Role of Scl39a13/ZIP13 in cardiovascular homeostasis

Figures

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  Fig 1. Effects of zinc level alteration and Dox stimulation on PNCs. (a) Effect of either zinc depletion or supplementation on the morphology of PNCs cultured in media containing 10 μM TPEN or 100 μM ZnSO4 for 24 h. Representative images are shown (n = 3–4; scale bar = 10 μm). (b) Effect of Dox treatment on Il-1β and Zip13 expression in PNCs. PNCs were cultured with or without Dox (10 ng/mL) for 24 h. (c) Effect of Dox treatment on Il-1β and Zip13 expression levels in heart tissues of mice. C57BL/6J mice were intraperitoneally injected with Dox (15 mg/kg), and heart tissue samples were collected two weeks after Dox injection. All data were collected from three to four independent experiments. Significant differences were analyzed using the Student’s t-test for each experimental group (* p < 0.05).

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  Fig 2. ZIP13-deficient PNCs exhibited abnormal morphology and impaired beating rhythm. (a) Morphological (top) and beating (bottom) of PNCs in WT mice. PNCs were cultured as described in the Materials and Methods. The heartbeats of cardiomyocytes were recorded under a bright field of microscopic observation as a movie file. The data were analyzed using ImageJ software. Beating was quantified as grayscale color change at the edge of the cardiomyocytes. (b) Morphological and beating observations of PNCs in Zip13-KO mice. Aberrant shrinking morphology (upper) and irregular beating (lower) were observed. Representative images are shown (n = 3–4; scale bars = 10 μm). (c) MA-plot of RNA-seq data of WT- and Zip13-KO-derived PNCs samples. The obtained total RNA was subjected to RNA-seq analysis. (d) Heat map analysis of the CPM of 607 genes in WT and Zip13-KO derived samples. (e) GO terms enriched in genes upregulated in PNC samples of Zip13-KO mice. The grey bar represents the log (p-value) of each GO term. The black lines indicate the number of genes. GO terms enriched in downregulated genes were downregulated in the Zip13-KO samples.

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  Fig 3. Cardiac analysis of Zip13-KO mice in vivo. (a) Histological analysis of steady-state heart tissue from WT and Zip13-KO mice. Heart tissue samples were sectioned longitudinally and stained with hematoxylin and eosin (n = 4). (b) Heart tissue weights of WT and Zip13-KO mice injected with or without Dox (normalized to body weight). n = 3 to 4. (c) Representative heartbeat analysis in either WT or Zip13-KO mice injected with or without Dox was monitored using the telemetry ECG system, as described in Materials and Methods. n = 3 to 4. Scale bar 100 ms.

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  Fig 4. Gene expression profiling of Zip13-KO mice heart tissues. The expression of genes related to cardiomyocyte function (a: BNP; b: eNOS, c: Trp53, d: HDAC4, e: Col1a1, f: Col1a2, g: MMP9, and h: GM130) was analyzed in heart tissue samples derived from WT and Zip13-KO mice, with or without Dox administration. Each gene was quantitatively analyzed using qPCR. Significant differences were analyzed using the Student’s t-test, *p < 0.05, n = 3–4.