Korean J Physiol Pharmacol.  2024 Jul;28(4):335-344. 10.4196/kjpp.2024.28.4.335.

The NADPH oxidase inhibitor diphenyleneiodonium suppresses Ca²⁺ signaling and contraction in rat cardiac myocytes

• Le QA
• Trinh TN
• Luong PK
• Anh VTV
• Tran HN
• Kim JC
• Woo SH

• KMID: 2557206
• DOI: http://doi.org/10.4196/kjpp.2024.28.4.335

Abstract

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca²⁺ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca²⁺ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC₅₀ of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content by 20%–30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca²⁺ transients by DPI. The L-type Ca²⁺ current (ICa) was decreased concentration-dependently by DPI (IC₅₀ of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca²⁺ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca²⁺ and Na+. Mitochondrial superoxide level was reduced by DPI at 3–100 µM. Our data suggest that DPI may suppress L-type Ca²⁺ channel and RyR, thereby attenuating Ca²⁺-induced Ca²⁺ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

Keyword

Cardiac myocytes; Ca²⁺ release; Contraction; Diphenyleneiodonium; L-type Ca²⁺ current

Figure

• 

  Fig. 1 Negative inotropy induced by diphenyleneiodonium (DPI) in rat ventricular myocytes. (A) Representative contraction traces recorded immediately before (“Control”) and after the exposure to different concentrations of DPI in field-stimulated rat ventricular myocytes at 1 Hz. The traces were selected when a maximal decrease in cell shortening by DPI was observed. (B) Concentration-dependent decrease in cell shortenings (% suppression) by the extracellular application of DPI; 0.01 μM, n = 3, p > 0.05; 0.05 μM, n = 4, p < 0.01; 0.1 μM, n = 4, p < 0.01; 1 μM, n = 6, p < 0.001; 10 μM, n = 6, p < 0.0001; 10 μM, n = 4, p < 0.001. Paired t-test was used. The sigmoidal curve represents the fit of the Hill equation.

• 

  Fig. 2 Alterations of global Ca2+ signaling in the presence of diphenyleneiodonium (DPI). (A) Ca2+ transients measured in a representative ventricle cell followed by caffeine (10 mM)-induced Ca2+ signals in the absence and presence of 3 μM DPI. (B, C) Summary of mean values of the diastolic and systolic Ca2+ levels, and in the magnitude, time-to-90%-peak (TP,90) and half decay time of Ca2+ transient in the absence (Con) and presence of DPI (3 μM) (n = 15). ****p < 0.0001, **p < 0.01 vs. Con (Paired t-test). (D) Comparison of magnitudes of the caffeine-induced Ca2+ transients and fractional release measured before (Control) and after application of DPI (3 μM) (n = 15). ****p < 0.0001 vs. Con (Paired t-test).

• 

  Fig. 3 Suppression of Ca2+ current (ICa) by diphenyleneiodonium (DPI). (A) Superimposed ICa recorded at 0 mV (holding potential at –40 mV) before and after applications of 3 μM, 30 μM, 100 μM, and 200 μM DPI in the representative ventricular myocytes, showing concentration-dependent inhibition in ICa by DPI. Scale bars indicate 30 ms in x axis and 2 pA/pF in y axis. (B) Comparison of mean peak ICa measured under control conditions and after applications of different concentrations of DPI (0.3 μM, n = 3; 3 μM, n = 6; 30 μM, n = 8; 100 μM, n = 4; 200 μM, n = 3). **p < 0.01, *p < 0.05 vs. control (Con). Paired t-test. (C) Concentration-dependence curve for % inhibition in ICa versus concentrations of DPI. Plot was fit with Hill equation (Hill coefficient = 0.68). (D) Superimposed ICa measured at voltage steps ranging –40 to +70 mV (holding at –40 mV) with 10-mV-increment before (Control) and after application of 30 μM DPI in a representative ventricle cell. (E) Current-voltage relationships of averaged ICa at the step potentials in the control condition and after exposure to 30 μM DPI (n = 3). Cells were dialyzed with 15 mM EGTA containing internal solutions.

• 

  Fig. 4 Suppression of Ca2+ sparks by diphenyleneiodonium (DPI) in the absence of external Na+ and Ca2+. (A) A series of sequential confocal Ca2+ images recorded at 30 Hz from a representative resting rat ventricular myocyte in the control condition (“1”) and after brief exposure to zero Na+ and zero Ca2+ external solutions (0 Na, 0 Ca) for 10 s (“2”), followed by additional application of DPI (0 Na, 0 Ca, DPI) for 3 min (“3”). The images were selected from the periods marked with the boxes correspondingly numbered (“1”, “2”, and “3”) in the panel B. After 3-min DPI application, DPI was removed from the zero Na+ and zero Ca2+ solutions (2 min; (0 Na, 0 Ca, DPI)`), which was followed by the exchange of external solution with control solutions (5 min; “Wash”). Arrows indicate distinct Ca2+ sparks. (B) Plots of the total numbers of sparks occurred in each frame (33 frames/s) versus 2-s-long recording periods under each condition labeled above the plots. (C) Summary of mean spark frequency detected under each condition indicated underneath the graphs. Paired t-test was used.

• 

  Fig. 5 Reduction of mitochondrial superoxide by diphenyleneiodonium (DPI). (A) Plots of averaged Mito-SOX fluorescence, normalized to the levels (F0) just prior to the application of DPI (3 μM, n = 4; 30 μM, n = 5; 100 μM, n = 4), versus recording time, showing decrease of mitochondrial superoxide levels by DPI in a concentration-dependent manner. (B) Summary of Mito-SOX fluorescence ratio measured at 100-s after the onset of DPI applications, showing DPI-induced signal reductions at 3 μM, 30 μM, and 100 μM DPI. **p < 0.01, *p < 0.05 vs. control (Con) (paired t-test).

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