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. 2023 Aug 17;12:e83975. doi: 10.7554/eLife.83975

Figure 5. Cardiac arrest and resumption of heartbeats with GtCCR4-3.0, KnChR, CrChR2[T159C], and GtACR1.

(A) Cardiac arrest rates of 4-dpf Tg larvae expressing GtCCR4-3.0-EYFP, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, or GtACR1-EYFP in cardiomyocytes. The heart area was irradiated with appropriate light (520 nm for GtCCR4 and GtACR1; 470 nm for KnChR and CrChR2) for 100 ms at a strength of 0.5 mW/mm2. Sibling larvae that did not express the rhodopsins were used as controls. Six consecutive stimulation trials were analyzed for four rhodopsin-expressing larvae and four control larvae of each Tg line, Wilcoxon rank-sum test (GtCCR4-3.0-EYFP, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, and GtACR1-EYFP, p=0.0131). (B, C) Latency to cardiac arrest (B) and time to resumption of HBs (C) after light stimulation with GtCCR4-3.0-EYFP, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, or GtACR1-EYFP. HB data were obtained from the experiments described above (A). One-way ANOVA with Tukey’s post hoc test (latency to cardiac arrest KnChR-3.0-EYFP vs. GtACR1-EYFP, p=0.00144; CrChR2[T159C]-mCherry vs. GtACR1-EYFP, p=0.0190; time to resumption of heartbeat GtCCR4-3.0-EYFP vs. GtACR1-EYFP, p=0.000786; KnChR-3.0-EYFP vs. GtACR1-EYFP, p=0.0189; CrChR2[T159C]-mCherry vs. GtACR1-EYFP, p=0.000236). (D) Light intensity dependence of cardiac arrest rates of 4-dpf Tg larvae expressing GtCCR4-3.0-EYFP, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, GtACR1-EYFP in cardiomyocytes. The heart area was irradiated with light (520 nm for GtCCR4 and GtACR1; 470 nm for KnChR and CrChR2) for 100 ms at a strength of 0.5, 0.2, or 0.05 mW/mm2. Six consecutive stimulation trials were analyzed for four rhodopsin-expressing larvae of each Tg line. One-way ANOVA with Tukey’s post hoc test (GtCCR4-3.0-EYFP: 0.5 mW/mm2 vs. 0.05 mW/mm2; p=0.0222). (E, F) Changes in fluorescence intensity of GCaMP6s (ΔF/F) in the heart of 4-dpf Tg larvae expressing KnChR-3.0-EYFP and GCaMP6s (E), or GtACR1-EYFP and GCaMP6s (F). Sibling larvae that did not express the rhodopsins were used as the control. The heart area of Tg larvae was stimulated with a fluorescence detection filter (excitation 470–495 nm, emission 510–550 nm). Two rhodopsin-expressing larvae (green) and two control larvae (black) were analyzed for each rhodopsin. Three trials were analyzed for each larva. The linear mixed-effects model was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. Means and SEMs are indicated.

Figure 5—source data 1. Data for Figure 5, cardiac arrest and resumption of heartbeats with ChRs.
elife-83975-fig5-data1.xlsx (188.5KB, xlsx)

Figure 5.

Figure 5—figure supplement 1. Cardiac arrest induced with channelrhodopsins (ChRs) by light of various intensities.

Figure 5—figure supplement 1.

Latency to cardiac arrest (A) and time to resumption of heartbeats (B) after light stimulation with ChRs. The heart area of 4-dpf Tg larvae expressing GtCCR4-3.0-EYFP, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, or GtACR1-EYFP was irradiated with light (520 nm for GtCCR4, GtACR1; 470 nm for KnChR, CrChR2) for 100 ms at a strength of 0.5, 0.2, or 0.05 mW/mm2. Six consecutive stimulation trials were analyzed for four rhodopsin-expressing larvae of each Tg line. One-way ANOVA with Tukey’s post hoc test (time to resumption of heartbeat GtACR1-EYFP 0.5 mW/mm2 vs. 0.05 mW/mm2, p=0.0240). *p<0.05, ns: not significant. Means and SEMs are indicated.
Figure 5—figure supplement 1—source data 1. Data for Figure 5—figure supplement 1, cardiac arrest induced with ChRs by light of various intensities.
Figure 5—figure supplement 2. Cardiac arrest induced with CoChR and ChrimsonR.

Figure 5—figure supplement 2.

(A) Cardiac arrest rate. The heart area of 4-dpf Tg larvae expressing CoChR-tdTomato or ChrimsonR-tdTomato was irradiated with light (470 nm for CoChR; 520 nm for ChrimsonR) for 100 ms at a strength of 0.5, 0.2, or 0.05 mW/mm2. Six consecutive stimulation trials were analyzed for four rhodopsin-expressing larvae of each Tg line. (B, C) Latency to cardiac arrest (B) and time to resumption of heartbeats (C) after light stimulation with optogenetic tools. One-way ANOVA with Tukey’s post hoc test was used for statistical analysis. ns: not significant. Means and SEMs are indicated.
Figure 5—figure supplement 2—source data 1. Data for Figure 5—figure supplement 2, cardiac arrest induced with CoChR and ChrimsonR.
Figure 5—video 1. Ca2+ imaging in the heart of a larva expressing KnChR-3.0-EYFP and GCaMP6s in cardiomyocytes.
Download video file (514.1KB, mp4)
The heart area of Tg(myl7:GAL4FF);Tg(UAS-hsp70l:KnChR-3.0-EYFP, myl7:mCherry);Tg(UAS-hsp70l:GCaMP6s) was stimulated with a fluorescence detection filter (excitation 470–495 nm, emission 510–550 nm). GCaMP6s fluorescence was monitored with the same filter set. The fluorescence of GCaMP6s gradually increased.
Figure 5—video 2. Ca2+ imaging in the heart of a larva expressing GtACR1-EYFP and GCaMP6s in cardiomyocytes.
Download video file (465.9KB, mp4)
The heart area of Tg(myl7:GAL4FF);Tg(UAS-hsp70l:GtACR1-EYFP, myl7:mCherry);Tg(UAS-hsp70l:GCaMP6s) was stimulated with a fluorescence detection filter (excitation 470–495 nm, emission 510–550 nm). GCaMP6s fluorescence, which was monitored with the same filter set, gradually decreased.