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. 2021 Jun 2;15(6):e0009421.
doi: 10.1371/journal.pntd.0009421. eCollection 2021 Jun.

A novel substrate for arrhythmias in Chagas disease

Artur Santos-Miranda  1 , Julliane V Joviano-Santos  1 , Jaqueline O Sarmento  1 , Alexandre D Costa  2 , Allysson T C Soares  3 , Fabiana S Machado  3 , Jader S Cruz  2   3 , Danilo Roman-Campos  1
Affiliations

A novel substrate for arrhythmias in Chagas disease

Artur Santos-Miranda et al. PLoS Negl Trop Dis. .

Abstract

Background: Chagas disease (CD) is a neglected disease that induces heart failure and arrhythmias in approximately 30% of patients during the chronic phase of the disease. Despite major efforts to understand the cellular pathophysiology of CD there are still relevant open questions to be addressed. In the present investigation we aimed to evaluate the contribution of the Na+/Ca2+ exchanger (NCX) in the electrical remodeling of isolated cardiomyocytes from an experimental murine model of chronic CD.

Methodology/principal findings: Male C57BL/6 mice were infected with Colombian strain of Trypanosoma cruzi. Experiments were conducted in isolated left ventricular cardiomyocytes from mice 180-200 days post-infection and with age-matched controls. Whole-cell patch-clamp technique was used to measure cellular excitability and Real-time PCR for parasite detection. In current-clamp experiments, we found that action potential (AP) repolarization was prolonged in cardiomyocytes from chagasic mice paced at 0.2 and 1 Hz. After-depolarizations, both subthreshold and with spontaneous APs events, were more evident in the chronic phase of experimental CD. In voltage-clamp experiments, pause-induced spontaneous activity with the presence of diastolic transient inward current was enhanced in chagasic cardiomyocytes. AP waveform disturbances and diastolic transient inward current were largely attenuated in chagasic cardiomyocytes exposed to Ni2+ or SEA0400.

Conclusions/significance: The present study is the first to describe NCX as a cellular arrhythmogenic substrate in chagasic cardiomyocytes. Our data suggest that NCX could be relevant to further understanding of arrhythmogenesis in the chronic phase of experimental CD and blocking NCX may be a new therapeutic strategy to treat arrhythmias in this condition.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Prolonged action potential (AP) duration in chagasic cardiomyocytes.
(A) Representative AP recorded from control (black traces) and infected (red traces) isolated cardiomyocytes paced at 0.2 Hz and 1 Hz. (B) Time required to 50% (T50) and 90% (T90) of full AP repolarization at 0.2 Hz and 1 Hz. Data were analyzed using Two-way ANOVA. n represents the number of cells. *p<0.05.
Fig 2
Fig 2. Membrane potential instability due to after-depolarization events in the chronic phase of experimental Chagas disease.
(A) Eight to nine consecutive recorded action potentials from control and infected isolated cardiomyocytes paced at 0.2 Hz and 1 Hz. (B) The fraction of cells displaying membrane potential instability triggered by after-depolarizations, either during membrane repolarization (exemplified by the blue arrow) or after full membrane repolarization (exemplified by the green arrow). Data were compared using Fisher’s exact test. n represents the number of cells. *p<0.05.
Fig 3
Fig 3. Ni2+ shortens action potential duration in chronic phase of experimental Chagas disease.
(A) Representative AP recorded from isolated cardiomyocytes from control and infected mice, before and after perfusion of Ni2+ (5 mM). (B) Time required to reach 90% (T90) of full AP repolarization before and after exposure to Ni2+ (5 mM). (C) AP repolarization (APR) fractional reduction taken at 90% after challenge control and chagasic cardiomyocytes with Ni2+ at 5mM. Data were compared using one-way ANOVA (B) and Student’s t test (C). n represents the number of cells. * comparing Chagasic to control prior Ni2+, # comparing Chagasic prior and after Ni2+, $ comparing Chagasic to control after Ni2+ (p<0.05).
Fig 4
Fig 4. SEA0400 shorts action potential waveform in the chronic phase of experimental Chagas disease.
(A) Representative AP recorded from cardiomyocytes isolated from control and infected mice, before and after perfusion of SEA0400 (1 μM). (B) Time required to 90% (T90) of full AP repolarization before and after exposure to SEA0400 (1 μM). (C) AP repolarization (APR) fractional reduction taken at 90% after challenge cells with SEA0400 (1 μM). Data were compared using one-way ANOVA (B) and Student’s t test (C). n represents the number of cells. * comparing Chagasic without SEA0400 to all other groups (p<0.05).
Fig 5
Fig 5. Enhanced susceptibility for the appearance of transient inward current (ITi) in the chronic phase of experimental Chagas disease.
The stimulation protocol is depicted at the top of the figure. (A) Current traces following 100 pre-conditioning pulses and a single 500 ms pause using a short pulse (left) and a long pulse (right). Black arrows indicate ITi. (B) and (C) are the percentage of cells showing any identifiable ITi, using short and long pulses, respectively. (D) and (E) are bar graphs summarizing, only for those cells that developed ITi, the calculated integral of the ITi responses recorded with short and long pulses, respectively. Data were compared using Fisher’s exact test (B and C) and Student’s t test for (D and E). n represents the number of cells. *p<0.05.
Fig 6
Fig 6. Ni2+ attenuates transient inward current (ITi) in the chronic phase of experimental Chagas disease.
The protocol is depicted at the top of the figure. (A) Current traces following 100 pre-conditioning pulses and a single 500 ms pause using long pulse, before (red) and after perfusion of 5 mM Ni2+ (green traces). (B) Composite data representing the calculated area during ITi responses recorded from isolated cardiomyocytes from control and infected mice using long AP-simulating pulses, before and after Ni2+ perfusion. Data were compared using OneWay ANOVA. n represents the number of cells. *p<0.05.
Fig 7
Fig 7. SEA0400 attenuates transient inward current (ITi) in the chronic phase of experimental Chagas disease.
The protocol is depicted at the top of the figure. (A) Current traces following 100 pre-conditioning pulses and a single 500 ms pause using long pulse, before (red) and after perfusion of SEA0400 (1 μM). (B) Composite data representing the calculated integral of the ITi responses recorded from isolated cardiomyocytes from control and infected mice using long pulses, before and after SEA0400 (1 μM). Data were compared using OneWay ANOVA. n represents the number of cells. *p<0.05.
Fig 8
Fig 8. Ni2+-sensitive Na+/Ca2+ exchange current (INCX) density does not change in experimental Chagas disease.
(A) Representative INCX vs. Membrane Potential relationships before (Ni (-)) and after (Ni (+)) perfusion of 5 mM Ni2+ measured in isolated cardiomyocytes from control (Panel A, left) and infected (Panel A, right) mice. Orange and green traces represent the difference current obtained by digitally subtracting traces before and after Ni2+ application. (B) Average Ni2+-subtracted Current Density versus Membrane Potential in control (orange squares) and chagasic (green circles) cardiomyocytes were not different. Data were analyzed using Two-way ANOVA. n indicates the number of cells.
Fig 9
Fig 9. Reduced sarcoplasmic reticulum Ca2+ content measured by Na+/Ca2+ exchange current in the chronic phase of experimental Chagas disease.
(A) Representative recordings of caffeine-induced Na+/Ca2+ exchange current (INCX) in cardiomyocytes from control (black) and infected (red) mice. (B) Area of inward current measured in the presence of 10 mM caffeine. Data were compared using Student’s t test. n indicates the number of cells. *p<0.05.
Fig 10
Fig 10. Detection of T. cruzi in isolated left ventricular cardiomyocytes from mice.
RT-PCR analysis was performed using isolated cells obtained from mice 180 to 200 days after infection and age matched controls. The results of RT-PCR amplification of 18S T. cruzi (A), threshold cycle (B), and relative expression (C; represented as ΔCt values) were demonstrated. Data were normalized to mice 18S endogenous (isolated left ventricular cardiomyocytes). N = 5 for both groups. Data were compared using Student’s t test. ****p<0.0001.
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Grants and funding

DRC received funding from the Fundação de Amparo à Pesquisa do Estado de Sao Paulo (https://fapesp.br/) (grant no. 2014/09861-1 and grant no. 2019/21304-4). ASM received a postdoctoral fellowship from the Fundação de Amparo à Pesquisa do Estado de Sao Paulo (https://fapesp.br/bolsas/pd) (grant no. 2018/22830-9). JVJS received a postdoctoral fellowship from the Fundação de Amparo à Pesquisa do Estado de Sao Paulo (https://fapesp.br/bolsas/pd) (grant no. 2018/20777-3). JOS received a master student fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (https://fapesp.br/bolsas/ms) (grant no. 2020/09403-4). JSC received funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico (https://www.gov.br/cnpq/pt-br) (grant no. 312474/2017-2 and grant no. 437969/2018-5). DRC received funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico (https://www.gov.br/cnpq/pt-br) (grant no. 304257/2020-6). FSM received a grant from National Institute for Science and Technology in Dengue and Host-microbial interactions (http://labs.icb.ufmg.br/inctemdengue/) (grant no. APQ-03606-17). FSM received a grant from Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Rede Mineira de Imunobiológicos (https://fapemig.br/pt/) (grant no. 00140-16). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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