Glen F. Tibbits

Effect of temperature and temperature acclimation on the ryanodine sensitivity of the trout myocardium

The influence of acute temperature change and temperature acclimation on the sensitivity of contracture development to ryanodine were examined in the rainbow trout myocardium using two preparations: in vitro isolated ventricular strips and in situ working perfused hearts. Ryanodine effects in vitro were dependent on test temperature (8 and 18 °C), pacing frequency (0.2–1.5 Hz) and acclimation temperature (8 and 18 °C). At a pacing frequency of 0.2 Hz and a test temperature of 18 °C, ryanodine depressed isometric tension development in ventricular strips both from trout acclimated to 8 and 18 °C but the decrease was significantly greater in strips from 8 °C-acclimated trout. No ryanodine effect was observed in either acclimation group at a test temperature of 8°C. The effect of ryanodine in vitro was reduced or lost at pacing frequencies greater than 0.2 Hz and at 0.6 Hz ryanodine depressed tension development at 18 °C only in strips from 8 °C-acclimated trout. Ryanodine did not affect tension development at stimulation rates above 0.6 Hz in any test group. Likewise, ryanodine did not significantly impair cardiac performance of in situ working perfused heart preparations which operated at intrinsic beat frequencies in excess of 0.6 Hz. These results suggest that the sarcoplamic reticulum calcium release channel of the trout myocardium is expressed but is not functionally involved in beat-to-beat regulation of contractility at either (1) low temperature (8 °C), or (2) at routine physiological heart rate (>0.6 Hz). However, under conditions in which involvement of the sarcoplasmic reticulum is observed (18 °C and a heart rate < 0.6 Hz), prior acclimation to low temperature results in either a greater capacity of the sarcoplasmic reticulum to store releasable calcium or an increase in the amount of calcium that is in releasable form.

6 - Excitation–Contraction Coupling in the Teleost Heart

This chapter describes the excitation–contraction coupling in the teleost heart. Cardiac output, the product of heart rate and stroke volume, must be regulated in vertebrates to maintain appropriate blood flow to exercising muscle, the brain, and other tissues under a wide variety of conditions. Stroke volume, in turn, is controlled primarily by the modulation of two important parameters: the preload or end-diastolic volume and myocardial contractility. The knowledge of cytosolic Ca 2+ buffering in the teleost heart is critical to the understanding of the regulation of contractility. Tissue acidosis is another factor known in mammalian cardiac myofilaments to profoundly affect Ca 2+ sensitivity. Decreasing pH from 7.0–6.5 decreases TnC affinity for Ca 2+ several fold. The effect of pH is enhanced when TnC is complexed to TnI, suggesting that TnC–TnI interactions may be involved in the inhibition by pH. While tissue acidosis would occur in fish under the same conditions as in mammals, there are also the effects of temperature on intracellular pH to consider. Electron microscopic observations of a number of poikilothermic hearts, including those of the teleosts, demonstrate both paucity and a lack of organizational complexity of sarcoplasmic reticulum in comparison to the mammalian heart.

Familial hypertrophic cardiomyopathy-related cardiac troponin C mutation L29Q affects Ca2+ binding and myofilament contractility

The cardiac troponin C (cTnC) mutation, L29Q, has been found in a patient with familial hypertrophic cardiomyopathy. We previously showed that L29, together with neighboring residues, Asp2, Val28, and Gly30, plays an important role in determining the Ca(2+) affinity of site II, the regulatory site of mammalian cardiac troponin C (McTnC). Here we report on the Ca(2+) binding characteristics of L29Q McTnC and D2N/V28I/L29Q/G30D McTnC (NIQD) utilizing the Phe(27) --> Trp (F27W) substitution, allowing one to monitor Ca(2+) binding and release. We also studied the effect of these mutants on Ca(2+) activation of force generation in single mouse cardiac myocytes using cTnC replacement, together with sarcomere length (SL) dependence. The Ca(2+)-binding affinity of site II of L29Q McTnC(F27W) and NIQD McTnC(F27W) was approximately 1.3- and approximately 1.9-fold higher, respectively, than that of McTnC(F27W). The Ca(2+) disassociation rate from site II of L29Q McTnC(F27W) and NIQD McTnC(F27W) was not significantly different than that of control (McTnC(F27W)). However, the rate of Ca(2+) binding to site II was higher in L29Q McTnC(F27W) and NIQD McTnC(F27W) relative to control (approximately 1.5-fold and approximately 2.0-fold respectively). The Ca(2+) sensitivity of force generation was significantly higher in myocytes reconstituted with L29Q McTnC (approximately 1.4-fold) and NIQD McTnC (approximately 2-fold) compared with those reconstituted with McTnC. Interestingly, the change in Ca(2+) sensitivity of force generation in response to an SL change (1.9, 2.1, and 2.3 mum) was significantly reduced in myocytes containing L29Q McTnC or NIQD McTnC. These results demonstrate that the L29Q mutation enhances the Ca(2+)-binding characteristics of cTnC and that when incorporated into cardiac myocytes, this mutant alters myocyte contractility.

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.

Cloning, expression, and characterization of the trout cardiac Na+/Ca2+exchanger

Isoform 1 of the cardiac Na+/Ca2+exchanger (NCX1) is an important regulator of cytosolic Ca2+ concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7°C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout ( Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca2+ binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at ∼23°C demonstrated Na+-dependent inactivation and Ca2+-dependent activation in a manner qualitatively similar to that for NCX1 currents.

Hyperpolarization‐activated cyclic nucleotide‐modulated ‘HCN’ channels confer regular and faster rhythmicity to beating mouse embryonic stem cells

The hyperpolarization‐activated cation current (If), and the hyperpolarization‐activated cyclic nucleotide‐modulated ‘HCN’ subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell‐derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. If and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole‐cell voltage‐ and current‐clamp techniques. Our data show that the proportion of cells expressing If, and the density of If in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The If blocker ZD7288 (0.3 μm) reduced If and the beating rate of embryoid bodies. Taken together, the activation kinetics of If and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of If to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation.

Calcium handling in zebrafish ventricular myocytes

The zebrafish is an important model for the study of vertebrate cardiac development with a rich array of genetic mutations and biological reagents for functional interrogation. The similarity of the zebrafish (Danio rerio) cardiac action potential with that of humans further enhances the relevance of this model. In spite of this, little is known about excitation-contraction coupling in the zebrafish heart. To address this issue, adult zebrafish cardiomyocytes were isolated by enzymatic perfusion of the cannulated ventricle and were subjected to amphotericin-perforated patch-clamp technique, confocal calcium imaging, and/or measurements of cell shortening. Simultaneous recordings of the voltage dependence of the L-type calcium current (I(Ca,L)) amplitude and cell shortening showed a typical bell-shaped current-voltage (I-V) relationship for I(Ca,L) with a maximum at +10 mV, whereas calcium transients and cell shortening showed a monophasic increase with membrane depolarization that reached a plateau at membrane potentials above +20 mV. Values of I(Ca,L) were 53, 100, and 17% of maximum at -20, +10, and +40 mV, while the corresponding calcium transient amplitudes were 64, 92, and 98% and cell shortening values were 62, 95, and 96% of maximum, respectively, suggesting that I(Ca,L) is the major contributor to the activation of contraction at voltages below +10 mV, whereas the contribution of reverse-mode Na/Ca exchange becomes increasingly more important at membrane potentials above +10 mV. Comparison of the recovery of I(Ca,L) from acute and steady-state inactivation showed that reduction of I(Ca,L) upon elevation of the stimulation frequency is primarily due to calcium-dependent I(Ca,L) inactivation. In conclusion, we demonstrate that a large yield of healthy atrial and ventricular myocytes can be obtained by enzymatic perfusion of the cannulated zebrafish heart. Moreover, zebrafish ventricular myocytes differed from that of large mammals by having larger I(Ca,L) density and a monophasically increasing contraction-voltage relationship, suggesting that caution should be taken upon extrapolation of the functional impact of mutations on calcium handling and contraction in zebrafish cardiomyocytes.

Gene structure evolution of the Na+-Ca2+ exchanger (NCX) family.

BackgroundThe Na+-Ca2+ exchanger (NCX) is an important regulator of cytosolic Ca2+ levels. Many of its structural features are highly conserved across a wide range of species. Invertebrates have a single NCX gene, whereas vertebrate species have multiple NCX genes as a result of at least two duplication events. To examine the molecular evolution of NCX genes and understand the role of duplicated genes in the evolution of the vertebrate NCX gene family, we carried out phylogenetic analyses of NCX genes and compared NCX gene structures from sequenced genomes and individual clones.ResultsA single NCX in invertebrates and the protochordate Ciona, and the presence of at least four NCX genes in the genomes of teleosts, an amphibian, and a reptile suggest that a four member gene family arose in a basal vertebrate. Extensive examination of mammalian and avian genomes and synteny analysis argue that NCX4 may be lost in these lineages. Duplicates for NCX1, NCX2, and NCX4 were found in all sequenced teleost genomes. The presence of seven genes encoding NCX homologs may provide teleosts with the functional specialization analogous to the alternate splicing strategy seen with the three NCX mammalian homologs.ConclusionWe have demonstrated that NCX4 is present in teleost, amphibian and reptilian species but has been secondarily and independently lost in mammals and birds. Comparative studies on conserved vertebrate homologs have provided a possible evolutionary route taken by gene duplicates subfunctionalization by minimizing homolog number.

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.

Effects of active oxygen generated by DTTFe2+ on cardiac Na+Ca2+ exchange and membrane permeability to Ca2+

Sarcolemmal vesicles isolated from bovine heart were preincubated at 37 degrees C with an oxygen radical generating system consisting of 1 mM dithiothreitol (DTT) and 50 microM FeSO4. Exposure of the vesicles for 1 to 40 mins stimulated Na+/Ca2+ exchange about 2.5-fold. The DTT/Fe2+ treatment decreased the apparent Km for Ca2+ of Nai+-dependent Ca2+ uptake by 80% (from 63 to 13 microM). The effect on Vmax was much smaller however. The resulting stimulation of exchange activity was diminished by the presence of desferrioxamine (95%) or catalase (60%). In contrast, superoxide dismutase and sodium formate did not prevent the effects of DTT/Fe2+ on the exchanger. Neither Zn2+ nor Ga3+ could replace Fe2+ in the stimulation of Na+/Ca2+ exchange. Passive Ca2+ efflux was determined by first allowing Na+/Ca2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. Ca2+ leakage from the vesicles was slightly but significantly (P less than 0.05) increased by the action of DTT/Fe2+, the rate constants for the passive Ca2+ efflux being 0.22 and 0.26/min in control and treated groups, respectively. The calcium loading observed in myocytes in ischemia/reperfusion injury suggests that the stimulation of Na+/Ca2+ exchange by active oxygen may moderate the myocardial response to oxygen mediated injuries including ischemia/reperfusion injury. However, the clinical relevance of these phenomena is far from clear as the stimulation depends in part on the Km for Ca2+ prior to treatment.