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Altered mechanical properties and intracellular calcium signaling in cardiomyocytes from annexin 6 null-mutant mice¹

GUOJIE SONG^*, SIAN E. HARDING, MICHAEL R. DUCHEN, RICHARD TUNWELL, PETER O’GARA, TIM E. HAWKINS and STEPHEN E. MOSS^*2

^* Division of Cell Biology, Institute of Ophthalmology, University College London, London EC1V 9EL, UK;
Cardiac Medicine, NHLI, Imperial College School of Medicine, London SW3 6LY, UK; and
Department of Physiology, University College London, London WC1E 6BT, UK

²Correspondence: Division of Cell Biology, Institute of Ophthalmology, University College London, 11–43 Bath St., London EC1V 9EL, UK. E-mail: s.moss{at}ucl.ac.uk

SPECIFIC AIM

The aim of this work was to investigate the role of annexin 6 in stimulus-response coupling in cardiomyocytes. Using mice containing a targeted disruption of the annexin 6 gene, we demonstrate that loss of annexin 6 leads to a significant increase in the rates of shortening and relengthening accompanied by faster diastolic Ca²⁺ removal from the cytoplasm.

PRINCIPAL FINDINGS

1. Loss of annexin 6 leads to an increase in contraction-relaxation dynamics in isolated cardiomyocytes
To examine the role of annexin 6 in cardiac function, we used isolated myocytes from mice containing a targeted disruption of the annexin 6 gene and compared them with cells from wild-type (WT) control mice at 12 wk of age. Morphological investigation of isolated ventricular myocytes revealed that cell lengths were not significantly different between annexin 6^-/- mice [133 µm±5, mean±SE, n=6 animals (133 cells)] and controls [122 µm±3, n=6 animals (132 cells)]. Contraction was monitored at a calcium concentration of 1 mM and a steady-state stimulation rate of 1 Hz. Figure 1 A shows averaged data from 6 pairs of mice, where amplitudes obtained from up to 30 cells for each mouse have been pooled to give a single value per animal. Steady-state amplitude was increased by 83% in the annexin 6^-/- mice (P<0.01). Mouse ventricular myocytes have a strong post-rest potentiation of contraction and amplitude after 1 min of rest is close to maximum. Figure 1A shows the amplitude of the first beat after 1 min rest (PR B1), which also increased, in this case by 53% (P<0.01). As is normal for this species, spontaneous contractions were observed during rest in a proportion of myocytes. Of 111 myocytes from control animals, 42 contracted spontaneously during rest vs. 68 of 121 for annexin 6^-/- mice (P<0.005, ² test). The average frequency of spontaneous contraction was 8.8/min for control and 6.6/min for annexin 6^-/- mice, although these figures were variable. In cells where contraction occurred during the rest period, it is possible that post-rest amplitude was submaximal. This may explain why the increase in amplitude in annexin 6^-/- mice was slightly lower for B1 than steady-state values and the variation slightly greater. Velocities of contraction (-dL/dT) and relaxation (+dL/dT) also increased significantly (Fig. 1B ), but largely in proportion to the increase in amplitude. Consistent with this, time-to-peak contraction (TTP) and time-to-50% relaxation (R50) were unaffected (Fig. 1C ). However, the late phase of relaxation, defined by the time-to-90% relaxation (R90), was significantly accelerated in the annexin 6^-/- animals.

View larger version (23K): [in this window] [in a new window]   Figure 1. Altered contractile dynamics in cardiomyocytes lacking annexin 6. Ventricular myocytes were isolated by enzymatic dissociation, placed into a Perspex cell bath on the stage of an inverted microscope, and stimulated at a basal rate of 1 Hz by bipolar pulses through platinum electrodes placed along the bath. After electrical stimulation for at least 10 min at 1 mM Ca2+, contraction was measured in up to 32 cells from each preparation. Each myocyte was allowed a 1 min rest period, and the amplitude of the first post-rest beat was recorded. A) Contraction amplitude of isolated ventricular myocytes (% shortening) at steady-state 1 Hz (SS) and after 1 min rest (PR B1). B) Contraction (-dL/dT) and relaxation (dL/dT) velocities at 1 Hz. C) Times to peak contraction (TTP) to 50% relaxation (R50) and to 90% relaxation (R90). Mean ± SE, n = 6 animals, *P < 0.05, **P < 0.01 vs. control.

2. Loss of annexin 6 leads to accelerated calcium clearance after caffeine stimulation
To investigate whether the changes in contractility in the annexin 6 null mutant animals could be explained by alterations in Ca²⁺ handling, we loaded isolated cardiomyocytes with Fura-2 AM and performed ratiometric analysis of Ca²⁺ fluxes during the contraction-relaxation cycle (Fig. 2 ). The diastolic intracellular Ca²⁺ level, shown as the baseline [Ca²⁺] signal, in myocytes isolated from annexin 6^-/- mice was identical to that in controls under our experimental conditions (0.96±0.04 vs. 0.95±0.04, KO vs. WT). The amplitude of the [Ca²⁺]_c signal in response to emptying SR stores with caffeine reached similar levels in the two groups (the increases in Fura-2 ratio (DR) were 0.51 ± 0.198 vs. 0.56 ± 0.15, KO vs. WT, n = 15 and 13, respectively). There was no significant difference in the TTP of the calcium signal in the two experimental groups, demonstrating that the velocity of calcium release from the SR to cytoplasm is similar under these experimental conditions. However, the rate of recovery of the calcium signal was significantly faster in cells from annexin 6^-/- mice, with a mean time constant of decay some twofold faster than the controls (time constant of 5.13±0.49 (n=15, SE) vs. 10.35 ± 0.79 s^-1 (n=13) for KO vs. WT, P < 0.05). The difference between the two genotypes was also observed at the 90% decline point of the Ca²⁺ signal (T90) and the 50% decline point (T50). To examine whether the anomalies in calcium handling might be explained by changes in the levels of expression of major Ca²⁺ regulatory proteins, we immunoblotted cardiomyocyte extracts for ryanodine receptors and SERCA pumps (Fig. 2D ). The use of extracts pooled from several mice in each case (n=6) provides an averaged but nonquantitative assessment of protein expression, revealing in this case no obvious up- or down-regulation in expression of the proteins under investigation. Thus, the accelerated clearance of cytosolic Ca²⁺ cannot be explained by simple elevation of expression of extrusive intracellular Ca²⁺ pumps.

View larger version (23K): [in this window] [in a new window]   Figure 2. Statistical analysis of cardiomyocyte intracellular Ca2+ dynamics. Cells were loaded with Fura-2 by incubation with 5 µM Fura-2 AM and 0.02% pluronic for 30 min at room temperature and imaged using an epifluorescence microscope equipped with a photomultiplier tube. Caffeine (2 mM) was applied locally to Fura-2-loaded cells to release SR Ca2+ and so explore the dynamics of [Ca2+]c signaling (indicated by the bar). Analysis of the resulting [Ca2+]c transients revealed that the mean baseline, time-to-peak, and peak amplitudes were not significantly different in the two populations of cells. However, the time constant of recovery to baseline was significantly shorter in the annexin 6-/- cells. Examples of individual responses from control (A) and knockout. C) Summary of data (x axis indicates units in Fura-2 ratio for baseline and peak signals and s-1 for the time constant . D) Immunoblots of cardiomyocyte microsomal extracts from control (+/+) and knockout mice (-/-) probed with antibodies specific to protein components of the Ca2+ regulating system.

CONCLUSION

In considering the mechanistic effects of annexin 6 in the light of published data, one might predict that the absence of annexin 6 in myocytes would lead to a decrease in the opening probability and the mean open time of SR ryanodine-sensitive calcium release channels. Yet the kinetics and amplitude of Ca²⁺ release were normal in the mutant cells. Alternatively, loss of annexin 6 in myocytes may influence Na⁺/Ca²⁺ exchanger activity, which would be consistent with published work showing that annexin 6 is a potent modulator of the Na⁺/Ca²⁺ exchange activity of cardiac sarcolemma vesicles. A role for annexin 6 in this context would be expected to lead to augmentation of Ca²⁺ efflux and uptake (Fig. 3 ), as was indeed observed. Further investigations using annexin 6 antibody or inhibitors of cardiomyocyte Ca²⁺ channels, pumps, and exchangers are required to determine the specific target(s) of annexin 6 responsible for the regulation of intracellular Ca²⁺.

View larger version (37K): [in this window] [in a new window]   Figure 3. Regulation of intracellular Ca2+ dynamics by annexin 6. The model depicts the established cycle of cardiac myofilament contraction and relaxation. Cytosolic Ca2+ derived from both the extracellular milieu and sarcoplasmic reticulum (SR) stores stimulates contraction of actomyosin, followed by rapid clearance of Ca2+ via energy-dependent Ca2+ pumps on the SR and the plasma membrane Na+/Ca2+ exchanger. The accelerated removal of cytosolic Ca2+ in annexin 6 null cardiomyocytes suggests that annexin 6 might act as a regulator of the Na+/Ca2+ exchanger and/or SR Ca2+-ATPase.

Cardiomyocytes isolated from transgenic mice with cardiac-specific overexpression of annexin 6 display impaired myocyte contractility with depression of intracellular Ca²⁺ transients. Consistent with this, annexin 6 mRNA and protein levels were found to be decreased in end-stage congestive heart failure in humans, whereas expression levels of annexins 2 and 5 were increased. These findings, together with those reported here, indicate that annexin 6 has the potential to influence ion fluxes vital to heart function. In conclusion, we have identified an important role for annexin 6 in excitation-contraction coupling in mouse cardiomyocytes. Loss of annexin 6 does not affect intracellular calcium levels, but the temporal anomalies in Ca²⁺ clearance and the amplified contractile properties suggest a negative inotropic role for annexin 6 in regulating intracellular calcium and cardiomyocyte mechanical function.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0892fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0892fje

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This Article Full Text (PDF) All Versions of this Article: 16/6/622 01-0892fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SONG, G. Articles by MOSS, S. E. Search for Related Content PubMed PubMed Citation Articles by SONG, G. Articles by MOSS, S. E.