Sinoatrial tissue of crucian carp heart has only negative contractile responses to autonomic agonists

Abstract

Background: In the anoxia-tolerant crucian carp (Carassius carassius) cardiac activity varies according to the seasons. To clarify the role of autonomic nervous control in modulation of cardiac activity, responses of atrial contraction and heart rate (HR) to carbacholine (CCh) and isoprenaline (Iso) were determined in fish acclimatized to winter (4 degrees C, cold-acclimated, CA) and summer (18 degrees C, warm-acclimated, WA) temperatures.

Results: Inhibitory action of CCh was much stronger on atrial contractility than HR. CCh reduced force of atrial contraction at an order of magnitude lower concentrations (EC50 2.75-3.5.10-8 M) in comparison to its depressive effect on HR (EC50 1.23-2.02.10-7 M) (P < 0.05) without differences between winter and summer acclimatized fish. Inhibition of nitric oxide synthase with 100 microM L-NMMA did not change the response of the sinoatrial tissue to CCh. Reduction of atrial force was associated with a strong shortening of action potential (AP) duration to approximately 50% (48 +/- 10 and 50 +/- 6% for CA and WA fish, respectively) and 11% (11 +/- 3 and 11 +/- 2% for CA and WA fish, respectively) of the control value at 3.10-8 M and 10-7 M CCh, respectively (P < 0.05). In atrial myocytes, CCh induced an inwardly rectifying K+ current, IK,CCh, with an EC50 value of 3-4.5.10-7 M and inhibited Ca2+ current (ICa) by 28 +/- 8% and 51 +/- 6% at 10-7 M and 10-6 M, respectively. These currents can explain the shortening of AP. Iso did not elicit any responses in crucian carp sinoatrial preparations nor did it have any effect on atrial ICa, probably due to the saturation of the beta-adrenergic cascade in the basal state.

Conclusion: In the crucian carp, HR and force of atrial contraction show cardio-depressive responses to the cholinergic agonist, but do not have any responses to the beta-adrenergic agonist. The scope of inhibitory regulation by CCh is increased by the high basal tone of the adenylate cyclase-cAMP cascade. Higher concentrations of CCh were required to induce IK,CCh and inhibit ICa than was needed for CCh's negative inotropic effect on atrial muscle suggesting that neither IK,CCh nor ICa alone can mediate CCh's actions but they might synergistically reduce AP duration and atrial force production. Autonomic responses were similar in CA winter fish and WA summer fish indicating that cardiac sensitivity to external modulation by the autonomic nervous system is not involved in seasonal acclimatization of the crucian carp heart to cold and anoxic winter conditions.

Figure 1

Effects of CCh on contractility of crucian carp heart. Concentration dependent effects of CCh on heart rate and atrial contractility in warm-acclimated and cold-acclimated crucian carp at the acclimation temperatures of the fish. (A) Depression of heart rate and force of atrial contraction (F_max) by CCh. Note the difference in CCh sensitivity between heart rate and force of contraction. The inset indicates the effect of 3·10^-6 M atropine and 100 μM L-NMMA on the responses. (B) Effect of CCh on the duration of atrial contraction (DC). The inset shows blockade of the response in the presence of atropine. The results are means ± SEM of 15 and 19 fish for cold-acclimated and warm-acclimated fish, respectively.

Figure 2

Effects of Iso on crucian carp heart. Concentration dependent effects of Iso on heart rate and atrial contractility in warm-acclimated and cold-acclimated crucian carp at the acclimation temperatures of the fish. Effect of Iso on heart rate and force of contraction (F_max) (A) and on the duration of atrial contraction (DC) (B) in the absence and presence (inset) of 3·10^-6 M timolol. Note the absence of responses to Iso. Results are means ± SEM of 15 and 17 fish for cold-acclimated and warm-acclimated fish, respectively.

Figure 3

CCh shortens atrial action potential duration. The bars show duration of atrial action potential (APD50%) of the crucian carp heart at 50% of repolarization level in the presence of 3·10^-8 and 1·10^-7 M CCh. The inset shows representative action potential recordings from the warm-acclimated fish heart. The results are means of 10-15 stable impalements from 5 and 3 hearts for CA and WA fish, respectively. Asterisks (**) indicate a statistically significant difference (P < 0.01) from the value in the absence of CCh.

Figure 4

Activation of the inward rectifier potassium current by CCh. Induction of the I_K,CCh by CCh in atrial myocytes from warm-acclimated and cold-acclimated crucian carp heart. Concentration-dependent increases in density of the I_K,CCh for outward current (left) and representative recordings of the CCh-induced potassium current (red line) from a warm-acclimated atrial myocyte at 18°C, comparison with the background inward rectifier current, I_K1 (blue line). Note the weak inward rectification of I_K,CCh in comparison to I_K1. The experiments were conducted at 4°C and 18°C for both acclimation groups. The results are means ± SEM of 10 myocytes (from 4 fish) for both acclimation groups. Asterisk (*) indicates a statistical difference (P < 0.05) in density of I_K1 current between cold-acclimated and warm-acclimated fish at 18°C.

Figure 5

Inhibition of I_Ca by CCh. Effect of 10^-7 and 10^-6 M CCh on atrial I_Ca from warm-acclimated and cold-acclimated crucian carp were measured using the whole-cell patch-clamp. (Left) A representative recording showing concentration-dependent inhibition of I_Ca by CCh and partial recovery (R) of I_Ca upon washout of CCh. Mean results ± SEM from 12 myocytes (from 3 fish) are shown on the right. CCh induced a small but statistically significant increase of outward current, which disappeared upon washout of CCh. The experiments were conducted on atrial myocytes of warm-acclimated crucian carp at 18°C. Asterisk (*) indicates a statistically significant different value (P < 0.05) from the control (C).

Figure 6

Depression of atrial contraction force by blockade of I_Ca. To estimate the contribution of I_Ca inhibition to CCh's response in the intact sinoatrial tissue, I_Ca was specifically blocked with 0.84·10^-6 M nifedipine, a concentration that causes about 30% inhibition of I_Ca i.e. similar I_Ca inhibition as CCh causes at the concentration of 10^-7 M. Representative recordings of contraction from warm-acclimated and cold-acclimated fish hearts (left) and mean responses of force (F_max), duration of contraction (DC) and heart rate (HR) to nifedipine. Nifedipine causes 34% and 41% inhibition of contractile force for warm-acclimated and cold-acclimated fish, respectively, and slight reduction in duration of contraction. Furthermore, nifedipine slightly depressed heart rate (HR) in cold-acclimated fish but not in warm-acclimated fish. The results are means ± SEM of 6 preparations for both acclimation groups.

Figure 7

I_Ca is not stimulated by Iso. 10^-6 M Iso was rapidly applied to atrial myocytes of warm-acclimated and cold-acclimated crucian carp to see if beta-adrenergic activation could increase I_Ca. Iso did not enhance I_Ca. Subsequent exposure of the cells to 10^-6 M CCh reduced I_Ca by about 40%. Experiments were made at 18°C. The results are means ± SEM of 10 myocytes (from 4 fish) for both acclimation groups. Asterisk (*) indicates a statistically significant difference (P < 0.05) between control value and the value in the presence of CCh.

Figure 8

Putative signaling pathways of autonomic nervous responses. Muscarinic cholinergic and beta-adrenergic pathways involved in autonomic nervous system responses of crucian carp atrial myocytes. Activation of muscarinic cholinergic receptors induces I_K,CCh and inhibits I_Ca via βγ and α_i/o-subunits of the trimeric G protein, respectively. Inhibition of I_Ca is dependent on the tonic activation of adenylate cyclase (AC) under basal conditions, i.e. in the absence of agonist binding to beta-adrenergic receptors (β). Nitric oxide synthase (NOS) is not involved in modulating the responses. Cholinergic activation of I_K,CCh and inhibition of I_Ca results in shortening of action potential and depression of atrial contraction force. GIRK, G protein activated inward rectifier potassium channel; LCC, L-type calcium channel; M2, muscarinic cholinergic receptor; PDE2, phosphodiesterase; PKA, protein kinase A.