapia NCX-TL1.0 currents showed mammal-like temperature dependence, with peak and steady-state current activation energies of 53 +/- 9 and 67 +/- 21 kJ/mol, respectively. Using comparative sequence analysis, we highlighted 10 residue positions in the N-terminal domain of the NCX that, in combination, may confer exchanger temperature dependence through subtle changes in protein flexibility. Tilapia NCX-TL1.0 represents the first non-mammalian NCX to exhibit a mammalian temperature dependence phenotype and will prove to be a useful model in defining the interplay between molecular flexibility and stability in NCX function.

Alternative Splicing↗
PubMed2005

Increasing mammalian cardiomyocyte contractility with residues identified in trout troponin C.

The Ca2+ sensitivity of force generation in trout cardiac myocytes is significantly greater than that from mammalian hearts. One mechanism that we have suggested to be responsible, at least in part, for this high Ca2+ sensitivity is the isoform of cardiac troponin C (cTnC) found in trout hearts (ScTnC), which has greater than twice the Ca2+ affinity of mammalian cTnC (McTnC). Here, through a series of mutations, the residues in ScTnC responsible for its high Ca2+ affinity have been identified as being Asn2, Ile28, Gln29, and Asp30. When these residues in McTnC were mutated to the trout-equivalent amino acid, the Ca2+ affinity of the molecule, determined by monitoring the fluorescence of a Trp inserted for a Phe at residue 27, is comparable to that of ScTnC. To determine how a McTnC mutant containing Asn2, Ile28, Gln29, and Asp30 (NIQD McTnC) affects the Ca2+ sensitivity of force generation, endogenous cTnC in single, chemically skinned rabbit cardiomyocytes was replaced with either wild-type McTnC or NIQD McTnC. Our results demonstrate that the cardiomyocytes containing NIQD McTnC were approximately twice as sensitive to Ca2+, illustrating that a McTnC mutant with similar Ca2+ affinity as ScTnC can be used to sensitize mammalian cardiac myocytes to Ca2+.

Amino Acid Sequence↗
PubMed2005

Phylogeny of Na+/Ca2+ exchanger (NCX) genes from genomic data identifies new gene duplications and a new family member in fish species.

The Na+/Ca2+ exchanger (NCX) is a member of the cation/Ca2+ antiporter (CaCA) family and plays a key role in maintaining cellular Ca2+ homeostasis in a variety of cell types. NCX is present in a diverse group of organisms and exhibits high overall identity across species. To date, three separate genes, i.e., NCX1, NCX2, and NCX3, have been identified in mammals. However, phylogenetic analysis of the exchanger has been hindered by the lack of nonmammalian NCX sequences. In this study, we expand and diversify the list of NCX sequences by identifying NCX homologs from whole-genome sequences accessible through the Ensembl Genome Browser. We identified and annotated 13 new NCX sequences, including 4 from zebrafish, 4 from Japanese pufferfish, 2 from chicken, and 1 each from honeybee, mosquito, and chimpanzee. Examination of NCX gene structure, together with construction of phylogenetic trees, provided novel insights into the molecular evolution of NCX and allowed us to more accurately annotate NCX gene names. For the first time, we report the existence of NCX2 and NCX3 in organisms other than mammals, yielding the hypothesis that two serial NCX gene duplications occurred around the time vertebrates and invertebrates diverged. In addition, we have found a putative new NCX protein, named NCX4, that is related to NCX1 but has been observed only in fish species genomes. These findings present a stronger foundation for our understanding of the molecular evolution of the NCX gene family and provide a framework for further NCX phylogenetic and molecular studies.

Amino Acid Sequence↗
PubMed2004

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes.

Much less is known about the contributions of the Na(+)/Ca(2+) exchanger (NCX) and sarcoplasmic reticulum (SR) Ca(2+) pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca(2+) decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I(NCX)) and the Ca(2+) transient. We found that the integral of I(NCX), an indicator of SR Ca(2+) content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca(2+) extrusion by NCX (V(NCX)) was linear with [Ca(2+)] and did not indicate saturation kinetics until [Ca(2+)] reached 1-3 microM for each age group. There was a significantly greater time delay between the peaks of I(NCX) and the Ca(2+) transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age.

Animals↗
PubMed2004

Effect of temperature on the structure of trout troponin C.

Adaptation for life at different temperatures can cause changes in many aspects of an organism. One example is the expression of different protein isoforms in species adapted to different temperatures. The calcium regulatory protein cardiac troponin C (cTnC), from rainbow trout (Oncorhynchus mykiss), is a good model for studying temperature effects, both because of its low physiological temperature and because mammalian cTnC, extensively studied at higher temperatures, can be used for comparison. We determined the structure and studied the backbone dynamics of the regulatory domain of trout cardiac troponin C (ScNTnC) with one Ca(2+) bound at 7 and 30 degrees C, using nuclear magnetic resonance spectroscopy (NMR). The overall fold of the regulatory domain of trout cTnC at both temperatures is similar to the regulatory domain of mammalian (human, bovine, and porcine isoform) cTnC bound to one Ca(2+). By comparing the trout structures at the two temperatures, we identify differences between the positions of the helices flanking the calcium binding loops, and the overall structure at 7 degrees C is more compact than that at 30 degrees C. The structure at 7 degrees C is more similar to the mammalian cTnC, which was determined at 30 degrees C, indicating that they have the same conformation at their respective physiological temperatures. The dynamic properties of the regulatory domain of trout cTnC are similar at the two temperatures that were used in these studies.

Adaptation, Physiological↗
PubMed2004

Deconvolution of confocal images of dihydropyridine and ryanodine receptors in developing cardiomyocytes.

Colocalization of dihydropyridine (DHPR) and ryanodine (RyR) receptors, a key determinant of Ca(2+)-induced Ca2+ release, was previously estimated in 3-, 6-, 10-, and 20-day-old rabbit ventricular myocytes by immunocytochemistry and confocal microscopy. We now report on the effects of deconvolution (using a maximum-likelihood estimation algorithm) on the calculation of colocalization indexes. Clusters of DHPR and RyR can be accurately represented as point sources of fluorescence, which enables a model of their relative distributions to be constructed using images of point spread functions to simulate their fluorescence inside a cell. This model was used to investigate the effects of deconvolution on colocalization as a function of separation distance. Deconvolution resulted in significant improvements in both axial and transverse resolutions, producing significant increases in clarity. Comparisons of intensity profiles (full-width half-maximum) pre- and postdeconvolution showed decreased dispersion of the fluorescent signal and a corresponding decrease in false colocalization as determined by fluorescence modeling. This hypothesis was extended to physiological data previously collected. The number of colocalized voxels was quantified after deconvolution, and the degree of colocalization of DHPR with RyR decreased significantly after deconvolution in all age groups: 3 days (62 +/- 2% before deconvolution, 43 +/- 3 after deconvolution) to 20 days old (79 +/- 1% before deconvolution, 63 +/- 2% after deconvolution). The data demonstrate that confocal images should be deconvolved before any quantitative analysis, such as colocalization index determination, to minimize the detrimental effects of out-of-focus light in coincident voxels.

Algorithms↗
PubMed2003

Effect of temperature and the F27W mutation on the Ca2+ activated structural transition of trout cardiac troponin C.

The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.

Amino Acid Sequence↗
PubMed2003

Triggering of sarcoplasmic reticulum Ca2+ release and contraction by reverse mode Na+/Ca2+ exchange in trout atrial myocytes.

Whole cell patch clamp and intracellular Ca(2+) transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca(2+) trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca(2+) carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca(2+)/pF, giving an SR Ca(2+) release rate of 279 amol Ca(2+). pF(-1). s(-1) or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca(2+) release, intracellular Ca(2+) transient, and cell shortening at 10 mV. Although 100 microM CdCl(2) abolished this local maximum, it had no effect on SR Ca(2+) release elicited by a depolarization to 110 or 150 mV, and the SR Ca(2+) release was proportional to the membrane potential in the range -50 to 150 mV with 100 microM CdCl(2). Increasing the intracellular Na(+) concentration ([Na(+)]) from 10 to 16 mM enhanced SR Ca(2+) release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca(2+) release was potentiated with 16 mM but not 10 mM pipette [Na(+)]. Comparison of the total sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR gave a gain factor of 18.6 +/- 7.7. Nifedipine (Nif) at 10 microM inhibited L-type Ca(2+) current (I(Ca)) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca(2+) release was 16.0 +/- 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl(2). The gain of the Nif-insensitive but NiCl(2)-sensitive SR Ca(2+) release was 14.8 +/- 7.1. Thus both reverse-mode Na(+)/Ca(2+) exchange (NCX) and I(Ca) can elicit Ca(2+) release from the SR, but I(Ca) is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca(2+) released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca(2+) release.

Animals↗
PubMed2003

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II.

Cardiac myofibrils isolated from trout heart have been demonstrated to have a higher sensitivity for Ca(2+) than mammalian cardiac myofibrils. Using cardiac troponin C (cTnC) cloned from trout and mammalian hearts, we have previously demonstrated that this comparatively high Ca(2+) sensitivity is due, in part, to trout cTnC (ScTnC) having twice the Ca(2+) affinity of mammalian cTnC (McTnC) over a broad range of temperatures. The amino acid sequence of ScTnC is 92% identical to McTnC. To determine the residues responsible for the high Ca(2+) affinity, the function of a number of ScTnC and McTnC mutants was characterized by monitoring an intrinsic fluorescent reporter that monitors Ca(2+) binding to site II (F27W). The removal of the COOH terminus (amino acids 90-161) from ScTnC and McTnC maintained the difference in Ca(2+) affinity between the truncated cTnC isoforms (ScNTnC and McNTnC). The replacement of Gln(29) and Asp(30) in ScNTnC with the corresponding residues from McNTnC, Leu and Gly, respectively, reduced Ca(2+) affinity to that of McNTnC. These results demonstrate that Gln(29) and Asp(30) in ScTnC are required for the high Ca(2+) affinity of site II.

Amino Acid Sequence↗
PubMed2003

Na+/H+ exchange inhibition with HOE 642 improves recovery of the injured neonatal rabbit heart.

BACKGROUND: The effects of inhibition of the Na+/H+ exchanger (NHE) on postischemic recovery of the injured neonatal rabbit heart were examined. The NHE may be an important mechanism for reperfusion injury in the neonate heart. The effects of two NHE inhibitors, HOE 642 (HOE) and 5-(N,N-dimethyl)-amiloride (DMA), given during hypothermic cardioplegic arrest, were evaluated. METHODS: Isolated working crystalloid-perfused neonatal rabbit hearts were subjected to 10 min of normothermic ischemia to cause injury before undergoing 4 h of hypothermic (10 degrees C) cardioplegic arrest with a single dose of crystalloid solution (controls, n=21) or with the addition of 0.5 micromol/L HOE (n=24) or 30 micromol/L DMA (n=15). RESULTS: Hearts subjected to HOE had improved recoveries of aortic flow when compared with controls at 15 min and 30 min of reperfusion (35.7+/-1.3 mL/min versus 26.2+/-1.4 mL/min, respectively, at 15 min, P<0.0001; 36.5+/-1.5 mL/min versus 23.6+/-1.6 mL/min, respectively, at 30 min, P<0.0001) and with DMA at 30 min (36.5+/-1.5 mL/min versus 29.9+/-1.9 mL/min, P=0.0214). Cardiac output and systolic pressure were also improved at 30 min in HOE hearts versus controls (P<0.0001). CONCLUSIONS: NHE inhibition with HOE during cardioplegic arrest resulted in improved functional recovery of injured hearts. Further studies in blood-perfused neonatal preparations are warranted.

Animals↗
PubMed2002

Beating the cold: the functional evolution of troponin C in teleost fish.

The sensitivity of the cardiac myocyte contractile element for Ca(2+) decreases with temperature. As myocyte contractility is regulated by changes in cytosolic [Ca(2+)], this desensitizing effect represents a challenge for temperate fish such as the rainbow trout, Oncorhynchus mykiss, living in environments where temperatures are low and variable. To allow cardiac function in a temperate environment it is thought that the comparatively high Ca(2+) sensitivity of trout cardiac myocytes compensates for the effects of low temperature on myocyte contractility. The high Ca(2+) sensitivity of the trout myocyte is due, at least in part, to changes in the amino acid sequence of the thin filament protein, cardiac troponin C (cTnC). cTnC is the Ca(2+)-activated switch that triggers myocyte contraction. The isoform of cTnC cloned from trout ventricle (ScTnC) is 92% identical to mammalian cTnC (McTnC) and is significantly more sensitive to Ca(2+). This result suggests that ScTnC has evolved in trout to allow cardiac function at low temperatures. cTnC also appears to play a role in maintaining cardiac function when temperatures change. Increasing myofibrillar pH according to alpha-stat regulation, as would occur when temperature decreases, increases Ca(2+) sensitivity. A similar increase in pH also sensitizes cTnC to Ca(2+). ScTnC therefore appears critical in maintaining cardiac function in trout at low temperatures as well as during changes in temperature.

Acclimatization↗
PubMed2002

Ontogeny of excitation-contraction coupling in the mammalian heart.

The neonate mammalian heart is phenotypically different from the adult heart in many respects. Understanding these phenotypic differences are a fundamental component of understanding the mechanisms of congenital heart disease and its treatment. Differences in excitation-contraction (E-C) coupling of the neonatal heart from that of the adult include less reliance on intercellular sources of Ca(2+) such as that from sarcoplasmic reticulum (SR). Electron micrographs indicate that these immature cardiomyocytes lack transverse tubules and the SR is sparse. This paper focuses on the changes in the phenotype of E-C coupling during ontogeny in the mammalian heart and the molecular mechanisms underlying these changes.

Animals↗
PubMed2002

Determinants of cardiac Na(+)/Ca(2+) exchanger temperature dependence: NH(2)-terminal transmembrane segments.

The cardiac Na(+)/Ca(2+) exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30 degrees C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the NH(2)-terminal region of the molecule. Specifically, we found that approximately 75% of the Na(+)/Ca(2+) exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site.

Amino Acid Sequence↗