Increases in [IP3]i aggravates diastolic [Ca2+] and contractile dysfunction in Chagas' human cardiomyocytes.

Chagas cardiomyopathy is the most severe manifestation of human Chagas disease and represents the major cause of morbidity and mortality in Latin America. We previously demonstrated diastolic Ca2+ alterations in cardiomyocytes isolated from Chagas’ patients to different degrees of cardiac dysfunction. In addition, we have found a significant elevation of diastolic [Na+]d in Chagas’ cardiomyocytes (FCII>FCI) that was greater than control. Exposure of cardiomyocytes to agents that enhance inositol 1,4,5 trisphosphate (IP3) generation or concentration like endothelin (ET-1) or bradykinin (BK), or membrane-permeant myoinositol 1,4,5-trisphosphate hexakis(butyryloxy-methyl) esters (IP3BM) caused an elevation in diastolic [Ca2+] ([Ca2+]d) that was always greater in cardiomyocytes from Chagas’ than non- Chagas’ subjects, and the magnitude of the [Ca2+]d elevation in Chagas’ cardiomyocytes was related to the degree of cardiac dysfunction. Incubation with xestospongin-C (Xest-C), a membrane-permeable selective blocker of the IP3 receptors (IP3Rs), significantly reduced [Ca2+]d in Chagas’ cardiomyocytes but did not have a significant effect on non-Chagas’ cells. The effects of ET-1, BK, and IP3BM on [Ca2+]d were not modified by the removal of extracellular [Ca2+]e. Furthermore, cardiomyocytes from Chagas’ patients had a significant decrease in the sarcoplasmic reticulum (SR) Ca2+content compared to control (Control>FCI>FCII), a higher intracellular IP3 concentration ([IP3]i) and markedly depressed contractile properties compared to control cardiomyocytes. These results provide additional and convincing support about the implications of IP3 in the pathogenesis of Chagas cardiomyopathy in patients at different stages of chronic infection. Additionally, these findings open the door for novel therapeutic strategies oriented to improve cardiac function and quality of life of individuals suffering from chronic Chagas cardiomyopathy (CC).


Introduction
Chagas disease (American trypanosomiasis) is caused by the protozoa parasite Trypanosoma cruzi (T. cruzi), which is transmitted to humans by blood-sucking triatomine bugs and by non-vectorial mechanisms, such as contaminated blood transfusion, organ transplantation, and congenital infection [1,2]. Chagas disease is a significant public health burden and the leading cause of death and morbidity in Latin American and Caribbean regions [3]. Worldwide, 10 million people are estimated to be infected with T. cruzi, and more than 120 million inhabitants are at risk of infection [4]. As a neglected disease, Chagas' disease is associated with malnutrition, poverty, and inadequate sanitation [5], and it is part of a self-propagating cycle of poverty in many endemic regions. Human migrations due to economic hardship, political problems, or both, have spurred an exodus from Chagas-endemic countries to geographical areas where the disease was not endemic [6][7][8][9]. Individuals with Chagas disease have been identified in non-endemic countries in Europe, Canada, and the USA [7,10], and an estimated 300,000 persons are suffering from this disease who live in the US, especially in Texas and along the Gulf coast [11,12]. Chagas' disease has become a potentially severe emerging threat to several countries throughout the world.
Chagas' disease is a multifactorial illness that consists of two sequential phases, an initial acute phase, followed by a chronic phase that can be categorized into a cardiac or digestive form [13]. The initial acute phase lasts for about 2 months after infection, and it is limited to a febrile episode, headache, enlarged lymph glands, muscle pain, and abdominal or chest pain [14]. In the chronic phase, 20-40% of the infected patients go on to develop cardiomyopathy or digestive damage (typical enlargement of the esophagus or colon) [15][16][17]. Chagas cardiomyopathy (CC) is an important form of chronic Chagas' disease which has a high morbidity and mortality and a significant medical and social impact. CC is associated with myocarditis, rhythm disturbances, depressed heart function, congestive failure, thromboembolism, and sudden death [14,18]. The most important prognostic marker in CC is the severity of myocardial contractile dysfunction [19].
Despite the extensive characterization of the clinical manifestations of CC, the mechanisms underlying the pathogenesis of this disease are still poorly understood. Earlier studies with non-human models [20][21][22] have shown there is a possible link between Chagas' infection and alteration in phospholipase-C/phosphoinositide signaling pathway. We recently demonstrated that cardiomyocytes isolated from Chagas patients have an intracellular Ca 2+ overload, which appears to be associated with changes in the inositol 1,4,5 trisphosphate (IP 3 ) signaling pathway [23]. IP 3 is a second messenger generated by hydrolysis of membrane lipid phosphatidylinositol 4,5-bisphosphate by phospholipase C in response to G protein-coupled receptor activation [24]. Once generated, IP 3 causes Ca 2+ release from the sarcoplasmic reticulum (SR) and the nuclear envelope via the IP 3 receptors (IP 3 Rs) [24]. In the heart, IP 3 Rs are thought to play an important role by modulating Ca 2+ signals during excitation-contraction coupling (ECC) and cardiac gene expression. IP 3 Rs activation is characterized by increasing action potential amplitude, and spontaneous Ca 2+ transient frequency, and decreasing resting membrane potential [25][26][27]. However, the role of IP 3 Rs in cardiac ECC is controversial due to lower expression levels in ventricular cardiomyocytes compared to other cell types [28,29].
The present study was undertaken to further investigate the involvement of IP 3 in the diastolic Ca 2+ and contractile dysfunctions observed in cardiomyocytes isolated from Chagas' patients.

Ethics statement
Written consent from all patients involved in this study was obtained prior to processing the samples. Invasive cardiac studies were performed after the patient provided written informed consent, and approval was granted by the Bioethics Committee of Hospital Pérez Carreño (No. 073/17), Caracas, Venezuela. Data on human subjects were analyzed anonymously, and clinical investigations have been conducted according to the Declaration of Helsinki.

Patient's study population
This study was conducted in 33 Chagas' patients with CC (see Table 1). Chagas patients had an abnormal electrocardiogram at rest (rhythm disturbance and conduction defects), positive blood culture and enzyme-linked immunosorbent assay (ELISA) for the Chagas disease. None of them had congestive heart failure or ischemic heart disease. Patients were grouped based on the New York Heart Association (NYHA) classification system, which considers the patient's clinical manifestations and risk factors that affect mortality: early (functional class I (FCI), intermediate (functional class II (FCII), and late (functional class III (FCIII). According to the NYHA 18 patients of the Chagas' patients fell within functional class I (FCI), and 15 patients in FCII, according to the NYHA. Besides, 17 non-Chagas' subjects (considered as control) with mild mitral stenosis and negative blood culture, and ELISA for Chagas disease served as control (see Table 1). Potential subjects (control or Chagas' patients) were excluded from the study if they had a history of alcoholism.

Endomyocardial biopsy
Left ventricular endomyocardial biopsies were obtained from the Chagas' patients using fluoroscopic as part of routine evaluation for Chagas patients at the Cardiology Department at Hospital Miguel Perez Carreño (Caracas, Venezuela). The Chagas' patients were pretreated with aspirin 800 mg twice daily on the day preceding the examination and 800 mg before the procedure to reduce the thromboembolic risk. Biopsies from control subjects were obtained during mitral valve replacement surgery. Although not all patients included in this study were taking medications at that time, those who were stopped their medications 48 h before the endocardial biopsies. Upon removal, the endomyocardial biopsies were immediately immersed in ice-cold, oxygenated, low Ca 2+ -solution supplemented with 2,3-butanedione monoxime (BDM) to prevent Ca 2+ -induced hypercontraction (see solutions). BDM reduces the activity of the myosin ATPase, inhibits Ca 2+ -induced force development [30], and decreases reoxygenation injury [31]. The connective tissue was removed from the biopsy specimens with the aid of a dissecting microscope, and the tissue was cut into small pieces. Calcium tolerant cardiomyocytes were isolated enzymatically following the technique described by Peeters et al. 1995 [32]. The isolated cardiomyocytes were settled for 10 min sequentially in a buffer solution containing 50 μM, 100 μM, 500 μM and 1.8 mM Ca 2+ , and at each step the injured cells (spontaneous contractile activity was discarded). The yield of Ca 2+ -tolerant ventricular cardiomyocytes (rod-shaped) was significantly higher in control samples (75%) than in cardiomyocytes from Chagas' patients (64% from FCI and 55% from FCII). This difference may due to the increased fibrosis and plasma membrane damage observed in cardiomyocytes from Chagas' patients [15].

Criteria for selecting cardiomyocytes
Cardiomyocytes were studied if they had sharp outlines and rod-shaped, clearly visible striations, without developing subsarcolemmal blebs, and showing spontaneous contractile activity in the presence of 1.8 mM extracellular [Ca 2+ ]. In some experiments, cell integrity was further determined by the ability of the cardiomyocyte to exclude the dye trypan blue.

Ca 2+ and Na + -selective microelectrodes
Double-barreled Ca 2+ and Na + selective microelectrodes were prepared as described previously [33]. Each ion-selective microelectrode was individually calibrated before and after the determination of diastolic Ca 2+ concentration ([Ca 2+ ] d ) and diastolic Na + concentration ([Na + ] d ) as described before [33]. Only those Ca 2+ selective microelectrodes with a linear relationship between pCa 3 and 7 (Nernstian response 30.5 mV/pCa unit at 37˚C, respectively) were used experimentally. The Na + selective microelectrodes gave virtually Nernstian responses at free [Na + ] e between 100 and 10 mM. However, although at concentrations between 10 and 1 mM [Na + ] e , the microelectrodes had a sub-Nernstian response (40-45 mV), their response was of sufficient amplitude to be able to measure [Na + ] d . The response of the

PLOS NEGLECTED TROPICAL DISEASES
Ca 2+ and Na + -selective microelectrodes were not directly affected by any of the drugs used in the present study.

Measurements of [Ca 2+ ] d in human cardiomyocytes
Within 1-2 h after isolation, human Ca 2+ tolerant cardiomyocytes were transferred to poly-Llysine-coated coverslips for 45 minutes in a small Plexiglas chamber filled with normal Tyrode solution containing 20 mM BDM at 37˚C. Only rod-shaped cardiomyocytes without any signs of deterioration and spontaneous activity at rest were used for experiments [23,34]. Cardiomyocytes from control and Chagas' patients were impalements with the doubled-barreled Ca 2 + selective microelectrodes with the aid of an inverted microscope fitted with an x10 eyepiece and an x40 oil objective. The potentials from the 3 M KCl barrel -resting membrane potential (Vm)-and the Ca 2+ barrel (V CaE ) were recorded via a high-impedance amplifier (model FD-223; WPI, Sarasota, FL). The potential of the voltage microelectrode (Vm) was subtracted electronically from the potential of the Ca 2+ electrode (V CaE ) to obtain the differential signal (V Ca ) representing the resting [Ca 2+ ] d . Vm and VCa potentials were acquired at a frequency of 1,000 Hz with AxoGraph software (version 4.6; Axon Instruments, Foster City, CA), and stored in a computer for further analysis. Two criteria were used as key elements to accept or to reject individual [Ca 2+ ] d measurements performed in cardiomyocytes from control and Chagas' patients: i) polarize resting membrane potential -more negative than -80 mV in control and more than -75 in Chagas cardiomyocytes-and ii) stable recording potentials for no less than 40 seconds (Vm, V Ca ).

Sarcoplasmic reticulum Ca 2+ content
To estimate the total amount of Ca 2+ stored in the sarcoplasmic reticulum (SR), control and Chagas' cardiomyocytes were loaded with 5 μm Fluo-4-AM for 30 min at 37˚C. Fluo-4 loaded cardiomyocytes were transferred to a small Plexiglas chamber filled normal Tyrode solution containing 20 mM BDM and placed on Plexiglass chamber on the stage of an inverted microscope equipped with epifluorescence illumination (XCite1 Series 120 or Lambda DG4) equipped with a CCD cooled camera (Retiga 2000R or Stanford Photonics 12 bit digital). The excitation wavelength of the argon-ion laser was set to 488 nm, and fluorescence emission was measured at wavelengths >515 nm. The experiments were conducted in a Ca 2+ -free solution to prevent the Ca 2+ uptake by the SR. The Ca 2+ transient elicited by 10 mM caffeine (2 min stimulus) was used as an index of the Ca 2+ content of the SR, which was estimated by taking the area under the curve of the signal induced by caffeine [35]. The experiments were carried out in a blinded fashion to validate our results.

Determination of cytosolic [IP 3 ]
Intracellular [IP 3 ] was determined in cardiomyocytes biopsies from control subjects and Chagas' patients using a competitive radioligand binding assay, as previously described [36]. In brief, ventricular myocytes from control or Chagas' patients were suspended in normal Tyrode solution maintained at 38˚C. Each sample was pre-incubated for 10 min, with 10 mM LiCl to inhibit inositol phosphate metabolism [37]. The tubes were maintained in ice for 20 min, then centrifuged, and the pellet was kept for protein determination by the Lowry method [38]. The supernatant was neutralized to pH 7.0 with 1.5 M KOH containing 60 mM HEPES. The intracellular IP 3 concentration was determined using the IP 3 assay kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions.

Cardiomyocyte contractility studies
Contractile properties of Chagas' and control cardiomyocytes were studied in a customdesigned Perspex chamber with a glass-bottom filled with normal Tyrode solution, using a video-based edge-detection system (IonOptix, Milton, MA). The cardiomyocytes were field stimulated through a pair of platinum electrodes at a frequency of 1 Hz (2 ms pulse duratioñ 1.5x threshold voltage). Myocyte edges were continuously tracked during contraction and relaxation, displayed as a voltage signal proportional to the changes in myocyte length, and sent to a PC for future analyses of different contraction and relaxation parameters (IonOptix, Milton, Massachusetts). The following parameters were measured: i) diastolic sarcomere length which was determined after a 30-s stimulation (2 ms pulse duration~1.5x threshold voltage) in quiescent cardiomyocytes; ii) peak shortening (PS), indicative of peak ventricular contractility; iii) maximal velocity of shortening (+dL/dt), indicative of ventricular pressure rise; iv) maximal velocity of relengthening (−dL/dt), indicative of ventricular pressure fall. Only rod-shaped cardiomyocytes with good striation and edges were used. Experiments were conducted at 37˚C.

Statistical analysis
All values are expressed as mean±SD; n represents the number of cardiomyocytes (control or Chagas) in which a successful measurement of [Ca 2+ ] d was carried out. The area-under-thecurve for the caffeine-induced release of Ca 2+ from the SR was calculated by the trapezoid rule (GraphPad Prism software 7.0). Statistical analysis was performed using a two-tailed paired and unpaired t-test or one-way analysis of variance coupled with either Tukey's or Dunnett's t-test for multiple measurements to determine significance. Significance was accepted at p<0.05 level. Statistical analysis was done using GraphPad Prism 7.03 (GraphPad Software, Inc.).

[Ca 2+ ] d and [Na + ] d in cardiomyocytes from Chagas' patients
We previously observed a significant increase in [Ca 2+ ] d in CC patients, which correlate directly with the extent of their cardiac dysfunction (NYHA class) regardless of gender [23]. Fig 1A, 1B  The partial depolarization observed in cardiomyocytes isolated from Chagas' patients correlates with the level of cardiac dysfunction determined by the NYHA classification. We found a 6% reduction in average Vm values in cardiomyocytes from FCI patients, and 11% in cardiomyocytes from FCII patients compared to control. These results confirm and extend our previous report demonstrating a diastolic Ca 2+ dysfunction in human cardiomyocytes from patients with Chagas' disease [23].
A significant difference for [Na + ] d was observed in cardiomyocytes isolated from FCI and FCII Chagas patients compared to control. In control [Na + ] d was 8±0.1 mM (n = 13) compared to 12±1 mM (n = 16) and 17±1.2 mM (n = 17) in FCI and FCII cardiomyocytes respectively (p�0.001 compared to control) ( Fig 2B). These results demonstrate that there is a diastolic Ca 2+ and Na + overload in chagasic cardiomyocytes compared to control cells.

Bradykinin elevates [Ca 2+ ] d
To further test the role of IP 3 , we investigated the effects of bradykinin (BK), a peptide that induces IP 3 and diacylglycerol formation in cardiomyocytes through activation of the G-protein-coupled receptor and phospholipase C (PLC) [43] which has been implicated in the pathogenesis of CC [44]. Incubation of control and Chagas' cardiomyocytes in 10 nM of BK for 10

Xestospongin C partially restores [Ca 2+ ] d
The effects of xestospongin C (Xest-C), a membrane-permeable selective blocker of the IP 3 R [45], were investigated on the observed increase in diastolic Ca 2+ in Chagas' cardiomyocytes.

Sarcoplasmic reticulum Ca 2+ loading
The level of the SR Ca 2+ store was determined by exposing Fluo-4-AM loaded-control and Chagas' cardiomyocytes to 10 mM caffeine [33]. Under these conditions, the total Ca 2+ released was significantly smaller in Chagas' cardiomyocytes compared with control cardiomyocytes. Quantitative analysis of the Ca 2+ signal indicates that the Ca 2+ SR loading was 37% lower in FCI (n = 8) than control cardiomyocytes (n = 10) (p�0.001), and in the FCII was reduced by 61% (n = 9) (p�0.001) (Fig 5). Moreover, treatment with 5 μM Xest-C for 15 min, partially restored the SR Ca 2+ content in FCI and FCII Chagas' cardiomyocytes ( Fig 5). SR Ca 2 + content was increased by 25% in FCI (n = 11) (p�0.001 compared to untreated cells) and by 71% in FCII (n = 9) (p�0.001 compared to untreated cells) in Chagas' cardiomyocytes. No significant difference was observed in control cardiomyocytes after Xest-C treatment (n = 11) (p>0.05). These results suggest that the reduction in the SR Ca 2+ levels appears to be mediated by an IP 3 Rs-Ca 2+ leak from the SR.

Intracellular [IP 3 ]
Levels of intracellular IP 3 , as determined by the competitive radioligand-binding assay were significantly higher in ventricular cells in patients with Chagas' disease than in control. The basal level of [IP 3 ] i was 5.4±0.6 pmol/mg protein (n = 11) in control cardiomyocytes (Fig 6), while in Chagas' cardiomyocytes classified as FCI [IP 3 ] i was 8.1±0.8 pmol/mg protein (n = 14) (p�0.001 compared to control and FCII values) and in those classified as FCII [IP 3 ] i was 14±2 pmol/mg protein (n = 10) (p�0.001 compared to control and FCI values) (Fig 6).  PLOS NEGLECTED TROPICAL DISEASES untreated cells), In FCI from 261±39 nM (n = 24) to 172±31 nM (n = 20) (p�0.001 compared to untreated cells) and in FCII from 377±44 nM (n = 15) to 207±33 nM (n = 18) (p�0.001 compared to untreated cells) (Fig 7A). Removal of extracellular [Ca 2+ ] did not modify significantly the effect of IP 3 BM, ET-1, and BK on [Ca 2+ ] d in Chagas' and control cardiomyocytes (Fig 7B, 7C and 7D) (p>0.05). These data indicate that the robust elevation of [Ca 2+ ] d elicited by IP 3 BM, ET-1, and BK in Chagas' and control cardiomyocytes is coming from an intracellular store rather than an extracellular Ca 2+ influx. Furthermore, that Ca 2+ entry from extracellular space plays a role in the perturbed cytosolic Ca 2+ regulation observed Chagas' cardiomyocytes.

Discussion
The current study reinforces our previous finding that a progressive deterioration of cardiac function in CC is associated at the cellular level with a defective intracellular Ca 2+ regulation. CC is the most severe and life-threatening manifestation of human Chagas disease and is one of the most common causes of heart failure and sudden death in Latin America. This disease has become a public health concern that is not limited to populations in Latin America but also poses a global problem because of migration of infected individuals for economic and/or political reasons to developed countries, mainly Europe and the United States.
The present study confirms that human cardiomyocytes isolated from Chagas' patients have an increase in [Ca 2+ ] d and a partial membrane potential depolarization, which corresponds with the degree of cardiac dysfunction determined by the NYHA classification [23]. In this report, we demonstrated, for the first time that IP 3 R activators, e.g., IP 3 BM, ET-1, and BKinduced a greater elevation of [Ca 2+ ] d in Chagas' compared to non-Chagas' human cardiomyocytes, which was not modified by the removal of [Ca 2+ ] e . Furthermore, Chagas' cardiomyocytes had a reduced SR Ca 2+ loading and a higher level of intracellular IP 3 with compromised contractile properties compared to control. Treatment with Xest-C, an IP 3 R blocker, improves [Ca 2+ ] d, increased SR-Ca 2+ loading and ameliorates contractile dysfunction in Chagas' cardiomyocytes.
Calcium is a central player in the regulation of cardiac contractility, and several cardiac pathologies have been associated directly or indirectly with changes in intracellular Ca 2+ handling. Normal functioning of multiple mechanisms like plasma-membrane exchanger (Na + / Ca 2+ exchanger) and pumps (PMCa 2+ and SERCA-ATPase pumps) which control Ca 2+ influx-efflux and reuptake allow for maintaining proper [Ca 2+ ] d during the rest period of the cardiac cycle (diastole) within a physiological range (~100 nM) [23,34]. The [Ca 2+ ] d values obtained from the control cardiomyocytes concur with previous estimations of the diastolic Ca 2+ level in human ventricular myocytes using Ca 2+ -selective microelectrodes [23] and fluorescent Ca 2+ indicator fluo-3 [47][48][49]. The magnitude of diastolic Ca 2+ elevation observed in Chagas cardiomyocytes correspond with the patients' functional class (NYHA). Perturbed intracellular Ca 2+ regulation in Chagas cardiomyocytes favors an intracellular Ca 2+ overload with direct consequences to systolic and diastolic function and also promotes arrhythmias, which have observed in patients suffering from CC [23,50,51]. Furthermore, chronic elevations in [Ca 2+ ] d as observed in Chagas' cardiomyocytes is deleterious to muscle cell function Cardiomyocytes were isolated from control (CTR) and Chagas patients (FCI and FCII) and observed using a video-based edge-detection system. (A) Resting sarcomere length was determined following 30 s of field stimulation at a frequency of 1 Hz (2 ms pulse duration,~1.5x threshold voltage) in quiescent cardiomyocytes; (B) Peak shortening (PS), (C) maximal velocity of shortening (+dL/dt), and (D) maximal velocity of relengthening (−dL/dt) were determined using steady-state twitches from 1 Hz electrical stimulation (2 ms pulse duration,~1.5x threshold voltage). Cardiomyocytes were obtained from 7-9 control individuals, 9-11 Chagas' FCI, and 7-9 Chagas' FCII patients. n represents the number of cardiomyocytes in which a successful measurement was carried out. Data are expressed as means ± S.D. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison tests. �� p�0.01, ��� p�0.001.
The changes in the [Ca 2+ ] d found in Chagas' cells are qualitatively similar to those reported in human epithelial cells infected with T. cruzi [54]. We consider that the elevation of [Ca 2+ ] d observed in Chagas' patients is related to the CC and not a resultant side effect from the patient's pharmacological treatment because all medications were suspended 48 h before the endomyocardial biopsy . The observed partial depolarization in Chagas' cardiomyocytes from FCI and FCII patients may relate to a diastolic Na + overload found in human Chagas' cardiomyocytes (1.4-fold in FCI and 2.1-fold in FCII compared to control). A membrane depolarization associated with intracellular Na + overload has been described in skeletal muscle cells [55]. Besides, an elevated [Na + ] d can contribute to a further intracellular Ca 2+ overload through the reverse mode of sarcolemmal Na + /Ca 2+ exchanger [56].
We previously presented evidence of a possible link between Chagas' infections and altered cellular Ca 2+ homeostasis and the intracellular messenger IP 3 [23]. Treatment with U-73122, a ß-phospholipase C inhibitor, and 2-APB partially reduced the elevated [Ca 2+ ] d in the Chagas' cardiomyocytes [23]. IP 3 -dependent Ca 2+ release represents the major pathway of intracellular Ca 2+ release in electrically non-excitable cells [24]. Although type 1 and 2 IP 3 receptors have been identified in several areas of cardiac cells and an IP 3 -Ca 2+ release has been well documented [57], the role of IP 3 in excitation-contraction coupling and cardiac function in the mammalian heart has remained controversial [58]. Several studies suggest that IP 3 may be involved in the regulation of the gene transcription [59], the amplification of ryanodine receptor signals [60], and the regulation of Ca 2+ influx through the modulation of transient receptor potential channel (TRPC) [34]. In contrast to the physiological condition, a more pronounced role of IP 3 has been suggested under various cardiac pathologies (e.g., cardiac hypertrophy, ischemic dilated cardiomyopathy, atrial fibrillation, failing myocardium and hypertension) [26,61,62]. Thus, increased expression of IP 3 Rs in the perinuclear compartment has been observed hypertrophied and failing hearts, which have associated with altered nucleoplasmic Ca 2+ regulation and an increase in diastolic [Ca 2+ ] d [63]. In this context, Harzheim et al. [25] have suggested that an increase in IP 3 Rs expression is a general mechanism that underlies remodeling of Ca 2+ signaling during heart disease, and in particular, in triggering arrhythmia during hypertrophy. Moreover, IP 3 -induced Ca 2+ release is increased in SR microsomes prepared from hypertrophic myocytes [64]. Additionally, elevated IP 3 R levels and increased InsP 3 binding has been reported in the left ventricle during human heart failure [29].
Further support for the IP 3 involvement in CC was obtained by showing that exposure of cardiomyocytes to agents that enhance endogenous generation or concentration of IP 3 like IP 3 BM, ET-1 or BK [39,40] caused an elevation in [Ca 2+ ] d which was always greater in cardiomyocytes from Chagas' patients than non-Chagas' subjects and related to the degree of cardiac dysfunction (FCII>FCI). The differential pharmacological effect of IP 3 BM on [Ca 2+ ] d in Chagas' cardiomyocytes persists up to a concentration of 30 μM, where the [IP 3 ] i levels would be equivalent between control and Chagas' cardiomyocytes, suggesting a greater IP 3 Rs expression in Chagas cardiomyocytes compared to control. The IP 3 BM, ET-1, or BK effects on [Ca 2+ ] d were not modified by the removal of extracellular Ca 2+ , but it was inhibited by Xest-C, suggesting that their pharmacological action is mediated through IP 3 -dependent Ca 2+ release. These results reinforce the notion that increased [Ca 2+ ] d observed in Chagas' cardiomyocytes is mediated in part by activation of IP 3 Rs.
The fact that incubation in L-IP 3 PM did not induce any change in [Ca 2+ ] d either in control or Chagas' cardiomyocytes indicates that the action of IP 3 BM was highly specific. Individuals with CC had increased levels of ET-1 in plasma [42], plasma ET-1 levels are elevated in mice infected with T. cruzi, and there is an increased expression of myocardial mRNA for ET-1 [65]. These findings represent the first report of an IP 3 -enhanced release of intracellular Ca 2+ induced by IP 3 BM-, ET-1-, or BK in human Chagas' cardiomyocytes.
In Chagas' cardiomyocytes, chronic elevated [Ca 2+ ] d may enhance the IP 3 sensitivity of IP 3 Rs [66] and could well synergize with the other factors that further elevate [Ca 2+ ] d . An increase in IP 3 Rs expression has been reported in atrial myocytes of humans and dogs during atrial fibrillation and in human heart failure [67,68]. The IP 3 R expression is significantly elevated in rat cardiac tissue from aorta-banded hypertrophic mice and human ischemic heart with dilated cardiomyopathy [25,26,29]. An elevated IP 3 R expression may represent a plausible explanation for the increased [Ca 2+ ] d observed in Chagas' cardiomyocytes.
In cardiomyocytes isolated from Chagas' patients [IP 3 ] i was higher compared to those from control subjects. It has been previously shown in various types of cells that elevation of IP 3 production, which release Ca 2+ from intracellular stores [24,69] may lead to an increase of [Ca 2 + ] d [69] and a robust Ca 2+ release upon exposure to IP 3 BM-, ET-1-, or BK [69]. The elevated intracellular [IP 3 ] can have two possible sources i) the plasma membrane of parasites in intracellular forms, such as amastigotes [70] and ii) IP 3 derived from the plasma membrane of the host changes due to changes in IP 3 synthesis and/or degradation [71,72]. Furthermore, an elevated [IP 3 ] i may provoke an increase in Ca efflux from the SR, which could end in a depletion of intraluminal sarcoplasmic reticulum Ca 2+ content [24,73]. We have found in Chagas' cardiomyocytes a decrease in SR Ca 2+ content compared to control (Control>FCI>FCII), and blocking the IP 3 Rs with Xest-C results in a significant increase in SR-Ca 2+ content in Chagas' cardiomyocytes which indicates that IP 3 Rs may play an intrinsic role in the intracellular Ca 2+ dysregulation in CC.
Chagas' cardiomyocytes exhibit markedly depressed contractile properties versus control across all parameters studied, such as peak shortening, maximal velocity of shortening (Con-trol>FCI>FCII), which may be related to a reduced SR Ca 2+ loading and subsequent intracellular Ca 2+ release. It is well established that Ca 2+ release directly regulates contractility of cardiomyocytes, and that a reduced release from intracellular stores decreases force development under heart failure [74,75]. Chagas' cardiomyocytes also showed an altered velocity of re-lengthening, which may be due to a defect of relaxation controlled by the SR-ATPase pump (SERCA), the NCX and/or the plasma membrane Ca 2+ pump (PMCA). The chronic elevation of the intracellular IP 3 levels in addition to the induced sustained increase in [Ca 2+ ] d , also elicits a Ca 2+ depletion of the SR, depressing the amount of Ca 2+ for release upon electrical stimulation [24,73]. Furthermore, a shorter resting sarcomere length was observed in Chagas' cardiomyocytes, which corresponds with chronic elevated [Ca 2+ ] d . Pretreatment with Xest-C partially reverse the contractile dysfunction in CC by significantly increasing PS, +dL/dt, and -dL/dt. The enhancement of contractile function induced by Xest-C may be related to the inhibition of IP 3 Rs and the prevention of SR Ca 2+ depletion.
An interesting observation was that depletion of extracellular Ca 2+ provoked a more significant reduction of [Ca 2+ ] d in Chagas than control cardiomyocytes. Several mechanisms of Ca 2+ entry non-voltage dependent have been described in cardiac cells; among them, the TRPC, a diversely regulated family of plasma membrane permeable cation channels, which are activated by diacylglycerol, by depletion of intracellular Ca 2+ stores or by stretch [76]. Biochemical and functional studies suggest a close coupling of some TRPC channels and InsP 3 R [77]. Further studies are necessary to establish the role of the TRPC channels in the CC.
In conclusion, patients suffering from CC have a chronic elevation of [Ca 2+ ] d that appears to be mediated by IP 3 Rs and is associated with the deterioration of cardiac function (FCII>FCI). Consistent with these results, agents that enhance intracellular IP 3 generation like ET-1, BK, or membrane-permeant IP 3 esters caused a further elevation in [Ca 2+ ] d more significant in cardiomyocytes from Chagas' than non-Chagas' subjects-and Xest-C an IP 3 Rs blocker decreased [Ca 2+ ] d , and improved cardiomyocytes contractile response from Chagas' patients. Furthermore, Chagas' cardiomyocytes had a higher level of intracellular [IP 3 ] with compromised SR-Ca 2+ loading compared to control.
These novel findings reveal an unmask mechanism by which IP 3 may play an essential role in the pathophysiology of CC and open the door for new therapeutic targets oriented at improving cardiac function and therefore, the quality of life of individuals suffering from CC. These discoveries are of paramount importance because there is still no highly effective cure available for those currently infected with T. cruzi, a third of which will develop potentially fatal cardiomyopathy.

Limitations of the study
The major limitation of this study is that downstream IP 3 cell-signaling and IP 3 Rs expressions in Chagas' cardiomyocytes were not studied. Scarcity and accessibility to human endomyocardial tissue were restrictions to carry out those experiments. Endomyocardial biopsies are conducted in patients under sedation via fluoroscopic guidance, and the tissue samples from each patient studied are limited in size (2 to 3 mm 3 ) and number (2 to 3 biopsies per patient). Furthermore, enzymatic isolation of intact ventricular cardiomyocytes from human heart biopsies is less successful than the retrograde perfusion of the whole heart used in experimental models. Determination of IP 3 cell-signaling and the expression of IP 3 Rs in the human cardiac cells have been conducted in explanted hearts from patients who underwent cardiac transplantation [25,29] or during coronary artery bypass surgery [68], where muscle size and tissue quantity are not limited. The observed changes in diastolic [Ca 2+ ] and intracellular [IP 3 ] in cardiomyocytes isolated from chagasic patients should be interpreted with caution. Both changes may occur as an epiphenomenon in a heart as a consequence of multiple pathological alterations observed in CC. However, despite the above limitations, we have confirmed the involvement of intracellular Ca 2+ dysregulation, and we unmask a thus far unrecognized involvement of IP 3 in the pathophysiology of CC.

S1 Fig. No effects of L-IP3PM on [Ca2+]d in cardiomyocytes from control and Chagas' patients.
[Ca 2+ ] d was measured using Ca 2+ -selective microelectrodes before and after treatments with L-myoinositol 1,4,5-trisphosphate hexakis(propionyloxy-methyl) ester (L-IP 3 PM). The incubation in L-IP 3 PM did not induce significant changes in [Ca 2+ ] d either in control (CTR) or Chagas' cardiomyocytes. Cardiomyocytes were obtained from 8-10 control individuals, 7-9 Chagas' FCI, and 6-8 Chagas' FCII patients, respectively; n represents the number of cardiomyocytes in which a successful measurement of [Ca 2+ ] d was carried out. Data are expressed as means ± S.D. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison tests, ��� p�0.001.