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First published online January 14, 2005
doi: 10.1242/10.1242/jcs.01614

Journal of Cell Science 118, 421-431 (2005)
Published by The Company of Biologists 2005
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Distinct subcellular location of the Ca2+-binding protein S100A1 differentially modulates Ca2+-cycling in ventricular rat cardiomyocytes

Patrick Most1,2,*, Melanie Boerries3,*, Carmen Eicher2,*, Christopher Schweda2, Mirko Völkers2, Thilo Wedel4, Stefan Söllner5, Hugo A. Katus2, Andrew Remppis2, Ueli Aebi3, Walter J. Koch1 and Cora-Ann Schoenenberger3,{ddagger}

1 Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
2 Department of Internal Medicine III, Division of Cardiology, University of Heidelberg, 69115 Heidelberg, Germany
3 Maurice E. Mueller Institute, Biozentrum, University of Basel, 4056 Basel, Switzerland
4 Institute of Anatomy, University of Lübeck, 23538 Lübeck, Germany
5 Department of Plastic Surgery, University of Lübeck, 23538 Lübeck, Germany



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  		Fig. 1. Subcellular location of overexpressed, endogenous and internalized S100A1. (A) Overexpressed S100A1 (AdS100A1; blue) is detected by an antibody that specifically reacts with S100A1. Western blot analysis comparing levels of S100A1 (upper) and cardiac actin (lower) expression in homogenates of Adcontrol and AdS100A1-transduced NVCMs. (B) Immunostaining of endogenous S100A1 (green) in untreated NVCMs reveals a fine granular network-like pattern (control). (C) Vesicular accumulation of internalized Rh-S100A1 protein (red) in the perinuclear region and in the cytosol of S100A1-treated cells. Western blot analysis comparing levels of S100A1 (upper) and cardiac actin (lower) in homogenates from S100A1-treated and control NVCMs. (D-F) AdS100A1-transduced cells were incubated for 1 hour with Rh-S100A1 24 hours post infection, and then immunolabeled with an antibody that specifically reacts with human S100A1. (D) The anti-human S100A1/Cy5-anti-rabbit antibody detects internalized Rh-S100A1 as well as overexpressed S100A1 protein (total exogenous S100A1). (E) Vesicular accumulation of internalized Rh-S100A1 protein in the perinuclear region and in the cytosol. (F) Overlay of both D and F shows that internalized Rh-S100A1 (violet) exhibits a distinct distribution compared with overexpressed S100A1 (blue). Bar, 10 µm.

 


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  		Fig. 2. Overexpressed and internalized S100A1 enhance cytosolic Ca2+-turnover in neonatal ventricular cardiomyocytes. (A-D) Adenoviral-transduced NVCMs overexpressing S100A1. (A) Superimposed tracings of calibrated Ca2+-transients in S100A1-overexpressing (AdS100A1, solid line) and control (Adcontrol, dashed line) NVCMs. Note the gain in systolic [Ca2+]i in S100A1-overexpressing NVCMs (bar). (B-D) Effects of increased S100A1 protein level on Ca2+-transients. Compared with control cells expressing endogenous levels of S100A1, S100A1 overexpression significantly increases the Ca2+-transient amplitude (AdS100A1 454±22 nM vs. Adcontrol 311±11 nM; 1B), lowers diastolic [Ca2+]i (AdS100A1 181±14 nM vs. Adcontrol 219±12 nM; 1C), and accelerates the decay of the Ca2+-transient ({tau}, AdS100A1 172±14 ms vs. Adcontrol 223±11 ms; 1D). n=150 cells from three different cell preparations. Data are given as mean±s.e.m. (A'-D') NVCMs treated with 1 µM human recombinant S100A1. (A') Superimposed tracings of calibrated Ca2+-transients in S100A1-treated (S100A1, solid line) and mock-treated (control, dashed line) NVCMs. The decrease in diastolic [Ca2+]i in S100A1-treated NVCMs is indicated by the bar. (B') S100A1-uptake increases the Ca2+-transient amplitude (S100A1-treated 442±23 nM vs. control 324±15 nM). (C') The effects of an increased S100A1 level are a reduction of diastolic [Ca2+]i (S100A1-treated 143±13 nM vs. control 236±14 nM) and (D') an accelerated decay of the Ca2+-transient ({tau}, S100A1-treated 182±16 ms vs. control 232±10 ms). Pre-incubation with monodansylcadaverine (MDC, an inhibitor of clathrin-mediated endocytosis) abolished the effects of extracellularly added S100A1 on the Ca2+-turnover (* P<0.01 vs. no inhibitor).

 


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  		Fig. 3. Overexpressed but not internalized S100A1 colocalizes with SERCA2a at the SR. Immunolocalization of S100A1 and SERCA2a. (A) Endogenous S100A1 labeled with a monoclonal anti-S100A1/Cy5-anti-mouse antibody in control cells. (A') Overexpressed S100A1 detected with a polyclonal anti-human S100A1/Cy5-anti-rabbit antibody in AdS100A1-transduced NVCMs. (A") Internalized Rh-S100A1. (B) Immunolabeling of SERCA2a in control, (B') AdS100A1-infected, (B") and Rh-S100A1-treated NVCMs. (C) Overlay of A and B, and C', A' and B' depicts colocalization of endogenous and overexpressed S100A1 with SERCA2a (violet). (C") Internalized Rh-S100A1 does not colocalize. Bar, 10 µm (C') and 5 µm (C,C").

 


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  		Fig. 4. Inhibition of SR Ca2+-fluxes abolishes enhanced Ca2+-cycling in S100A1-overexpressing but not in S100A1-treated NVCMs. (A) Addition of the SERCA2a inhibitor thapsigargin (1 µM) abolishes the S100A1-induced increase in the Ca2+-transient amplitude in overexpressing (AdS100A1) but not S100A1-treated NVCMs. (B) Thapsigargin reduces diastolic [Ca2+]i in control and S100A1-overexpressing cells, whereas S100A1-treated cells are not significantly affected. (C) SERCA2a inhibition raises the decay-constant {tau} in control and AdS100A1-infected NVCMs but does not significantly alter {tau} in S100A1-treated NVCMs. (D) Tetracaine (100 µM), an inhibitor of the SR Ca2+-release channel (RyR), abrogates the increased Ca2+-transient amplitude in S100A1-overexpressing NVCMs but does not significantly reduce the augmented amplitude of the Ca2+-transient in S100A1-treated NVCMs relative to tetracaine-treated control cells. *P<0.01 vs. control. Measurements represent n=150 cells from three independent cell preparations. Data are given as mean±s.e.m.

 


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  		Fig. 5. Sarcolemmal Ca2+-regulatory proteins modulate Ca2+-cycling in S100A1-treated NVCMs. (A) Caffeine-induced Ca2+-transient amplitudes reveal that overexpressed S100A1 (AdS100A1) enhances SR Ca2+-content, whereas SR Ca2+-levels are reduced in S100A1-treated cells compared with control cells exposed to caffeine. (B) The elimination of caffeine-releasable SR Ca2+ indicated by the decay-constant {tau} is similar in caffeine-treated control cells and S100A1-overexpressing cells. However, the decay of the Ca2+-transient is accelerated in S100A1-treated NVCMs compared with control cells. (C) Switching to sodium/calcium-free medium abrogates decay acceleration in S100A1-treated cells. *P<0.01 vs. control. n=50 cells in AdS100A1 and S100A1-treated cells, control (mock- and Adcontrol-treated NVCMs) n=100 cells, cells from each group were obtained from three independent cell preparations. Data are given as mean±s.e.m.

 


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  		Fig. 6. Ca2+-cycling in S100A1-treated NVCMs is regulated via PLC-PKC activation of NCX. (A) Inhibition of NCX activity by myr-FRCRCFa (30 µM) abrogated the S100A1-mediated decline in diastolic [Ca2+]i in NVCMs. (B,C) The S100A1-mediated decrease in diastolic [Ca2+]i is abolished trough inhibition of PLC and PK C by U-73122 and calphostin-c, respectively. *P<0.01 vs. control. Measurements represent n=50 cells from three independent cell preparations. Data are given as mean±s.e.m.

 


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  		Fig. 7. Increased level of S100A1 does not alter expression of Ca2+-regulatory proteins. Western blots of extracts from control, S100A1-treated, AdS100A1- and Adcontrol-transduced cells probed with specific antibodies. Levels of NCX and SERCA2a remain constant despite the increased levels of S100A1.

 


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  		Fig. 8. Differential effects of endocytosed and overexpressed S100A1 on sarcolemmal and sarcoplasmic Ca2+-cycling. (A) Simplified scheme of intracellular Ca2+-fluxes in a ventricular cardiomyocyte. Endogenous S100A1 (hatched oval) is located at the SR where it interacts with SERCA2a and RyR2. (1) Electrical depolarization of the transverse tubule membrane (T-tubulus) activates inward Ca2+-flux through L-type voltage-gated Ca2+-channels (LCC), which (2) triggers the release of Ca2+ from SR stores via ryandine receptors (RyR2). As a result, contractile filaments are activated. (3) For relaxation to occur, the cytosolic [Ca2+]i must decline. This process is mainly mediated by the SR Ca2+-ATPase (SERCA2a), which resequesters cytosolic Ca2+ in the SR. (4) At the sarcolemma, Ca2+ is extruded primarily via the sodium-calcium exchanger (NCX). (B) Extracellularly added S100A1 is internalized and subsequently routed to the endosomal compartment, where it increases the activity of PLC and PKC (both associated with the endosomal compartment). Through activation of this signaling pathway, internalized S100A1 eventually modulates intracellular Ca2+-flux through an enhanced sarcolemmal Ca2+-extrusion via NCX. The increased Ca2+-extrusion leads to a decreased SR Ca2+-load. (C) Effects of overexpressed S100A1 on the intracellular Ca2+-cycling. Overexpressed S100A1 is located at the SR, where it associates with the SR-regulatory proteins SERCA2a and RyR2. As a result, intracellular Ca2+-cycling is enhanced leading to an increased SR Ca2+-uptake and SR Ca2+-load. This, in turn, gives rise to an enhanced Ca2+-induced SR Ca2+-release (CICR).

 

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© The Company of Biologists Ltd 2005