Original article Evidence for regulation of mitochondrial function by the L-type Ca 2+ channel in ventricular myocytes Helena M. Viola, Peter G. Arthur, Livia C. Hool ⁎ a School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Australia b The Western Australian Institute for Medical Research, Australia abstract article info Article history: Received 13 November 2008 Received in revised form 12 December 2008 Accepted 16 December 2008 Available online 7 January 2009 Keywords: Ca 2+ channel Mitochondria Calcium Cytoskeleton Mitochondrial membrane potential Mitochondrial NADH Superoxide The L-type Ca 2+ channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca 2+ channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(-) or voltage-clamp of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calcium- dependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltage- clamp of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the α 1C -subunit of the channel (thereby preventing movement of the β-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca 2+ channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton. © 2008 Elsevier Inc. All rights reserved. 1. Introduction In the heart calcium influx through the L-type Ca 2+ channel or dihydropyridine receptor (DHPR) is a requirement for calcium release from the ryanodine receptor (RyR) in sarcoplasmic reticulum (SR) stores (a process known as Ca 2+ -induced Ca 2+ - release). In skeletal muscle, depolarization of the plasma mem- brane causes SR calcium release and contraction and the DHPR is thought to be the membrane potential sensor that transmits the signal to the RyR to release calcium [1]. Calcium influx is not a requirement. The intracellular loop between domains II and III of the skeletal muscle DHPR is thought to be important in transmit- ting the signal because peptides directed against the loop can alter RyR gating in skeletal muscle [2,3]. Therefore a direct coupling between DHPR–RyR in skeletal muscle has been proposed. Similarly, in cardiac muscle a coupling between cardiac DHPR and RyR may exist because peptides directed against the cardiac II–III loop of DHPR also alter RyR gating and the DHPR agonist BayK8644 can rapidly increase resting calcium spark frequency independent of depolarization or calcium entry in ferret ventricular myocytes [4,5]. However it is proposed that a DHPR–RyR coupling plays only a minor role in the heart [4]. In cardiac myocytes mitochondria are responsible for meeting the cellular energy demands required to maintain excitation and contraction on a beat to beat basis. It has been proposed that the mitochondria can rapidly track changes in cytosolic calcium from beat to beat [6,7]. The mitochondria rely on calcium to activate key dehydrogenases in the tricarboxylic acid cycle. This accelerates production of NADH that provides a driving force for increase in proton motive force that maintains ATP production [8]. A rapid uptake mechanism capable of responding to changes in cytosolic calcium has not been identified in the mitochondria and alternative mechanisms for regulating mitochondrial function have been sought. Cardiac myocytes are dynamic cells. L-type Ca 2+ channels are anchored to F-actin networks by subsarcolemmal stabilizing proteins that also tightly regulate the function of the channel [9–11]. Cytoskeletal proteins stabilize cell structure but also regulate the subcellular distribution of mitochondria [12]. Therefore L-type Ca 2+ Journal of Molecular and Cellular Cardiology 46 (2009) 1016–1026 ⁎ Corresponding author. Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway Crawley, WA, 6009, Australia. Tel.: +61 8 6488 3307; fax: +61 8 6488 1025. E-mail address: lhool@cyllene.uwa.edu.au (L.C. Hool). 0022-2828/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.12.015 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc