Pacing rate, halothane, and BDM affect fura 2reporting of [Ca2+]i in intact rat trabeculae

Experiments weredone on intact trabeculae from rats. Fura 2 in the salt form wasmicroinjected directly into the myoplasm. The experiments wereconducted at 30°C, with 2 mM extracellular Ca²⁺ concentrationand pacing at either 0.5 or 5 Hz. The aims were to establish a newmethod for in vivo calibration of fura 2 and to determine the effect ofautofluorescence changes on intracellular Ca²⁺ concentration([Ca²⁺]_i)reported by fura 2. Autofluorescence was recorded under optimal conditions for fura 2 fluorescence (emission at 510 nm). By alteration of the oxidation-reduction state, it was shown that NADH is the maincomponent of autofluorescence in heart. An increase in pacing frequencycaused a decrease in autofluorescence. Both halothane and2,3-butanedione monoxime (BDM) at 5-Hz pacing produced a substantial rise in autofluorescence, approaching the levels observed at 0.5-Hz pacing. The values for the dissociation constant (678 nM) and maximumfluorescence ratio of fura 2 forCa²⁺ for the in vivo calibrationare 3.4 times larger and 2.6 times smaller, respectively, than thosefound in vitro. Using the parameters obtained in vivo, we found thatthe diastolic and systolic[Ca²⁺]_iof a twitch at 30°C were 0.2 and 2.4 µM, respectively. Proper correction of the autofluorescence change unmasks the[Ca²⁺]_ielevation caused by 5-Hz pacing. It was concluded that autofluorescence is not constant and that interventions affecting autofluorescence needcorrection if fura 2 is used to report[Ca²⁺]_i.

     

The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.     

Effects of [Ca2+]o on contractility in the anoxic cardiac muscle of mammal and fish

Electrically paced atrial strips of hearts from rat and rainbow trout were exposed to increasing extracellular Ca2+ concentration, [Ca2+]o. This resulted in increases in the peak force in oxygenated atria from both species. During anoxia this response was suppressed for the rat, but accentuated for trout atrium.     

Synaptic transmission at high pressure: Effects of [Ca2+]o

• 1.1. The effects of pressure on synaptic currents were examined in crayfish abdominal muscles.
• 2.2. Helium pressure (10.1 MPa) considerably decreased extracellulariy-recorded excitatory junctional potentials associated with increased short-term facilitation.
• 3.3. These effects could be mimicked by a reduction of [Ca²⁺]_o, and partially compensated by an increase in [Ca²⁺]_o.
• 4.4. Pressure also reduced the amplitude of the extracellular nerve terminal potentials (ENTP) by up to 25%, and significantly increased synaptic delay in a [Ca²⁺]_o-dependent manner.
• 5.5. The interaction between compression and various [Ca²⁺]_o were analysed in terms of an existing model of transmitter release. The results were consistent with the hypothesis that high pressure decreases the maximal Ca²⁺ influx into nerve terminals.
• 6.6. The decreased ENTP and increased synaptic delay suggest that additional processes may be involved in pressure effects on synaptic transmission.

     

Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells

The Ca2+-sensitive photoprotein aequorin and the new green fluorescent protein-based fluorescent Ca2+ indicators 'ratiometric-pericam' were selectively expressed in the mitochondria, cytosol and/or nucleus of spontaneously beating ventricular myocytes from neonatal rats. This combined strategy reveals that mitochondrial [Ca2+] oscillates rapidly and in synchrony with cytosolic and nuclear [Ca2+]. The Ca2+ oscillations were reduced in frequency and/or amplitude by verapamil and carbachol and were enhanced by isoproterenol and elevation of extracellular [Ca2+]. An increased frequency and/or amplitude of cytosolic Ca2+ spikes was rapidly mirrored by similar changes in mitochondrial Ca2+ spikes and more slowly by elevations of the interspike Ca2+ levels. The present data unequivocally demonstrate that in cardiac cells mitochondrial [Ca2+] oscillates synchronously with cytosolic [Ca2+] and that mitochondrial Ca2+ handling rapidly adapts to inotropic or chronotropic inputs.     

Basis for late rise in fura 2R signal reporting [Ca2+]i during relaxation in intact rat ventricular trabeculae

Intact rat ventricular trabeculae were injected with the saltform of fura 2, and the fura 2 ratio signal (R) was used to reportintracellular Ca²⁺ concentration([Ca²⁺]_i).The fixed end relaxation phase of a twitch is associated with a slowingof the decay of the R signal, or even a reversal, to form a distinctbump, indicating a transient rise in[Ca²⁺]_i.The bump is most prominent at 30°C, and motion artifact is not itscause. Increasing doses of 2,3-butanedione monoxime caused progressiveattenuation of the twitch and bump. Increasing the bathingCa²⁺ concentration potentiated thetwitch and enhanced the bump. Imposed muscle shortening duringrelaxation caused a much quicker force decline, and this led to theappearance of a much more prominent associated bump. The amplitude ofthe bump depends on the amplitude of twitch force and the rate ofrelaxation. These findings can be explained, as in skeletal muscle, bymaking cross-bridge attachment andCa²⁺ binding to troponin Cstrongly cooperative; therefore, the bump during fast relaxation isproduced by a reversal of this cooperativity, leading to rapiddissociation of Ca²⁺ from troponinC into the myoplasm.

     

The effect of [Ca2+] and [H+] on the functional recovery of rat brain synaptosomes from anoxic insult in vitro.

The energy status (as measured by the ATP/ADP ratio), oxidative metabolism (14CO2 output) and neurotransmitter synthesis ( [14C]acetylcholine production) by rat brain synaptosomes utilizing [U-14C]glucose has been studied. The ability of anoxia in vitro to permanently alter these parameters was investigated with reference to external [Ca2+] and [H+]. It has previously been shown that anoxic damage to synaptosomal preparations is only apparent when their metabolism is stimulated by veratridine [Harvey, Booth & Clark (1982) Biochem. J. 206, 433-439]. It is concluded that low [Ca2+] ameliorates, and high [H+] exacerbates, the damage sustained by veratridine-stimulated anoxic synaptosomes. The combined effects of low pH, anoxia and veratridine stimulation on synaptosomal metabolism most closely approximated to the irreversible damage to brain metabolism observed during acute hypoxia in vivo [Booth, Harvey & Clark (1983) J. Neurochem. 40, 106-110]. Suitably treated synaptosomal preparations may therefore be usefully employed as models to study impaired neurotransmitter synthesis in vivo.     

FKBP12.6 overexpression decreases Ca2+ spark amplitude but enhances [Ca2+]i transient in rat cardiac myocytes

Ryanodine receptors/Ca2+-release channels (RyR2) from the sarcoplasmic reticulum (SR) provide the Ca2+ required for contraction at each cardiac twitch. RyR2 are regulated by a variety of proteins, including the immunophilin FK506 binding protein (FKBP12.6). FKBP12.6 seems to be important for coupled gating of RyR2 and its deficit and alteration may be involved in heart failure. The role of FKBP12.6 on Ca2+ release has not been analyzed directly, but rather it was inferred from the effects of immunophilins, such us FK506 and rapamycin, which, among other effects, dissociates FKBP12.6 from the RyR2. Here, we investigated directly the effects of FKBP12.6 on local (Ca2+ sparks) and global [intracellular Ca2+ concentration ([Ca2+]i) transients] Ca2+ release in single rat cardiac myocytes. The FKBP12.6 gene was transfected in single myocytes using the adenovirus technique with a reporter gene strategy based on green fluorescent protein (GFP) to check out the success of transfections. Control myocytes were transfected with only GFP (Ad-GFP). Rhod-2 was used as the Ca2+ indicator, and cells were viewed with a confocal microscope. We found that overexpression of FKBP12.6 decreases the occurrence, amplitude, duration, and width of spontaneous Ca2+ sparks. FK506 had diametrically opposed effects. However, overexpression of FKBP12.6 increased the [Ca2+]i transient amplitude and accelerated its decay in field-stimulated cells. The associated cell shortening was increased. SR Ca2+ load, estimated by rapid caffeine application, was increased. In conclusion, FKBP12.6 overexpression decreases spontaneous Ca2+ sparks but increases [Ca2+]i transients, in relation with enhanced SR Ca2+ load, therefore improving excitation-contraction coupling.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of caffeine on the intracellular [Ca2+] transient, membrane currents, and contraction

The effects of caffeine on tension, membrane potential, membrane currents, and intracellular [Ca2+], measured as the light emitted by the Ca2+-activated photoprotein aequorin, were studied in canine cardiac Purkinje fibers. An initial, transient, positive inotropic effect of caffeine was accompanied by a transient increase in the second component of the aequorin signal (L2) but not the first (L1). In the steady state, 4 or 10 mM caffeine always decreased twitch tension and greatly reduced both L1 and L2. At a concentration of 2 mM, caffeine usually reduced but occasionally increased the steady state twitch tension. However, 2 mM caffeine always reduced both L1 and L2. Caffeine eliminated the diastolic oscillations of intracellular [Ca2+] induced by high extracellular [Ca2+]. In voltage-clamp experiments, 10 mM caffeine reduced the transient outward current and the peak tension elicited by step depolarization from a holding potential of -45 mV. In the presence of 20 mM Cs+, 10 mM caffeine reduced slow inward current. However, the time course of this reduction was far slower than that in tension and light observed in separate experiments. The simplest explanation of the results is that caffeine inhibits the sequestration of Ca2+ by the sarcoplasmic reticulum. The results also suggest that in Purkinje fibers caffeine increases the sensitivity of the myofilaments to Ca2+.     

Measurement of sub-membrane [Ca2+] in adult myofibers and cytosolic [Ca2+] in myotubes from normal and mdx mice using the Ca2+ indicator FFP-18

The hypothesis that intracellular Ca(2+) is elevated in dystrophic (mdx) skeletal muscle due to increased Ca(2+) influx is controversial. As the sub-sarcolemmal Ca(2+) ([Ca(2+)](mem)) should be even higher than the global cytosolic Ca(2+) in the presence of increased Ca(2+) influx, we investigated [Ca(2+)](mem) levels in collagenase-isolated adult flexor digitorum brevis (FDB) myofibres and myotubes of mdx and normal mice with the near-membrane Ca(2+) indicator FFP-18. Confocal imaging showed strong localization of FFP-18 to the sarcolemma only. No significant difference in [Ca(2+)](mem) was found in FDB myofibres of normal (77.3+/-3.8 nM, n=68) and mdx (79.3+/-5.6 nM, n=21, p=0.89) mice using FFP-18. Increasing external Ca(2+) to 18 mM did not significantly affect [Ca(2+)](mem) in either the normal or mdx myofibres. In the myotubes, the FFP-18 was non-selectively incorporated, distributing throughout the cytoplasm, and FFP-18-derived [Ca(2+)] values were similar to values obtained with Fura-2. Nevertheless, in the mdx myotubes, the [Ca(2+)] measured with FFP-18 increased linearly to a level approximately 2.75 times that of controls as the time of culture was prolonged. In older mdx myotubes (>or=8 days in culture), 18 mM extracellular Ca(2+) increased the steady state cytosolic [Ca(2+)] to approximately 22 times greater level than controls. This study suggests that the sub-sarcolemmal Ca(2+) homeostasis is well maintained in isolated adult mdx myofibers and also further supports the hypothesis that cytosolic Ca(2+) handling is compromised in mdx myotubes.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of cardiotonic steroids on the intracellular [Ca2+] transient, membrane potential, and contraction

The [Ca2+]-activated photoprotein aequorin was used to measure [Ca2+] in canine cardiac Purkinje fibers during the positive inotropic and toxic effects of ouabain, strophanthidin, and acetylstrophanthidin. The positive inotropic effect of these substances was associated with increases in the two components of the aequorin signal, L1 and L2. On the average, strophanthidin at 10(-7) M produced steady, reversible increases in L1, L2, and peak twitch tension of 20, 91, and 240%, respectively. This corresponds to increases in the upper-limit spatial average [Ca2+] from 1.9 X 10(-6) M to 2.1 X 10(-6) M at L1 and from 1.4 X 10(-6) M to 1.8 X 10(-6) M at L2. Elevation of diastolic luminescence above the control level was not detected. At higher concentrations (5 X 10(-7) M), strophanthidin produced aftercontractions, diastolic depolarization, and transient depolarizations, all of which were associated with temporally similar changes in [Ca2+]. During these events, diastolic [Ca2+] rose from the normal level of approximately 3 X 10(-7) M up to 1-2 X 10(-6) M. The negative inotropic effect of 5 X 10(-7) M strophanthidin was not associated with a corresponding decrease in the [Ca2+] transient but was associated with a change in the relationship between [Ca2+] and tension. Assuming the Na+-lag mechanism of cardiotonic steroid action, we conclude the following: at low concentrations of drug, increased Ca2+ uptake by the sarcoplasmic reticulum prevents a detectable rise in cytoplasmic [Ca2+] during diastole, but this increased Ca2+ uptake results in increased release of Ca2+ during the action potential. At higher drug concentrations, observable [Ca2+] changes during diastole activate tension and membrane conductance changes.     

Impaired Orai1-mediated resting Ca2+ entry reduces the cytosolic [Ca2+] and sarcoplasmic reticulum Ca2+ loading in quiescent junctophilin 1 knock-out myotubes

In the absence of store depletion, plasmalemmal Ca(2+) permeability in resting muscle is very low, and its contribution in the maintenance of Ca(2+) homeostasis at rest has not been studied in detail. Junctophilin 1 knock-out myotubes (JP1 KO) have a severe reduction in store-operated Ca(2+) entry, presumably caused by physical alteration of the sarcoplasmic reticulum (SR) and T-tubule junction, leading to disruption of the SR signal sent by Stim1 to activate Orai1. Using JP1 KO myotubes as a model, we assessed the contribution of the Orai1-mediated Ca(2+) entry pathway on overall Ca(2+) homeostasis at rest with no store depletion. JP1 KO myotubes have decreased Ca(2+) entry, [Ca(2+)](rest), and intracellular Ca(2+) content compared with WT myotubes and unlike WT myotubes, are refractory to BTP2, a Ca(2+) entry blocker. JP1 KO myotubes show down-regulation of Orai1 and Stim1 proteins, suggesting that this pathway may be important in the control of resting Ca(2+) homeostasis. WT myotubes stably transduced with Orai1(E190Q) had similar alterations in their resting Ca(2+) homeostasis as JP1 KO myotubes and were also unresponsive to BTP2. JP1 KO cells show decreased expression of TRPC1 and -3 but overexpress TRPC4 and -6; on the other hand, the TRPC expression profile in Orai1(E190Q) myotubes was comparable with WT. These data suggest that an important fraction of resting plasmalemmal Ca(2+) permeability is mediated by the Orai1 pathway, which contributes to the control of [Ca(2+)](rest) and resting Ca(2+) stores and that this pathway is defective in JP1 KO myotubes.     

Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.     

Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.     

The significance of physiological [Ca2+] and [Mg2+] for in vitro experiments on synaptic transmission

Since 1932 $\text{in}$$\text{vitro}$ physiological and pharmacological studies on neuromuscular and other types of synaptic transmission have been carried out usually in Krebs' of similar balanced electrolyte solutions. It has been disregarded, however, that although the total calcium [Ca_t] (2.5 mM) and [Mg_t] (1.2 mM), are about the same in human plasma and in Krebs' solution, the physiologically important [Ca²⁺] and [Mg²⁺], primarily because of binding to plasma proteins, are much lower in plasma (1.1 and 0.6 mM) than in Krebs' solution (2.0 and 1.1 mM). We observed that in a modified Krebs' solution in which the [Ca_t] and [Mg_t] are 1.4 and 0.9 mM respectively and the [Ca²⁺] and [Mg²⁺] are about the same as in human plasma, the Ca²⁺ dependent volley output of acetylcholine is less and the inhibition of the electrically induced isometric twitch tension of the rat phrenic nerve - hemidiaphragm preparation by nondepolarizing neuromuscular blocking agents and certain antibiotics is greater than in conventional Krebs' solution, in which the [Ca²⁺] and [Mg²⁺] are higher than $\text{in}$$\text{vivo}$. Similarly, during electrical field stimulation of the guinea-pig myenteric plexus - longitudinal muscle preparation volley output of acetylcholine is lower and the inhibition of the isometric contraction of the muscle by normophine is greater in modified than in conventional Krebs' solution. It is suggested that for greater relevance to $\text{in}$$\text{vivo}$ conditions the [Ca²⁺] and [Mg²⁺] of balanced electrolyte solutions used in in vitro experiments on synaptic transmission should be the same as in human plasma or in the plasma of the species of the experimental animal.     

Stretch and quick release of rat cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions

Rapid shortening of active cardiac muscle [quick release (QR)] dissociates Ca2+ from myofilaments. We studied, using muscle stretches and QR, whether Ca2+ dissociation affects triggered propagated contractions (TPCs) and Ca2+ waves. The intracellular Ca2+ concentration was measured by a SIT camera in right ventricular trabeculae dissected from rat hearts loaded with fura 2 salt, force was measured by a silicon strain gauge, and sarcomere length was measured by laser diffraction while a servomotor controlled muscle length. TPCs (n = 27) were induced at 28 degrees C by stimulus trains (7.5 s at 2.65 +/- 0.13 Hz) at an extracellular Ca2+ concentration ([Ca2+]o) = 2.0 mM or with 10 microM Gd3+ at [Ca2+]o = 5.2 +/- 0.73 mM. QR during twitch relaxation after a 10% stretch for 100-200 ms reduced both the time between the last stimulus and the peak TPC (PeakTPC) and the time between the last stimulus and peak Ca2+ wave (PeakCW) and increased PeakTPC and PeakCW (n = 13) as well as the propagation velocity (Vprop; n = 8). Active force during stretch also increased Vprop (r = 0.84, n = 12, P < 0.01), but Gd3+ had no effect (n = 5). These results suggest that Ca2+ dissociation by QR during relaxation accelerates the initiation and propagation of Ca2+ waves.