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

Figures

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  Fig 1. Diastolic [Ca2+] is greater in cardiomyocytes from patients suffering from Chagas cardiomyopathy than control. Representative simultaneous measurements of the resting membrane potential (Vm) and diastolic Ca2+ concentration ([Ca2+]d) in cardiomyocytes isolated from control (CTR) and Chagas’ patients (FCI and FCII). (A) Recording of Vm = -83 mV and [Ca2+]d = 122 nM measured in a control cardiomyocyte; (B) Recordings of Vm and [Ca2+]d from a cardiomyocyte isolated from Chagas’ patient FCI (Vm = -75 mV and [Ca2+]d = 263 nM); (C) Recordings of Vm and [Ca2+]d from a cardiomyocyte isolated from Chagas’ patient FCII (Vm = -72 mV and [Ca2+]d = 364 nM).

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  Fig 2. Diastolic [Ca2+] and [Na+] are increased in Chagas’ cardiomyocytes. Summary of the recording of [Ca2+]d (2A) and [Na+]d (2B) in cardiomyocytes isolated from control and FCI and FCII Chagas’ cardiomyocytes. Cardiomyocytes were obtained from 17 control individuals, 18 Chagas’ FCI, and 14 Chagas’ FCII patients. n represents the number of cardiomyocytes in which a successful measurement of [Ca2+]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.

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  Fig 3. Effects of ET-1, BK and IP3BM on [Ca2+]d in cardiomyocytes from control and Chagas’ patients. [Ca2+]d was measured using Ca2+-selective microelectrodes before and after treatments with agents that enhance intracellular inositol 1,4,5 trisphosphate generation or concentration. (A) Effects of 10 μM membrane-permeant myoinositol 1,4,5-trisphosphate hexakis(butyryloxy-methyl) ester (IP3BM) on [Ca2+]d in cardiomyocytes isolated from control (CTR), FCI, and FCII patients. (B) Effects of 100 nM endothelin (ET-1) on [Ca2+]d in cardiomyocytes from control, FCI, and FCII patients. (C) Effects of 10 nM bradykinin (BK) on [Ca2+]d in cardiomyocytes from control individuals, FCI, and FCII patients. Cardiomyocytes were obtained from 9–12 control individuals, 9–11 Chagas’ FCI, and 6–10 Chagas’ FCII patients respectively. n represents the number of cardiomyocytes in which a successful measurement of [Ca2+]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.

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  Fig 4. Xestospongin-C reduces [Ca2+]d and prevents the effects of IP3BM, and ET-1 on diastolic Ca2+ concentration. [Ca2+]d was measured in cardiomyocytes isolated from control (CTR) and Chagas patients (FCI and FCII). (A) Pretreatment with Xest-C reduced significantly [Ca2+]d in FCI and FCII, but not in control cardiomyocytes; (B) Incubation in Xest-C prevented the elevation of [Ca2+]d induced by 10 μM myoinositol 1,4,5-trisphosphate hexakis(butyryloxy-methyl) ester (IP3BM) in all cells; (C) Xest-C inhibited the effect of 100 nM Endothelin (ET-1) on [Ca2+]d in control and FCI and FCII cardiomyocytes. Cardiomyocytes were obtained from 9–10 control individuals, 10–12 Chagas’ FCI, and 8–10 Chagas’ FCII patients, respectively. 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.

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  Fig 5. Decreases sarcoplasmic reticulum Ca2+ loading in chagasic cardiomyocytes. Control (CTR) and Chagas’ cardiomyocytes loaded with Fluo-4-AM were exposed to caffeine in Ca2+-free solution. Under these conditions, the total Ca2+ released was significantly smaller in Chagas’ cardiomyocytes (FCI<FIC) compared with the control cardiomyocytes (area under the curve: 49±7 in control versus 31±5 (p≤0.001) in FCI and 19±4 (p≤0.001) in FCII). Cardiomyocytes were obtained from 6 control individuals, 7 Chagas’ FCI, and 8 Chagas’ FCII patients respectively. 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.

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  Fig 6. Intracellular [IP3] in control and Chagas’ cardiac tissue. Levels of [IP3]i were determined by the competitive radioligand-binding assay. [IP3]i was significantly higher in ventricular cells in patients with Chagas’ disease (FCI and FCII) than in control (CTR). [IP3]i was elevated by 47% in FCI and 174% in FCII compared with control hearts. Heart samples were obtained from 10 control individuals, 11 FCI, and 10 FCII patients, respectively. n represents the number of determinations. 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.

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  Fig 7. Effects of extracellular Ca2+ on [Ca2+]i in control and Chagas’ cardiomyocytes. (A) Incubation of cardiomyocytes in Ca2+-free medium (see Materials and Methods) resulted in a significant reduction in [Ca2+]d in both groups of cells. However, the magnitude of [Ca2+]d decrease was more significant in Chagas (FCII>FCI) compared to the control (CTR) cardiomyocytes; (B) Removal of extracellular Ca2+ did not block the effect of IP3BM on [Ca2+]d in control and Chagas’ cardiomyocytes. (C) Withdrawal of extracellular Ca2+ did not inhibit the effect of ET-1 on [Ca2+]d in control and Chagas’ cardiomyocytes. (D) The effect of BK on [Ca2+]d in control and Chagas’ cardiomyocytes was not modified by free Ca2+ solution. Cardiomyocytes were obtained from 6 control individuals, 8 Chagas’ FCI, and 6 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.

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  Fig 8. The depressed contractile function of Chagas’ cardiomyocytes is improved by Xestospongin-C treatment. 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.