The Role of the Na+/Ca2+ Exchanger in Aberrant Intracellular Ca2+ in Cardiomyocytes of Chagas-Infected Rodents

Abstract

Chagas disease is produced by the parasite Trypanosoma cruzi (T. cruzi), which is the leading cause of death and morbidity in Latin America. We have shown that in patients with Chagas cardiomyopathy, there is a chronic elevation of diastolic Ca²⁺ concentration ([Ca²⁺]_d), associated with deterioration to further address this issue, we explored the role Na⁺/Ca²⁺ exchanger (NCX). Experiments were carried out in noninfected C57BL/6 mice and infected with blood-derived trypomastigotes of the T. cruzi Y strain. Anesthetized mice were sacrificed and the cardiomyocytes were enzymatically dissociated. Diastolic [Ca²⁺] ([Ca²⁺]_d) was measured using Ca²⁺ selective microelectrodes in cardiomyocytes from control mice (CONT) and cardiomyocytes from T. cruzi infected mice in the early acute phase (EAP) at 20 dpi, in the acute phase (AP) at 40 dpi, and in the chronic phase (CP) at 120 dpi. [Ca²⁺]_d was 1.5-times higher in EAP, 2.6-times in AP, and 3.4-times in CP compared to CONT. Exploring the reverse mode activity of NCX, we replaced extracellular Na⁺ in equivalent amounts with N-methyl-D-glucamine. Reduction of [Na⁺]_e to 65 mM caused an increase in [Ca²⁺]_d of 1.7 times in cardiomyocytes from CONT mice, 2 times in EAP infected mice, 2.4 times in AP infected mice and 2.8 in CP infected mice. The Na⁺ free solution caused a further elevation of [Ca²⁺]_d of 2.5 times in cardiomyocytes from CONT, 2.8 times in EAP infected mice, 3.1 times in AP infected mice, and 3.3 times in CP infected mice. Extracellular Ca²⁺ withdrawal reduced [Ca²⁺]_d in both CONT and cardiomyocytes from Chagas-infected mice and prevented the increase in [Ca²⁺]_d induced by Na⁺ depletion. Preincubation with 10µM KB-R7943 or in 1µM YM-244769 reduced [Ca²⁺]_d in cardiomyocytes from infected mice, but not control mice. Furthermore, both NCX blockers prevented the increase in [Ca²⁺]_d associated with exposure to a solution without Na⁺. These results suggest that Ca²⁺ entry through the reverse NCX mode plays a significant role in the observed [Ca²⁺]_d dyshomeostasis in Chagas infected cardiomyocytes. Additionally, NCX inhibitors may be a viable therapeutic approach for treating patients with Chagas cardiomyopathy.

Keywords: Na/Ca exchanger; Trypanosoma cruzi (T cruzi); calcium; cardiomyopathy; chagas disease.

Figure 1

Representative measurements of Vm and [Ca²⁺]_d in cardiomyocytes from control and Chagas infected mice. Simultaneous measurements of membrane potential (Vm) and [Ca²⁺]_d (A) Cardiomyocyte from control mice (Vm was -82 mV and [Ca²⁺]_d 122 nM). (B) Cardiomyocyte from EAP-infected mice (Vm was -76mV and [Ca²⁺]_d 200 nM). (C) Cardiomyocyte from AP-infected mice (Vm was -73 mV and [Ca²⁺]_d 312 nM). (D) Cardiomyocyte from CP-infected mice (Vm was -69 mV and [Ca²⁺]_d 414 nM). Vm, resting membrane potential and V_Cae, specific calcium potential; CONT, control mice; EAP, infected mice in the early acute phase; AP, infected mice in the acute phase; and CP, infected mice in the chronic phase. Calibration bar = 1 minute.

Figure 2

Resting membrane potential, diastolic [Ca²⁺] and [Na⁺] in cardiomyocytes from control and Chagas infected mice. (A) The resting membrane potential, (B) [Ca²⁺]_d, and (C) [Na⁺]_d were measured in quiescent cardiomyocytes from CONT mice (n_cells =24 and n_mice =3), EAP-infected mice (n_cells =19 and n_mice =3), AP-infected mice (n_cells =21 and n_mice =3) and CP-infected mice (n_cells =18 and n_mice =5). Data are expressed as means ± S.D. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparison tests, *p < 0.05; ***p < 0.001. CONT, control mice; EAP, early acute phase of infected mice; AP, acute phase of infected mice; and CP, chronic phase of infected mice.

Figure 3

Effects of reduction in [Na⁺]_e on [Ca²⁺]_d in cardiomyocytes from control and Chagas infected mice. [Ca²⁺]_d was measured before and after cardiomyocyte treatments with solutions in which [Na⁺]_e was partially or completely replaced with NMG. [Ca²⁺]_d was determined in quiescent cardiomyocytes from CONT mice (n_cells =13-24 and n_mice =4), EAP-infected mice (n_cells =12-19 and n_mice =5), AP-infected mice (n_cells =12-21 and n_mice =5) and CP-infected mice (n_cells =6-18 and n_mice =9). Data are expressed as means ± S.D. Statistical analysis was performed as described above, ***p < 0.001. CONT, control mice; EAP, early acute phase of infected mice; AP, acute phase of infected mice; and CP, chronic phase of infected mice.

Figure 4

Effect extracellular Ca²⁺ on Na⁺ depletion-induced elevation of [Ca²⁺]_d. [Ca²⁺]_d was measured in cardiomyocytes from CONT, EAP-, AP-, and CP-infected mice in normal Tyrode, Ca²⁺-free Tyrode solution, and Ca²⁺ and Na⁺ free Tyrode solution. [Ca²⁺]_d was determined in quiescent cardiomyocytes from CONT (n_cells =16-24 and n_mice =4), EAP-infect mice (n_cells =15-19 and n_mice =5), AP-infect mice (n_cells =18-21 and n_mice =6) and CP-infect mice (n_cells =15-18 and n_mice =8). Data are expressed as mean ± S.E. Statistical analysis was performed as described above, *** p < 0.001. CONT, control mice; EAP, early acute phase of infected mice; AP, acute phase of infected mice; and CP, chronic phase of infected mice. ns, no significant.

Figure 5

NCX blockers inhibited Na⁺ withdraw-induced [Ca²⁺]_d increases in cardiomyocytes from control and Chagas infected mice. (A) Effects of KB-R7943 and (B) YM-244769 on [Ca²⁺]_d increases caused by Na⁺ removal. [Ca²⁺]_d was determined in (i) normal Tyrode solution (ii) after 10 min of incubation with 10 μm KB-R7943) or 1 µM YM-244769 and (iii) after incubation, either KB-R7943 in Na⁺ free solution or YM-244769 in Na⁺ free solution. Experiments were carried out in cardiomyocytes from CONT (n_cells=15-24 and n_mice=6), EAP (n_cells=12-19 and n_mice=8), AP (n_cells=13-21 and n_mice=8) and CP cardiomyocytes (n_cells=11-18 and n_mice=9). Data are expressed as mean ± S.E. Statistical analysis was performed as described above, ***p < 0.001. CONT: control mice; EAP: early acute phase of infected mice; AP: acute phase of infected mice; and CP: chronic phase of infected mice.