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Peroxisomes contribute to intracellular calcium dynamics in cardiomyocytes and non-excitable cells

View ORCID ProfileYelena Sargsyan, Uta Bickmeyer, View ORCID ProfileChristine S Gibhardt, Katrin Streckfuss-Bömeke, Ivan Bogeski, View ORCID ProfileSven Thoms  Correspondence email
Yelena Sargsyan
1Department of Child and Adolescent Health, University Medical Center, Göttingen, Germany
Roles: Formal analysis, Investigation, Visualization, Writing—original draft, review, and editing
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  • ORCID record for Yelena Sargsyan
Uta Bickmeyer
1Department of Child and Adolescent Health, University Medical Center, Göttingen, Germany
Roles: Investigation, Writing—review and editing
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Christine S Gibhardt
2Molecular Physiology, Institute of Cardiovascular Physiology, University Medical Center, Göttingen, Germany
Roles: Investigation
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Katrin Streckfuss-Bömeke
3Clinic for Cardiology and Pneumology, University Medical Center, Göttingen, Germany
4Institute of Pharmacology and Toxicology, Würzburg University, Würzburg, Germany
6German Center of Cardiovascular Research (DZHK), Partner Site Göttingen, Germany
Roles: Resources, Supervision, Writing—original draft
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Ivan Bogeski
2Molecular Physiology, Institute of Cardiovascular Physiology, University Medical Center, Göttingen, Germany
Roles: Resources, Supervision, Writing—original draft
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Sven Thoms
1Department of Child and Adolescent Health, University Medical Center, Göttingen, Germany
5Department of Biochemistry and Molecular Medicine, Medical School, Bielefeld University, Bielefeld, Germany
6German Center of Cardiovascular Research (DZHK), Partner Site Göttingen, Germany
Roles: Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Project administration, Writing—original draft, review, and editing
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  • For correspondence: sven.thoms@med.uni-goettingen.de sven.thoms@uni-bielefeld.de
Published 30 July 2021. DOI: 10.26508/lsa.202000987
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    Figure 1. New sensors for peroxisomal Ca2+.

    (A) Genetically encoded calcium indicators (GECIs) targeted to peroxisomes. D3cpv-px and D1cpV-px are Förster resonance energy transfer (FRET) sensors with modified CaM sites. Pericam-px is a single fluorophore-based GECI that has M13 and CaM as Ca2+-binding sites. In the absence of Ca2+, the emission measured when the sensor is excited with 420 nm is higher than when excited with 505 nm. The ratio 505/420 is a measure for the Ca2+ concentration. (B) Subcellular localisation of GECIs used in this study. (C) Peroxisomal GECIs colocalise with the peroxisomal membrane marker PEX14 or PMP70. HeLa cells were transfected with the GECIs and stained with anti-PEX14 or anti-PMP70 antibodies. The images in the left part of the panel show one cell each (scale bar 10 µm). The cropped areas are marked and magnified in the right part of the panel (scale bar 2 µm). (D, E, F) D3cpv-px, D1cpv-px, and pericam-px are Ca2+ sensitive. Images false-colored with look-up table show representative cells before (left) and after (right) Ca2+ addition. Curves presented as mean ± SEM. Scale bar: 10 µm. (D) Addition of 1 mM Ca2+ to D3cpV-px expressing cells results in 1.5-fold FRET ratio increase, n = 60 cells from three independent experiments. (E) FRET ratio increases 1.08 times when 1 mM Ca2+ is added to D1cpv-px expressing cells, n = 33 cells from three experiments. (F) Ca2+ addition leads to 1.5-fold increase in 505/420 ratio with pericam-px, n = 75 cells from three experiments. (G) Measurement of D3cpV-px during cytosol washout. No change in signal is detected. (H) Measurement of pericam-px during cytosol washout. (G) No difference of signal before and after cytosol washout is detected, n = 43 cells for D3cpV-px in (G) and n = 45 cells for pericam-px in (H).

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    Figure 2. Measurement of peroxisomal Ca2+ in HeLa cells.

    (A) Quantification of D3cpV-px colocalisation with peroxisomes. Mander’s colocalisation coefficient was normalised to PMP70 and catalase colocalisation, which was set to 1. n = 5. (B) Förster resonance energy transfer ratio measured at different pH values for D3cpV (cyto) and D3cpV-px (pero). Cells were incubated in buffers with different pH values containing 10 µM nigericin. Cyto and pero show comparable results at physiological pH values of cytosol and peroxisomes. At the pH = 4 the Förster resonance energy transfer ratio decreases drastically because of the acceptor sensitivity. Cell numbers for cyto at pH 4 n = 51, 7.1 = 51, 7.35 = 51, 7.5 = 67, 7.65 = 48, 7.8 = 48; for pero at pH 4 n = 50, 7.1 = 50, 7.35 = 71, 7.5 = 71, 7.65 = 64, 7.8 = 64 from three independent experiments per condition. (C) Comparison of cytosolic and peroxisomal responses to ionomycin (Iono). In comparison to cytosol, peroxisomal signal increases gradually, n = 16 cells for D3cpV and n = 9 cells for D3cpV-px. (D) One-step experiment in HeLa cells with thapsigargin (Tg) addition in Ca2+-containing buffer. Cytosolic and peroxisomal Ca2+ increase upon Tg treatment. n = 31 (cyto), 30 (pero) from four (cyto) and five (pero) independent experiments. (E) Experimental paradigm of a two-step Ca2+ measurement in non-excitable cells. First peak after histamine (His) addition: ER-store depletion. Second peak, after addition of extracellular Ca2+: plasma membrane-based uptake. (F) Measurement of the effect of treatments from (E) on the acceptor. Neither cytosolic EYFP (cyto) nor peroxisomal Venus-ACOX3 (pero) undergo changes upon treatment, suggesting that the pH changes during the experiment will not affect the measurement with genetically encoded Ca2+ indicators. Data presented as mean, n = 44 (cyto), 44 (pero). (G) Measurement with D3cpV-px according to the paradigm in (E). Two Ca2+ peaks of the experimental paradigm are detectable with D3cpV-px, n ≥ 50 cells from three experiments. (H) In situ Kd calculation and the saturation curve of D3cpV and D3cpV-px. Curve fitting was performed with one-site model with Hill coefficient. (G, I) Absolute Ca2+ concentration dynamics calculated from the data in (G). (I, J) Basal and maximum (max) Ca2+ concentrations in peroxisomes based on (I). (K) Measurement with pericam-px according to the paradigm in (E), n = 27 cells from three experiments. (L) Simultaneous measurement of cytosolic (blue) and peroxisomal (green) Ca2+. No delay of signal increase after histamine addition, but a delayed drop of the signal in peroxisomes. Left y-axis: of D3cpV-px (peroxisomal sensor). Right y-axis: Fn/F0 ratio of R-GECO1 (cytosolic sensor), n = 35 cells from three experiments. (L, M) Decline of Fn/F0 ratio per minute (min) in the linear part of the curves in (L) (from second 65–115, t test). Kinetic delay in decrease in peroxisomal signal is seen. (A, B, C, G, H, I, K, M) Data presented as mean ± SEM. (I) Data presented as Tukey’s box plots. ***P < 0.001.

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    Figure 3. Comparison of peroxisomal with cytosolic and mitochondrial Ca2+ handling in HeLa cells.

    (A) Comparison of cytosolic, mitochondrial, and peroxisomal Ca2+ response measured following the two-step measurement described in Fig 1E. Characteristic two peaks present in all three compartments. (A, B) Basal levels of Ca2+ in peroxisomes are similar to mitochondria. Analysis performed based on the data from (A). (C) Peroxisomal Ca2+ increase upon ER-store depletion is smaller than that of cytosol or mitochondria. Analysis performed based on the data from (A). (A, D) Increase of Förster resonance energy transfer ratio per minute (min) in the linear part of the curves in (A) (from second 27–42). Slower increase in peroxisomal signal is seen. Data presented as mean ± SEM. (E) Peroxisomal Ca2+ increase upon plasma membrane-based cellular uptake of Ca2+ is comparable to mitochondria. Analysis performed based on the data from (A). (B, C, D, E) One-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ****P < 0.0001, Cyto: cytosolic, mito: mitochondrial, pero: peroxisomal. n = 83 (cyto), 116 (mito), 117 (pero) cells from six independent experiments. (B, C, E) Data presented as Tukey’s box plots.

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    Figure 4. Effect of mitochondrial calcium uniporter (MCU) knockdown on mitochondria and peroxisomal Ca2+ measured in the same cells.

    (A) mRNA expression of MCU-silenced (siMCU) HeLa cells in comparison to control (siCtrl), quantified by RT-qPCR. (B) Mitochondrial Ca2+ response in siCtrl and siMCU measured after the histamine addition with Ca2+-containing buffer. MCU knockdown results in decreased Ca2+ uptake to mitochondria. (C) Mitochondrial Ca2+ increase is smaller in siMCU. (B) Analysis performed based on the data from (B). (D) Peroxisomal Ca2+ response in siCtrl and siMCU measured following the histamine addition with Ca2+-containing buffer. (E) MCU knockdown does not affect maximum Ca2+ uptake to peroxisomes. (D) Quantification of data from (D). Cell numbers n = 73 (siCtrl), 61 (siMCU). (C, E) Data presented as Tukey’s box plots. ****P < 0.0001.

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    Figure 5. Measurement of peroxisomal Ca2+ in cardiomyocytes.

    (A) Experimental paradigm of Ca2+ measurement in excitable cells. The peak after thapsigargin (Tg) addition represents Ca2+ increase due to the sarcoplasmic/endoplasmic reticulum calcium ATPase inhibition and Ca2+ retention in the cytosol. (A, B) Cytosolic Ca2+ measurement in NRCMs following the experimental design in (A), n = 25 (Tg), 22 (control) from three experiments. Addition of Tg is compared with the addition of Tg-free buffer (control). (B, C) Basal levels are not different before the treatment in (B). (B, D) After Tg addition in (B) cytosolic Ca2+ increases. (A, E) Peroxisomal Ca2+ measurement in NRCMs following the experimental design in (A). Addition of Tg is compared with the addition of Tg-free buffer (control), n = 20 (Tg), 31 (control) from three experiments. (E, F) Basal levels of Ca2+ are not different before the treatment in (E). (E, G) Peroxisomal Ca2+ increases after Tg addition in (E). (H) Human-induced pluripotent stem cell (HiPSC)-CMs generation. Donor skin fibroblasts were reprogrammed to hiPSCs, which were then differentiated to CMs. (I) hiPSC-CMs were stained for cardiac troponin T (cTnT) and analysed by flow cytometry. Negative control without primary antibody. 94.8% of iPSC-CMs are cTnT-positive (cTNT+). (J) Immunofluorescence staining visualized α-actinin protein expression and regular sarcomeric organisation. Scale bar: 20 μm. (A, K) Cytosolic Ca2+ measurement in hiPSC-CMs with D3cpV following the experimental paradigm for excitable cells in (A). Addition of Tg is compared to the addition of Tg-free buffer (control) to avoid artefacts and false results of the mechanical effect on the cells because of the addition itself. n = 24 (Tg), 27 (control) from three experiments. (K, L) No difference is found between two groups before the treatment in (K). (K, M) Tg addition in (K) results in cytosolic Ca2+ increase. (A, N) Peroxisomal Ca2+ measurement in hiPSC-CMs with D3cpV-px following the experimental design for excitable cells depicted in (A). Addition of Tg is compared with the addition of Tg-free buffer (control). n = 26 (Tg), 33 (control) from three experiments. (N, O) Basal levels of Ca2+ are not different before the treatment in (N). (M, P) Peroxisomal Ca2+ increases after Tg addition in (M). (B, E, K, N) Data presented as means from three independent experiments. (C, D, F, G, L, M, O, P) Unpaired t test was used for the statistical analysis. ****P < 0.0001, Tukey’s box plots.

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    Figure 6. Relative localisation of peroxisomes and Ca2+ channels in human induced pluripotent stem cell–CMs.

    Human-induced pluripotent stem cell–CMs were either transfected with D3cpV-px (left panels) or stained with anti-Pex14 (right panels) as a peroxisomal marker. (A) Representative images from staining of L-type Ca2+ channel (LTCC) show occasional proximity of peroxisomes and LTCC. (B) Representative images from staining of ryanodine receptor (RyR2) show occasional yet more often contact of peroxisomes with the RyR2 than with LTCC. DAPI is shown in blue. Scale bar 5 μm.

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    Figure 7. Measurement of peroxisomal Ca2+ in paced cardiomyocytes.

    (A) D3cpV transfected NRCMs are stimulated with 1 Hz. Images are taken every 50 ms. Oscillations of Förster resonance energy transfer ratio are seen, n = 3. (A, B) FFT from the data in (A). Signal increases are rhythmic and correspond to the pacing frequency. (C) Förster resonance energy transfer ratio oscillates in D3cpV-px transfected NRCMs stimulated with 1 Hz. Images are taken every 100 ms, n = 3. (C, D) FFT from the data in (C). Signal increases are rhythmic and correspond to the pacing frequency.

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    Figure 8. Peroxisomal Ca2+ entry and cellular Ca2+ distribution.

    ER Ca2+ release triggers Ca2+ entry into the peroxisome. In this hypothetical model, ER–peroxisome proximity defines Ca2+ microdomains with locally elevated Ca2+ concentration shielded from the cytosol. As a result, Ca2+ entry to peroxisomes follows the local gradient but peroxisomal Ca2+ is eventually higher than in the cytosol. IP3Rc, IP3 receptor calcium release channel of the ER.

Tables

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    Table 1.

    Key properties of the genetically encoded Ca2+ indicators (GECIs) for cytosol and peroxisome.

    Cytosolic GECIsPeroxisomal GECIs (this study)
    Construct NameKd (in vitro)Dynamic range, DConstruct NameMaximal increase upon 1 mM Ca2+ addition
     D3cpV0.6 µMa5.0aD3cpV-px1.50×
     D1cpV60 µMb1.7cD1cpV-px1.08×
     Ratiometric-pericam1.7 µMd10.0dPericam-px1.50×
    • ↵a References: Palmer et al (2006).

    • ↵b Palmer et al (2004).

    • ↵c Greotti et al (2016).

    • ↵d Nagai et al (2001).

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Peroxisomes contribute to intracellular calcium dynamics
Yelena Sargsyan, Uta Bickmeyer, Christine S Gibhardt, Katrin Streckfuss-Bömeke, Ivan Bogeski, Sven Thoms
Life Science Alliance Jul 2021, 4 (9) e202000987; DOI: 10.26508/lsa.202000987

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Peroxisomes contribute to intracellular calcium dynamics
Yelena Sargsyan, Uta Bickmeyer, Christine S Gibhardt, Katrin Streckfuss-Bömeke, Ivan Bogeski, Sven Thoms
Life Science Alliance Jul 2021, 4 (9) e202000987; DOI: 10.26508/lsa.202000987
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Volume 4, No. 9
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