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The Shiels Laboratory

Excitation-contraction coupling in mammalian cardiomyocytes

We are investigating how electrical activity triggers contraction of cardiac muscle; this process is called excitation-contraction coupling. At each beat, an action potential leads to Ca entry into the cell mainly via L-type Ca channels. This Ca influx across the cell membrane triggers the release of more Ca from the sarcoplasmic reticulum (SR) by activating ryanodine receptors (RyRs) in the adjacent SR membrane (via the mechanism of Ca-induced Ca release). The rise of intracellular Ca that activates the contractile proteins - the systolic Ca transient – is the spatial and temporal sum of such local releases.

Relaxation is brought about by removal of Ca from the cell cytoplasm by 2 main routes: the SR Ca ATPase (SERCA), which is regulated by phospholamban (PLB), pumps Ca back into the SR, while Na/Ca exchange (NCX) uses the inwardly directed electrochemical gradient for Na to extrude Ca from the cell. The fraction of Ca removed from the cytoplasm by each of these pathways depends on their relative activities, although in the steady state the amount of Ca extruded from the cell must equal that entering during each beat. These events are summarized in the movie below.

 

We are particularly interested in the role and regulation of L-type Ca channels (CaV1.2 or a1C).

Cartoon showing the predicted structure of the major cardiac calcium channel (CaV1.2 or a1C and the auxiliary subunits).

Cartoon showing the predicted structure of the major cardiac calcium channel (CaV1.2 or a1C and the auxiliary subunits).
From Brette et al. (2006) Progress in Biophysics and Molecular Biology, 91(1-2), 1-82.

 

Ca as a charge carrier will depolarize the cell but also, as a second messenger, will trigger a larger release of Ca from the sarcoplasmic reticulum. Using electrophysiological recording techniques, we have shown that ICa is highly regulated by the Ca release from the SR (negative feedback). We are currently investigating how Ca, voltage and inotropic agents modulate the activity of ICa.

ICa recorded using various voltage conditions in a control myocyte (middle panel) and a myocyte where the t-tubule system has been disabled (lower panel).

ICa recorded using various voltage conditions in a control myocyte (middle panel) and a myocyte where the t-tubule system has been disabled (lower panel).
Modified from Brette et al. (2006) Biophysical Journal, 90(1), 381-389.

 

In collaboration with Prof Orchard (University of Bristol), we are also interested in the role of t-tubules (invaginations of the surface membrane of cardiac ventricular myocytes). Using electrophysiological techniques and confocal microscopy, we have shown that the t-tubules are the primary place for Ca flux in mammalian ventricular myocytes.

 When Science meets Art: confocal images of Ca variation in a control (left) and detubulated (right) ventricular myocyte. There is a striking resemblance with paintings from Mark Rothko, signature style which is characterized by floating rectangles aligned vertically against a colored ground (link to the NGA: (http://www.nga.gov/feature/rothko/rothkosplash.shtm)

When Science meets Art: confocal images of Ca variation in a control (left) and detubulated (right) ventricular myocyte. There is a striking resemblance with paintings from Mark Rothko, signature style which is characterized by floating rectangles aligned vertically against a colored ground (link to the NGA: (http://www.nga.gov/feature/rothko/rothkosplash.shtm)
Brette, unpublished data

 

Selected publications

Calcium current

Brette F., Sallé L. & Orchard C.H. (2006) Quantification of calcium entry at the t-tubules and surface membrane in rat ventricular myocytes. Biophysical Journal, 90(1), 381-389.

Brette F., Leroy J., Le Guennec J-Y. & Sallé L. (2006) Ca2+ currents in cardiac myocytes: old story, new insights. Progress in Biophysics and Molecular Biology, 91(1-2), 1-82. (review)

Brette F., Sallé L. & C.H. Orchard. (2004) Differential modulation of L-type Ca2+ current by SR Ca2+ release at the T-tubules and surface membrane of rat ventricular myocytes. Circulation Research, 95, e1-e7. (UltraRapid Communication).

Brette F., Lacampagne A., Sallé L., Findlay I. & Le Guennec J-Y. (2003) Intracellular Cs+ activates the PKA pathway revealing a fast reversible Ca2+ dependent inactivation of L-type Ca2+ current. American Journal of Physiology, 285(2), C310-C318.

Transverse-tubules

Brette F. and Orchard C.H. (2007) Resurgence of cardiac t-tubule research. Physiology, 22, 167-173. (review).

Brette F., Despa S., Bers D.M. & Orchard C.H. (2005) Spatiotemporal characteristics of SR Ca2+ uptake and release in detubulated rat ventricular myocytes. Journal of Molecular and Cellular Cardiology, 39(5), 831-839. An art picture of this article was chosen as the cover of this issue.

Despa S.*, Brette F.*, Orchard C.H. & Bers D.M. (2003) Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes. Biophysical Journal, 85(5), 3388-3396. (*Joint First co-author).

Brette F., Komukai K. & Orchard C.H. (2002) Validation of the use of formamide as a detubulation agent in isolated rat cardiac cells. American Journal of Physiology, 283(4), H1720-1728.

Other research interests

Brette F.,Luxan G, Cros C., Dixey H., Wilson C & Shiels H.A. (2008) Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio). Biochemical and Biophysical Research Communications, 374(1), 143-146.

Sallé L., Kharche S., Zhang H. & Brette F. (2008) Mechanisms underlying adaptation of action potential duration by pacing rate in rat myocytes. Progress in Biophysics and Molecular Biology, 96(1-3), 305-320.

Brette F. and Orchard C.H. (2006) No apparent requirement for neuronal sodium channels in excitation-contraction coupling in rat ventricular myocytes. Circulation Research, 98(5), 667-674. An art picture of this article was chosen as the cover of this issue.

Brette F., Calaghan S.C., Lappin S., White E., Colyer J. & Le Guennec J-Y. (2000) Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes. American Journal of Physiology, 279(4), H1963-H1971.