Human induced pluripotent stem cell-derived cardiomyocytes to study inflammation-induced aberrant calcium transient

Abstract

Heart failure with preserved ejection fraction (HFpEF) is commonly found in persons living with HIV (PLWH) even when antiretroviral therapy suppresses HIV viremia. However, studying this condition has been challenging because an appropriate animal model is not available. In this article, we studied calcium transient in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in culture to simulate the cardiomyocyte relaxation defect noted in PLWH and HFpEF and assess whether various drugs have an effect. We show that treatment of hiPSC-CMs with inflammatory cytokines (such as interferon-γ or TNF-α) impairs their Ca²⁺ uptake into sarcoplasmic reticulum and that SGLT2 inhibitors, clinically proven as effective for HFpEF, reverse this effect. Additionally, treatment with mitochondrial antioxidants (like mito-Tempo) and certain antiretrovirals resulted in the reversal of the effects of these cytokines on calcium transient. Finally, incubation of hiPSC-CMs with serum from HIV patients with and without diastolic dysfunction did not alter their Ca²⁺-decay time, indicating that the exposure to the serum of these patients is not sufficient to induce the decrease in Ca²⁺ uptake in vitro. Together, our results indicate that hiPSC-CMs can be used as a model to study molecular mechanisms of inflammation-mediated abnormal cardiomyocyte relaxation and screen for potential new interventions.

Keywords: calcium transient; heart failure with preserved ejection fraction; human; human immunodeficiency virus; human induced pluripotent stem cell-derived cardiomyocyte; infectious disease; inflammation; microbiology.

Figure 1.. Analyses of calcium transient in hiPSC-CMs treated with cytokines and/or MitoTempo.

Normalized downstroke time (A), decay time (B), and beating rate as assessed by beats per minute (C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) treated with bovine serum albumin (BSA), (control), TNF-α, IFN-γ, TNF-α plus mito-Tempo, and IFN-γ plus mito-Tempo. N = 5–14. Data were analyzed by ordinary one-way ANOVA and post hoc Tukey’s multiple-comparison test. Bars represent group mean, and error bars indicate standard error of the mean.

Figure 1—figure supplement 1.. A representative calcium transient curve in hiPSC-CMs and their mitochondrial potential.

(A) Representative image of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Scale bar represents 100 μm. (B) Representative curves of calcium transient and its derivative in hiPSC-CM. (C) Mitochondrial membrane potential (MMP) as assessed by tetramethylrhodamine ethyl ester (TMRE) in hiPSC-CM treated with various cytokines. Concentration of cytokines used is as follows: CCL3 1 nM, TNF-α 300 pg/ml, IL-6 1 ng/ml, IL-1β 200 pg/ml, IP-10 (or CXCL10) 500 pg/ml, IFN-γ 1 ng/ml. N = 4, Data were analyzed by ordinary one-way ANOVA and post hoc Tukey’s multiple-comparison test. Bars represent group mean , and error bars indicate standard error of the mean.

Figure 2.. Oxygen consumption rate (OCR) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) treated with cytokines and antioxidant agent N-acetylcysteine (NAC).

(A) OCR trace of hiPSC-CM treated with bovine serum albumin (BSA), TNF-α, IFN-γ, TNF-α + IFN-γ, TNF-α + NAC, IFN-γ + NAC, and TNF-α + IFN-γ + NAC. (B–D) Bar graph summary of data in (A) with OCR at baseline (B), after adding carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (C), and after adding antimycin A and rotenone (D). N = 5–6. Data were analyzed by ordinary one-way ANOVA and post hoc Tukey’s multiple-comparison test. Bars represent group mean, and error bars indicate standard error of the mean.

Figure 3.. Decay time in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) after treatment with TNF-α and various antiretroviral therapy (ART) drugs.

(A) Decay time after treatment with TNF-α, tenofovir, and TNF-α+tenofovir. (B) Decay time after treatment with TNF-α, darunavir, and TNF-α+darunavir. (C) Decay time after treatment with TNF-α, raltegravir, and TNF-α + raltegravir. (D) Decay time after treatment with TNF-α, emtricitabine, and TNF-α + emtricitabine. N = 5–21. Data were analyzed by ordinary one-way ANOVA and post hoc Tukey’s multiple-comparison test. Bars represent group mean, and error bars indicate standard error of the mean.

Figure 3—figure supplement 1.. Cell viability and ROS levels in hiPSC-CMs treated with anti-HIV drugs.

Cell viability (A) and cellular reactive oxygen species (ROS) levels (B) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) exposed to concentrations ranging from between 3 nM and 10 μM of tenofovir (a nucleotide-analog reverse transcriptase inhibitor), darunavir (a protease inhibitor), raltegravir, and elvitegravir (integrase inhibitors). N=1-6.

Figure 4.. Decay time in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) after treatment with TNF-α and riociguat, sildenafil citrate, PF-04447943, and dapagliflozin.

(A) Decay time after treatment with TNF-α, sGS agonist riociguat, and TNF-α + riociguat (N = 8–21). (B) Decay time after treatment with TNF-α, sildenafil citrate, and TNF-α + sildenafil citrate (N = 5–21). (C) Decay time after treatment with TNF-α, PF-04447943, and TNF-α+PF-04447943 (N = 7–21). (D) Representative curve of calcium transient in hiPSC-CM treated with TNF-α, dapagliflozin, and TNF-α + dapagliflozin. (E) Curve of derivative calculated with data in (D). (F) Decay time after treatment with TNF-α, dapagliflozin, and TNF-α+dapagliflozin (N = 5–21). (G) Downstroke time after treatment with TNF-α, dapagliflozin, and TNF-α + dapagliflozin(N = 5–21). (H) Representative curve of calcium transient in hiPSC-CM treated with IFN-γ, dapagliflozin, and IFN-γ + dapagliflozin. (I) Curve of derivative calculated with data in (H). (J) Decay time after treatment with INF-γ, dapagliflozin, and IFN-γ + dapagliflozin (N = 5–21). (K) Downstroke time after treatment with INF-γ, dapagliflozin, and IFN-γ + dapagliflozin (N = 5–21). Data were analyzed by ordinary one-way ANOVA and post hoc Tukey’s multiple-comparison test. Bars represent group mean, and error bars indicate standard error of the mean.

Figure 4—figure supplement 1.. Analyses of calcium transient in hiPSC-CMs treated with TNFa and/or 1 μM Dapagliflozin.

Decay time (A) and downstroke time (B) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) after treatment with TNF-α and 1 μM of dapagliflozin. N = 5–21, Data were analyzed by ordinary one-way ANOVA and post hoc Tukey’s multiple-comparison test. Bars represent group mean, and error bars indicate standard error of the mean.

Figure 5.. Ca²⁺-decay time of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and angiogenic function of ECs after treatment with serum from group 1 HIV+ patients with diastolic dysfunction (DD).

(A–C) Pooled normalized decay time (A), downstroke time (B), and beating rate (C) in hiPSC treated with 10% serum for 48 hr from group 1 HIV+ patients with DD (3 values from cells treated with each serum). (D–G) Angiogenic parameters, including total length of branching (D) (N = 3), number of junctions (E) (N = 3), total length (F) (N = 3), and number of meshes (G) (N = 3) in human umbilical vein endothelial cells (HUVEC) after treatment with 2% serum from group 1 patients for 20 hr. Data were analyzed by unpaired Student’s t-test. Bars represent group mean, and error bars indicate standard error of the mean.

Figure 5—figure supplement 1.. Representative images of formed tube structure with human umbilical vein endothelial cells (HUVEC) treated with 2% serum from HIV+ patients with high or low myocardial perfusion reserve (MPR) for 20 hr.

Scale bars represent 500 μm.

Figure 5—figure supplement 2.. Representative magnified images of formed tube structure with human umbilical vein endothelial cells (HUVEC) treated with 2% serum from HIV+ patients with high or low myocardial perfusion reserve (MPR) for 20 hr.

Scale bars indicate 250 μm.

Figure 6.. Diastolic function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and angiogenic function of ECs after treatment with serum from HIV+ patients with diastolic dysfunction (DD) (group 2).

(A, B) Individual normalized decay time 3 hr (N = 3–9) (A) or 24 hr (N = 3–13) (B) after treatment of hiPSC-CM with 2% serum from patients. (C, D) Pooled decay time data from patients in (A) and (B) with 3 hr (C) and 24 hr (D) after treatment with serum (N = 21–22 for (C) and N = 31-38 for (D)). (E) Representative image of formed tube structure with cultured human umbilical vein endothelial cells (HUVEC) treated with serum from HIV+ patients with DD. Scale bars indicate 250 μm. (F–I) Assessment of tube formation of HUVECs 20 hr after treatment with serum of patients, including number of meshes (F) (N = 8), number of junctions (G) (N = 8), number of segments (H) (N = 8), and total length of branching (I) (N = 8). Data were analyzed by unpaired Student’s t-test for (C, D, F, G, H, I). Bars represent group mean, and error bars indicate standard error of the mean.