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The main differences in the experimental conditions used by Joiner et al.7 and in our previous study3 were: the use of hypotonic shock to prepare mitoplasts (versus French press in our study), the presence of high Na+ concentration in recording solutions (versus Na+-free solutions), and the age of the mice (2–3 months versus 3–4 weeks).

Figure 1a shows mouse heart mitoplasts obtained by exposure of mitochondria to hypotonic shock. The measured average membrane capacitance (Cm) was 0.65 ± 0.03 pF (± s.e.m., n = 65), which correlates well with Cm measurements reported for heart mitoplasts obtained with French press3, as well as with measurements of the inner mitochondrial membrane surface area using electron microscopy8,9 and with estimated measurements of idealized cardiac mitochondria10. Therefore, the values reported by Joiner et al.7 seem to be abnormally high (5−9 pF), indicating inaccuracy in monitoring Cm leading to faulty values of IMCU densities throughout the paper.

Figure 1: Heart MCU current and CaMKII.
figure 1

a, Transmitted image of heart mitoplasts obtained by exposure of mitochondria to 5-min hypotonic shock. Both round (left) and ‘figure-8-shaped’ (right) mitoplasts were present in this preparation and used for electrophysiological experiments. Arrows indicate remnants of the outer mitochondrial membrane. The average diameter of heart mitoplasts in this preparation is ∼4.5 μm (n = 65), which corresponds well with the average membrane capacitance (Cm) measurements of 0.67 pF that we previously reported. b, Representative heart whole-mitoplast MCU currents (IMCU) recorded in the presence (left) or absence (middle) of 150 mM Na-gluconate in both the pipette and bath solutions. IMCU was recorded with different bath Ca2+ concentrations: 0.2 mM (red), 1 mM (blue) and 105 mM (green). IMCU was blocked by 50 nM RuR added to the 0.2 mM Ca2+ bath solution (control, black). Currents in left and middle panels are not normalized and were recorded from two different mitoplasts with comparable membrane capacitance (Cm = 0.80 pF and 0.84 pF, respectively). The voltage ramp protocol used to elicit IMCU is indicated at the top. With Na+ in the recording solutions we also observed a small outward current at high positive voltages. This current was absent in Na+-free conditions (middle and ref. 3). Pipette solution (in mM): 150 Na-gluconate, 40 HEPES, 2 NaCl, 1.5 EGTA, tonicity 450 mmol per kg with sucrose, pH 7.2 with NaOH. Bath Ca2+ solutions with 0.2 and 1 mM Ca2+ were prepared by addition of 1 M stock solution of CaCl2 into the bath solution containing (in mM): 150 Na-gluconate, 40 HEPES, tonicity 300 mmol per kg, pH 7.4 with NaOH. The bath solution with 105 mM Ca2+ contained 105 mM CaCl2 and 10 mM HEPES, pH 7.2 with Tris base. Right: histogram representing average MCU current densities (IMCU normalized to Cm) obtained in the presence (black) or absence (red) of 150 mM Na-gluconate in recording solutions with different bath Ca2+ concentrations (0.2, 1 and 105 mM). Current amplitudes were measured at 5 ms after stepping from 0 to –160 mV. IMCU densities were as follows: at bath 0.2 mM Ca2+, 3.3 ± 0.4 pA pF–1 (n = 8) with 150 Na-gluconate in recording solutions and 6 ± 0.7 pA pF–1 without Na-gluconate in recording solutions; at bath 1 mM Ca2+, 6.2 ± 0.7 pA pF–1 (n = 9) with Na-gluconate and 11.4 ± 0.7 pA pF–1 (n = 6) without Na-gluconate; at bath 105 mM Ca2+, 14.2 ± 0.7 pA pF–1 (n = 12) with Na-gluconate and 33.2 ± 2 pA pF–1 (n = 7) without Na-gluconate in the pipette solution. Statistical data are presented as mean ± s.e.m. c, Representative IMCU in control (left), in the presence of a constitutively active monomeric CaMKII (T287D mutant) in the patch pipette (middle), and in the presence of wild-type monomeric CaMKII previously activated (autophosphorylated) with Ca2+/calmodulin (CaM) and Mg2+/ATP (γ-thiol-ATP) (right) in the patch pipette. IMCU was elicited by a voltage ramp protocol (see panel b) in the presence of 0.2 and 105 mM Ca2+. IMCU amplitude was monitored for up to 35 min after formation of the whole-mitoplast configuration as in Joiner et al.7 (however, the calculated diffusion time15 for the 35-kDa monomer of CaMKII from the pipette into the mitoplast is only ∼25 s). Pipette solution contained (in mM): 150 Na-gluconate, 40 HEPES, 2 NaCl, 1.5 EGTA, tonicity 450 mmol per kg with sucrose, pH 7.2 with NaOH. The recombinant T287D and wild-type CaMKII were added to the control solution at 0.5 or 1 μM, in the presence of 2 mM Na2ATP and 3 mM MgCl2. (Addition of ATP and Mg2+ alone did not affect IMCU.) d, Histogram showing average IMCU current densities obtained in the absence (black, control) or presence of T287D (red) or wild-type monomeric CaMKII pre-autophosphorylated with thiol-ATP (blue) in the pipette. Currents were measured in 0.2 and 105 mM Ca2+ as described in c, and amplitudes were determined at 5 ms after stepping from 0 to –160 mV. IMCU densities were as follows: at bath 0.2 mM Ca2+, 3.2 ± 0.3 pA pF–1 (n = 17) in control, 3.2 ± 0.3 pA pF–1 (n = 14) for T287D, and 3.0 ± 0.3 pA pF–1 (n = 8) for autophosphorylated wild-type CaMKII; at bath 105 mM Ca2+, 16.4 ± 0.5 pA pF–1 (n = 16) in control, 17.9 ± 1.1 pA pF–1 (n = 11) for T287D, and 16.2 ± 0.5 pA pF–1 (n = 5) for autophosphorylated wild-type CaMKII. Statistical data are presented as mean ± s.e.m. e, Histogram showing average IMCU current densities in control (black) and in the presence of a constitutively active monomeric CaMKII (T287D mutant) in the patch pipette either alone (red) or with 1 μM CaM and 5–10 μM free Ca2+ (green). IMCU densities were as follows: at bath 0.2 mM Ca2+, 3.2 ± 0.3 pA pF–1 (n = 17) in control, 3.2 ± 0.3 pA pF–1 (n = 14) for T287D, and 2.8 ± 0.1 pA pF–1 (n = 5) for T287D in the presence of 1 μM CaM and 5–10 μM free Ca2+. Current amplitudes were measured at 5 ms after stepping from 0 to –160 mV. Statistical data are presented as mean ± s.e.m.

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We recorded IMCU from heart mitoplasts isolated by hypotonic shock with 150 mM Na-gluconate in the pipette and bath solutions (as in Joiner et al.7; Fig. 1b, left panel) and without Na+ (conditions previously used by us3; Fig. 1b, middle panel). IMCU recorded in the presence of Na-gluconate was significantly smaller than in its absence (Fig. 1b). Our data support the observation that elevated Na+ may regulate heart mitochondrial Ca2+ concentration11,12. Notably, the whole-mitoplast IMCU was about two orders of magnitude lower than the current reported by Joiner et al.7 (∼2 pA at −160 mV in 0.2 mM Ca2+ versus ∼180 pA) and did not exhibit high fluctuations as expected for a small-conductance channel. Also, the current reported by Joiner et al.7 was not inhibited by Ru360 in the same fashion as the IMCU (ref. 2). In 10 nM Ru360, IMCU shows no immediate inhibition upon stepping from 0 mV to −120 mV (ref. 2), and the inhibition develops slowly over time2, whereas the current of Joiner et al.7 was inhibited immediately upon stepping from 0 to −160 mV. All these observations indicate that Joiner et al.7 did not record IMCU. We suggest that either they did not record from inner mitochondrial membrane or the integrity of their mitoplasts was compromised.

Next, we tested whether IMCU is directly regulated by CaMKII, as claimed by Joiner et al.7, who reported that addition of a constitutively active monomeric form of CaMKII (T287D mutant) to the patch pipette potentiated their currents. When we applied T287D mutant CaMKII, we failed to observe any functional change in IMCU, either without (Fig. 1c, middle panel, and Fig. 1d) or with Ca2+ plus calmodulin (Fig. 1e). We further verified these results using wild-type monomeric CaMKII pre-autophosphorylated with thiol-ATP to prevent de-autophosphorylation and again observed no change in IMCU (Fig. 1c, right panel, and Fig. 1d).

The noisy currents presented by Joiner et al.7 do not appear to be carried by MCU, and their extremely high amplitude misrepresents the actual MCU activity in heart. Heart, with abundant mitochondria and frequently elevated cytosolic Ca2+, has very low MCU current3, which is probably critical for avoiding disruption of cytosolic Ca2+ signalling and preventing mitochondrial Ca2+ overload and cell death. Finally, our electrophysiological experiments with MCU currents did not indicate that MCU is regulated by CaMKII.

Methods

Electrophysiological experiments were performed as in ref. 3. Recombinant δ-human monomeric CaMKII (1–317) was purified from baculovirus using an amino-terminal 6×-HN tag and Ni chromatography followed by gel filtration. Activity of recombinant CaMKII was measured in Na-gluconate pipette solution using the peptide substrate AC-2 (ref. 13). Constitutive activity (no Ca2+/calmodulin) was undetectable for wild-type CaMKII and 4.6 µmol min–1 mg–1 for the T287D mutant. The Ca2+/calmodulin stimulated activity of T287D CaMKII was 9.7 µmol min–1 mg–1. Wild-type CaMKII was autophosphorylated in γ-thiol-ATP to promote Thr 287 autophosphorylation, which allows CaMKII to be active without Ca2+/calmodulin (that is, autonomous activity)14. The autonomous activity of wild-type CaMKII was 19.4 µmol min–1 mg–1 (∼91% of the Ca2+/calmodulin stimulated activity).