Homozygous male C57BL/10 with a bodyweight of 25 ± 0.45 g. and 12 weeks of age were housed at constant temperature (24 C) and a 12 h light/12 h dark cycle, with free access to water and fed a commercial pelletized diet.

Mice were randomly divided into two groups: Group A (Control) that were not inoculated with T. cruzi and Group B (infected mice) were infected intraperitoneally with 5x10^-2 blood-form trypomastigotes of the Y strain of T. cruzi (O’Daly et al., 1984). The Y strain of T. cruzi was originally obtained from patients in Brazil and was kindly provided by Dr. Jose O’Daly, who has maintained the strain over the years through serial passages in homozygous C57B1/6 mice. Parasitemia was monitored by counting the number of parasites/5 μl of blood obtained from the tail vein of infected mice. A positive parasitemia was found in all inoculated animals on days 12-15 post-infection (dpi). Non-infected and infected anesthetized (ketamine/xylazine) mice were sacrificed by cervical dislocation at 20 (early acute phase), 40 (acute phase), and 120-dpi (chronic phase), and the cardiomyocytes were enzymatically dissociated.

Control and infected mice were anesthetized (ketamine 100 mg/kg/xylazine 5 mg/Kg) and hearts were rapidly removed, attached to a cannula, and mounted on a Langendorff reverse coronary perfusion system for the enzymatic dissociation of ventricular cardiomyocytes (Liao and Jain, 2007). Only Ca²⁺ tolerant cardiomyocytes with rod-shaped, well-defined striation spacing and a resting sarcomere length of ≥1.75 µm (Wussling et al., 1987) were used and studied within 4-6 h after isolation. The isolation procedure was carried out at 37°C.

Double-barreled Ca²⁺ and Na⁺ selective microelectrodes were prepared as previously described (Eltit et al., 2013). Each ion-selective microelectrode was individually calibrated before and after the determination of [Ca²⁺]_d and the [Na⁺]_d as previously described (Mijares et al., 2014). Ca²⁺ selective microelectrodes with a Nernstian response between pCa3 and pCa7 (30.5 mV/pCa units at 37°C) and Na⁺ selective microelectrodes with Nernstian responses between 100 and 10 mM [Na⁺]_e, and an adequate response (40–45 mV) between 10 and 1 mM [Na⁺]_e were used experimentally (Lopez et al., 1983; Lopez et al., 2000; Eltit et al., 2013). The Ca²⁺- and Na⁺-microelectrode response was not affected by any of the drugs used in the present study.

Isolated cardiomyocytes from control mice and T. cruzi infected mice were impaled with double-barrel Ca²⁺ or Na⁺ microelectrodes to measure [Ca²⁺]_d and [Na⁺]_d, respectively, and potentials were recorded using a high impedance electrometer (WPI 773 electrometer, FL, USA) as previously described (Lopez et al., 2000; Lopez et al., 2017). The potential of the 3M KCl microelectrode barrel (Vm) was electronically subtracted from the potential recorded by the Ca²⁺ selective barrel (V_Ca) to produce a differential Ca²⁺ specific potential (V_CaE) that represents the cardiomyocyte [Ca²⁺]_d. A similar approach was used to produce the specific Na⁺ potential (V_NaE), which represents the cardiomyocyte [Na⁺]_d. The potentials were acquired at a frequency of 1,000 Hz with the AxoGraph software (version 4.6; Axon Instruments, CA, USA) and stored in a computer for further analysis.

Individual cardiomyocyte measurements of [Ca²⁺]_d and [Na⁺]_d from control and infected mice were accepted when there was i) an abrupt drop to a steady level of Vm equal to or more negative than -80 mV in healthy cells and -70 mV in unhealthy cardiomyocytes; ii) a stable recording of Vm and V_Ca potentials or Vm and V_Na potentials for more than 60 seconds and an abrupt return to baseline at the exit of the microelectrode from the cell. Vm was used as a biological indicator of the integrity of cardiomyocyte sarcolemma in real-time; therefore, any cardiomyocytes with a recorded Vm <-70 mV were discarded. These criteria were not met in 18% of total impalements performed in cardiomyocytes from control mice and 41% in infected mouse cardiomyocytes.

The effect of NCX (reverse mode) on [Ca²⁺]_d in cardiomyocytes from control and infected mice was determined by preincubating cardiomyocytes in a solution in which [Na⁺]e was partially or completely removed (see solutions below) and replaced by N-methyl-d-glucamine hydrochloride. Measurements of [Ca²⁺]_d were carried out before and after incubation in low Na⁺ solutions.

The equilibrium potentials for Ca²⁺ (E _Ca) and Na⁺ (E _Na) were calculated using the Nernst equation (E_ion= RT/zF x ln [ion]_i/[ion]_e) and [Ca²⁺]_d and [Na⁺]_d obtained in cardiomyocytes from CONT and infected mice in EAP, AP, and CP. The equilibrium potential of the Na⁺/Ca²⁺ exchanger (E _NCX) was estimated using the following equation: E_NCX = 3E_Na - 2E_Ca (with a stoichiometry of 3:1), where E _Na and E _Ca are the equilibrium potentials of Na⁺ and Ca²⁺ obtained from the Nernst equation.

All solutions were made with ultrapure water supplied by a Milli-Q system (Millipore, Bedford, MA). The Tyrode solution had the following composition (in mM): 130 NaCl, 5 KCl, 2.5 CaCl₂, 1 MgCl₂, 20 NaHCO₃, 0.33 NaH₂PO₄, and 10 glucose gassed with 95% O₂ and 5% CO₂, pH 7.4. A low- or Na⁺-free solution was prepared by the partial or total withdrawal of [Na⁺]_e, and replacement by an equivalent amount of impermeable cation N-methyl-d-glucamine (NMG) hydrochloride to maintain osmolarity. A Ca^{2 +}-free solution was prepared, omitting CaCl₂ and adding 1 mm of EGTA and 2 mm of MgCl₂. Solutions 2-(2-(4-(4-nitrobenzyloxy-phenyl-ethyl-isothiourea methanesulfonate (KB-R7943) and N- (3-aminobenzyl) -6- 4- [(3-fluorobenzyl-oxy] phenoxy nicotinamide (YM-244769) were made by adding the desired concentration of the reagent to the Tyrode solution. All experiments were carried out at 37°C.

Experimental results are expressed as means ± SD; n_mice represents the number of mice used and n_cells the number of successful measurements in cardiomyocytes isolated from CONT and infected mice used for statistical analysis. Data were analyzed using one-way analysis of variance ANOVA for repeated measures, followed by Tukey’s multiple comparison tests to determine significance. A p<0.05 was considered significant. GraphPad Prism 9 (GraphPad Software, CA, USA) was used for statistical analysis.

We previously reported a significant elevation in [Ca²⁺]_d in cardiomyocytes obtained from patients with chronic Chagas cardiomyopath, which was related to the extent of their cardiac dysfunction (Lopez et al., 2011; Mijares et al., 2020). Figure 1 shows representative records of simultaneous measurement of Vm and [Ca²⁺]_d in a single cardiomyocyte isolated from (A) CONT mice, (B) EAP-infected mice (20 dpi), (C) AP-infected mice (40 dpi), and (D) CP-infected mice (120 dpi). The cardiomyocytes of Chagas-infected mice showed partial depolarization and increased [Ca²⁺]_d, which aggravated as a function of days after infection. The mean Vm and [Ca²⁺]_d in quiescent cardiomyocytes from CONT mice were 81 ± 1 mV and 122 ± 3 nM. Respectively. In cardiomyocytes from EAP-infected mice, Vm was 78 ± 1.7 mV and [Ca²⁺]_d 194 ± 23 nM (p<0.001 compared to CONT cardiomyocytes); in cardiomyocytes from AP-infected mice, Vm was 76 ± 1.6 mV and [Ca²⁺]_d 320 ± 38 nM (p<0.001 compared to CONT cardiomyocytes) and in cardiomyocytes from CP-infected mice, Vm was 72 ± 1.8 mV and [Ca²⁺]_d 470 ± 43 nM (p<0.001 compared to CONT cardiomyocytes) ( Figures 2A, B ).

Similar differences in [Na⁺]_d were obtained in the measurements carried out in quiescent cardiomyocytes of Chagas-infected mice at different phases of infection. In cardiomyocytes from CONT mice [Na⁺]_d was 8 ± 0.2 mM; In cardiomyocytes from EAP-infected mice [Na⁺]_d was 9.4 ± 0.7 mM (p<0.001 compared to CONT cardiomyocytes); In cardiomyocytes from AP-infected mice [Na⁺]_d was 13.9 ± 0.8 mM (p<0.001 compared to CONT cardiomyocytes); and in cardiomyocytes from CP-infected mice [Na⁺]_d was 15.9 ± 1 mM (p<0.001 compared to CONT cardiomyocytes) ( Figure 2C ).

In a separate set of experiments, the reverse mode activity of NCX (Ca²⁺ entry in exchange for Na⁺ leaving the cell) was studied in quiescent cardiomyocytes isolated from control and infected mice by incubation in a solution in which Na⁺ was replaced in equivalent amounts by NMG. [Ca²⁺]_d was measured before and after incubation with a low or Na⁺ free solution. The reduction of [Na⁺]_e to 65 mM caused an increase in [Ca²⁺]_d in CONT cardiomyocytes to 212 ± 27 nm, in cardiomyocytes from EAP-infected mice to 400 ± 24 nM, in cardiomyocytes from AP infected mice to 790 ± 62 nM, and cardiomyocytes from CP-infected mice to 1,320 ± 137 nM (p<0.001 compared to cardiomyocytes from CONT, EAP, AP and CP-infected mice bathed in normal [Na⁺]_e) ( Figure 3 ).

Incubation in Na⁺ free solution caused a further increase in [Ca²⁺]_d in control and cardiomyocytes from infected mice. In CONT cardiomyocytes [Ca²⁺]_d increased significantly to 309 ± 42 nM, in cardiomyocytes from EAP-infected mice to 540 ± 69 nM, from AP-infected mice to 1,016 ± 64 nM, and from CP-infected mice to 1,523 ± 62 nM respectively (p<0.001 compared to cardiomyocytes from CONT, and EAP-, AP- and CP- infected mice bathed in normal [Na⁺]_e). ( Figure 3 ). The sustained increase [Ca²⁺]_d was maintained as long as Na⁺ was absent from the bath solution and reversed when Na⁺ was reintroduced into the Tyrode solution, except for cardiomyocytes from CP- infected mice where an irreversible elevation [Ca²⁺]_d associated with contracture was observed.

In a different set of experiments, we explored the contribution of the Ca²⁺ influx to the elevation of [Ca²⁺]_d elicited by the withdrawal of Na⁺ in quiescent cardiomyocytes from control and infected mice. Cardiomyocytes were incubated for 10 minutes in a Ca²⁺-free solution prior to the withdrawal of Na⁺ (see solutions). Incubation in a Ca²⁺-free solution significantly reduced [Ca²⁺]_d to 98 ± 5 nM in cardiomyocytes from CONT mice (p<0.001 compared to CONT cardiomyocytes in normal [Ca²⁺]_e), to 100 ± 11 nM in cardiomyocytes from EAP-infected mice (p<0.001 compared to EAP cardiomyocytes in normal [Ca²⁺]_e), to 123 ± 14 nM cardiomyocytes from AP-infected mice (p<0.001 compared to AP cardiomyocytes in normal [Ca²⁺]_e), and to 136 ± 11 nM in cardiomyocytes from CP-infected mice (p<0.001 compared to CP cardiomyocytes in normal [Ca²⁺]_e). ( Figure 4 ). Elevation of [Ca²⁺]_d induced by withdrawal of Na⁺ was abolished in the absence of extracellular Ca²⁺ in all cardiomyocyte groups ( Figure 4 ).

Using the Nernst equation and the actual values [Ca²⁺]_d and [Na⁺]_d found in the present study, the estimated E _Ca in cardiomyocytes from CONT mice was +133 mV, in cardiomyocytes from EAP-infected mice was +127 mV, in cardiomyocytes from AP-infected mice was +119 mV, and in cardiomyocytes from CP-infected mice were +116 mV. While the estimated E _Na in cardiomyocytes isolated from CONT mice was +74 mV, EAP-infected mice was +70 mV, AP-infected mice was +59 mV, and CP-infected mice was +56 mV. The equilibrium potential of the Na⁺/Ca²⁺ exchanger (E _NCX) was -44 mV in cardiomyocytes from CONT, -46 mV from EA-infected mice, -61 mV from AP-infected mice, and –64 mV from CP-infected mice, respectively.

To determine the mechanisms involved in the elevation of [Ca²⁺]_d caused by exposure to Na⁺ free medium, quiescent cardiomyocytes from control and infected mice were incubated in KB-R7943, a nonspecific blocker of the NCX reverse mode, which modified Na⁺ dependent binding (Iwamoto et al., 1996; Akabas, 2004). Cardiomyocyte preincubation with 10 μm KB-R7943 for 10 minutes significantly reduced [Ca²⁺]_d and inhibited the increase in [Ca²⁺]_d induced by the solution without Na⁺ ( Figure 5A ). KB-R7943 reduced [Ca²⁺]_d to 152 ± 16 nM in cardiomyocytes from EAP-infected mice, to 186 ± 21 nM from AP-infected mice, and 258 ± 30 nM from CP-infected mice ( Figure 5A ). No significant effect was observed in cardiomyocytes from CONT mice (116 ± 5 nM). Preincubation with KB-R7943 prevented the elevation of [Ca²⁺]_d caused by the Na⁺ free solution in cardiomyocytes of control and infected mice (compare Figures 3 , 5A ).

Due to the lack of specificity of KB-R7943 (Abramochkin and Vornanen, 2014), we also examined the effects of YM-244769, a high-affinity blocker of the NCX reverse mode (Iwamoto and Kita, 2006). Incubation with 1 μm YM-244769 for 10 minutes reduced [Ca²⁺]_d to 176 ± 20 nM in cardiomyocytes from EAP-infected mice, to 254 ± 19 nM from AP-infected mice, and to 336 ± 32 nM from CP-infected mice. YM-244769 did not significantly reduce [Ca²⁺]_d in cardiomyocytes of CONT and EAP-infected mice. YM-244769 fully inhibited the elevation of [Ca²⁺]_d induced by the Na⁺ free solution in CONT cardiomyocytes and infected mice (compare Figures 3 , 5B ).

These results provide, for the first time, evidence that NCX plays an important role in aberrant diastolic [Ca²⁺] observed in cardiomyocytes from infected mice (chronic>acute>early acute). These novel findings may open a new therapeutic approach to improve cardiac function in patients suffering from chronic Chagas cardiomyopathy because there is still no effective treatment. Unfortunately, Chagas cardiomyopathy remains largely ignored despite its medical and social relevance.

Despite the novelty of this study, some limitations should be pointed out. We did not determine whether the elevation of [Na⁺]_d in cardiomyocytes from infected mice could be related to an increased influx or decreased efflux of Na⁺. Furthermore, the expression of NCX and other proteins involved in intracellular Ca²⁺ regulation were not studied.