Cholesterol depletion alters coronary artery myocyte Ca(2+) signalling in a stimulus-specific manner

Abstract

Although there is evidence that caveolae and cholesterol play an important role in myocyte signalling processes, details of the mechanisms involved remain sparse. In this paper we have studied for the first time the clinically relevant intact coronary artery and measured in situ Ca(2+) signals in individual myocytes using confocal microscopy. We have examined the effect of the cholesterol-depleting agents, methyl-cyclodextrin (MCD) and cholesterol oxidase, on high K(+), caffeine and agonist-induced Ca(2+) signals. We find that cholesterol depletion produces a stimulus-specific alteration in Ca(2+) responses; with 5-HT (10microM) and endothelin-1 (10nM) responses being selectively decreased, the phenylephrine response (100microM) increased and the responses to high K(+) (60mM) and caffeine (10mM) unaffected. Agonist-induced Ca(2+) signals were restored when cholesterol was replenished using cholesterol-saturated MCD. In additional experiments, enzymatically isolated myocytes were patch clamped. We found that cholesterol depletion caused a selective modification of ion channel function, with whole cell inward Ca(2+) current being unaltered, whereas outward K(+) current was increased, due to BK(Ca) channel activation. There was also a significant decrease in cell capacitance. These data are discussed in terms of the involvement of caveolae in receptor localisation, Ca(2+) entry pathways and SR Ca(2+) release, and the role of these in agonist signalling.

Fig. 1

Experimental traces showing (A) sequential application of agonist (i) 10 nM ET-1, (ii) 100 μM PE and (iii) 10 μM 5-HT. (B) The effect of cholesterol depletion using MCD and (C) the effect of cholesterol depletion with cholesterol oxidase, on the response of coronary artery myocytes to (i) 10 nM ET-1, (ii) 100 μM PE and (iii) 10 μM 5-HT.

Fig. 2

Mean data showing the effect of cholesterol depletion (using MCD and cholesterol oxidase) on the response of coronary artery myocytes to 10 nM ET-1, 100 μM PE and 10 μM 5-HT. ***p < 0.0001 and *p < 0.05.

Fig. 3

Experimental trace showing (A) the effect of cholesterol depletion (using MCD) and subsequent cholesterol replenishment (using cholesterol-saturated MCD) on the response of coronary artery myocytes to 10 μM 5-HT and (B) effect of cholesterol-saturated MCD alone on the response of coronary artery myocytes to 10 μM 5-HT.

Fig. 4

Experimental trace showing (A) the effect of 10 μM nifedipine on the response to 10 μM 5-HT and (B) the susceptibility of the nifedipine-resistant portion of the response to treatment with MCD.

Fig. 5

Experimental trace showing (A) the effect of MCD on the response of coronary artery myocytes to 60 mM high K⁺ solution and (B) the effect of 10 mM caffeine on [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺. The Ca²⁺-free solution was perfused for 5 min prior to addition of caffeine.

Fig. 6

(A) Mean inward current–voltage relationship in the absence and presence of MCD. (B) Representative traces, recorded from the same cell, showing the outward current generated by a +50 mV depolarisation in the absence and presence of MCD. (C) Effect of MCD on outward K⁺ current and (D) Effect of MCD on cell capacitance.

Fig. 7

(A) Representative traces, recorded from the same cell, showing that the increase in outward K⁺ current induced by MCD is blocked by 100 nM iberiotoxin and (B) mean data showing the effects of 100 nM iberiotoxin, 100 nM TRAM-34 and 100 nM apamin on MCD-induced increases in outward K⁺ current.