The negative inotropic action of canrenone is mediated by L-type calcium current blockade and reduced intracellular calcium transients

Abstract

Background and purpose: Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis.

Experimental approach: Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca(2+) transients by fluorescence and Ca(2+) fluxes by electrophysiological techniques.

Key results: Canrenone (300-600 micromol L(-1)) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 micromol L(-1)) reduced cell shortening and L-type Ca(2+) channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca(2+) content of the sarcoplasmic reticulum, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) concentration. However, the time course of [Ca(2+)](i) decline during transients evoked by caffeine was not affected by canrenone.

Conclusion and implications: Canrenone reduced L-type Ca(2+) channel current, amplitude of intracellular Ca(2+) transients and Ca(2+) content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.

Figure 1

Effect of CRN on cardiomyocyte shortening and intracellular calcium transients. (A) Representative experimental record of changes in cardiomyocyte shortening (ΔShortening) as percentage of RCL during electrically induced twitches before and after 5 min of superfusion with 50 µM CRN. (B) Intracellular calcium transients (Δ[Ca²⁺]_i) of cardiomyocytes during electrically induced twitches before and after 5 min of superfusion with 50 µM CRN. (C) Left: representative experimental record showing total SR Ca²⁺ content, estimated from caffeine-induced calcium release in a Na⁺- and Ca²⁺-free medium before and after 5 min of superfusion with 50 µM CRN. Right: total SR Ca²⁺ content, estimated from caffeine-induced calcium release, before and after 5 min of superfusion with 50 µM CRN (n = 6). *P < 0.05. 0Na⁺, Na⁺-free; 0Ca²⁺, Ca²⁺-free; [Ca²⁺]_i, free intracellular Ca²⁺; Caff, caffeine; CON, control; CRN, canrenone; RCL, resting cell length; SR, sarcoplasmic reticulum.

Figure 2

Effect of CRN on L-type Ca²⁺ channel current (I_Ca-L). (A) Representative experimental record showing time course changes in I_Ca-L current amplitude before (a), during (b) and after superfusion with CRN (c – washout). Inset shows current traces recorded at the indicated times (a, b and c). The current was elicited from a holding potential of −70 to 0 mV with a prepulse to −40 mV for 25 ms, activated every 6 s, in rat ventricular myocytes. (B) Current–voltage relationship of the peak I_Ca-L elicited by test potentials ranging from −50 to +60 mV, before and after 4 min of perfusion with CRN (n = 12). (C) Mean ± SEM values of maximal I_Ca-L amplitude before, during and after CRN perfusion (n = 5). (D) Representative experimental record showing I_Ca-L amplitude in control conditions and after sequential addition of CRN (50 µM) and nicardipine (1 µM). *P < 0.05 and #P < 0.001 CRN versus CON. CON, control; CRN, canrenone.

Figure 3

Lack of effect of CRN on voltage-dependent characteristics of L-type Ca²⁺ channel current (I_Ca-L) in rat cardiomyocytes. (A) The steady-state activation curves were calculated as normalized conductance values from the current-voltage curves before and 2 min after exposure to CRN (50 µM; n = 7). (B) Steady-state inactivation curve, I_Ca-L at the test step to 0 mV was normalized to maximum current and plotted against the potential of a 1 s inactivating conditioning prepulse between −60 and +60 mV before and 2 min after exposure to CRN (n = 5). (C) Time constant of recovery from inactivation of I_Ca-L. Depolarizing pulses were applied every 6 s at a holding potential of −40 mV. The recovery from inactivation was fitted by a single exponential (n = 5). Recovery was complete in about 1 s. CON, control; CRN, canrenone.