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Complex regulation of ryanodine receptors by calsequestrin in cardiac muscle: the effect of doxorubicin

N.A. Beard,1 A.D. Hanna,2 M. Janzcura,1 C. Thekkedam,2 H. Willemse1 and A.F. Dulhunty,2 1Health Research Institute, The University of Canberra, Bruce, ACT 2614, Australia and 2John Curtin School of Medical Research, The Australian National University, Canberra, ACT 0200, Australia.

Ca2+ release from the internal sarcoplasmic reticulum (SR) Ca2+ store triggers muscle contraction and is an integral part of the excitation-contraction coupling process. Ca2+ flows through the SR Ca2+ release channel - the ryanodine receptor (RyR) - which forms a complex with three proteins within the SR lumen; calsequestrin (CSQ; the main SR Ca2+ binding protein) and triadin and junctin (which anchor CSQ to the RyR). In addition to providing a pool of Ca2+ for release through the RyR, CSQ acts as a luminal Ca2+ sensor for the channel, a role that is integral for stable RyR function. CSQ regulates the channel to ensure both robust Ca2+ release during systole, and minimal diastolic RyR2 leak (Dulhunty et al., 2012). Several therapeutic agents bind to CSQ and modify its structure in cardiac and skeletal muscle (Subra et al., 2012), namely the anti-psychotic agent trifluoperazine, the cholesterol lowering drugs statins and importantly doxorubicin, a chemotherapy drug used to treat breast cancer. Our aim was to determine how the binding of potent doxorubicin metabolite doxorubicinol (doxOL) to cardiac CSQ (CSQ2) impacts cardiac RyR (RyR2) function.

Sheep hearts were excised from anaesthetized ewes (5% pentobartitone (IV) then oxygen/hatothane), with SR prepared on a discontinuous sucrose gradient and CSQ2 purified by native preparative gel electrophoresis (Hanna et al., 2011). SR vesicles (containing RyR2 channels) were reconstituted into artificial planar lipid bilayers that separate two chambers that are equivalent to the cytoplasmic and SR luminal compartments of the myocytes. The impact of doxOL on CSQ2’s ability to bind Ca2+ was determined using a 45Ca2+ binding assay (Wium et al., 2012) and on formation of Ca2+-induced polymers (required for protein function) was tracked using a CSQ2 turbidity assay (Wium et al., 2012).

CSQ’s role as a luminal Ca2+ sensor for RyR2 is due to its ability to bind Ca2+, and to its Ca2+ dependent polymer structure, which promotes association with the channel. DoxOL (2.5μM) significantly reduced CSQ2’s ability to bind Ca2+, leading to a near 50% loss in the number of Ca2+ ions bound to the protein. In addition, the ability of CSQ2 to assemble into aggregated polymers was significantly impeded by 2.5μM doxOL in a time-dependent manner, which was reflected by a significant loss of CSQ2 association with the RyR2 complex over time. These data illustrate that doxOL alters both the Ca2+ binding capacity of CSQ2, and that long term doxOL exposure would depolymerize CSQ2, resulting in only monomers and dimers of CSQ2 attached to the RyR complex.

In lipid bilayers, native RyR2 activity increased as luminal Ca2+ was raised from 0.1mM to 1.5mM, which would allow robust release of Ca2+ during systole. Pre-treatment with 2.5μM doxOL completely abolished RyR2 sensitivity to changes in luminal [Ca2+]. To determine whether doxOL binding to CSQ2 was responsible for the loss of Ca2+ sensitivity, CSQ2 was selectively dissociated from the native RyR2 by high Cs+ prior to incubation with or without doxOL. The luminal Ca2+ response curve from CSQ2-dissociated RyR2 was identical with and without doxOL, and was in stark contrast to the lack of Ca2+ sensitivity in doxOL treated native RyR2. Together, these data provide compelling evidence that doxOL binding to CSQ2 mediates the loss of RyR2 luminal Ca2+ sensitivity, and that long term, cellular doxOL accumulation would disrupt CSQ2 polymer structure, undoubtedly leading to an additional dysfunction in RyR2 regulation.

Dulhunty AF, Wium E, Li L, Hanna AD, Mirza S, Talukder S, Ghazali NA & Beard NA (2012). Clin Exp Pharmacol Physiol 39, 477-484.

Hanna AD, Janczura M, Cho E, Dulhunty AF & Beard NA (2011.) Mol Pharmacol 80, 538-549.

Subra AK, Nissen MS, Lewis KM, Muralidharan AK, Sanchez EJ, Milting H & Kang CH (2012). Int J Mol Sci 13, 14326-14343.

Wium E, Dulhunty AF & Beard NA (2012). PLoS One 7, e43817.