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. 2024 Mar;300(3):105697.
doi: 10.1016/j.jbc.2024.105697. Epub 2024 Jan 30.

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1)

Zhuqing Liang  1 , Tyler Ralph-Epps  1 , Michael W Schmidtke  1 , Vikalp Kumar  1 , Miriam L Greenberg  2
Affiliations

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1)

Zhuqing Liang et al. J Biol Chem. 2024 Mar.

Abstract

Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. Recent studies have identified the inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS model systems. However, the molecular mechanisms linking aberrant CL remodeling, particularly the primary, direct consequence of reduced tetralinoleoyl-CL (TLCL) levels, to PDH activity deficiency are not yet understood. In the current study, we found that remodeled TLCL promotes PDH function by directly binding to and enhancing the activity of PDH phosphatase 1 (PDP1). This is supported by our findings that TLCL uniquely activates PDH in a dose-dependent manner, TLCL binds to PDP1 in vitro, TLCL-mediated PDH activation is attenuated in the presence of phosphatase inhibitor, and PDP1 activity is decreased in Tafazzin-knockout (TAZ-KO) C2C12 myoblasts. Additionally, we observed decreased mitochondrial calcium levels in TAZ-KO cells and treating TAZ-KO cells with calcium lactate (CaLac) increases mitochondrial calcium and restores PDH activity and mitochondrial oxygen consumption rate. Based on our findings, we conclude that reduced mitochondrial calcium levels and decreased binding of PDP1 to TLCL contribute to decreased PDP1 activity in TAZ-KO cells.

Keywords: Barth syndrome; calcium; caridiolipin; metabolism; pyruvate dehdrogenase phosphatase; pyruvate dehydrogenase; tafazzin.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
PDH activity is rescued by supplementation of C(18:2)4CL.A, phosphorylated PDH (p-PDH) levels were assayed in mitochondria obtained from WT and TAZ-KO myoblasts. Mitochondria were incubated on ice for 2.5 h with the indicated CL species. The signal intensities of protein bands were quantified using ImageJ software (bottom panel). The protein band was normalized to NDUFB6 and quantified relative to the WT control-treated sample. B and C, mitochondria from WT and TAZ-KO myoblasts were treated with or without C(18:2)4 CL, and PDH activity was assayed as described in “Experimental procedures”. Data points represent mean ±S.D. (error bars) for each individual biological replicate of each group. ∗ 0.01<p < 0.05, ∗∗ 0.001<p < 0.01, ∗∗∗ <p < 0.001.
Figure 2
Figure 2
C(18:2)4CL binds to PDP1 in vitro.A, PDP and PDK isoform mRNA levels were evaluated in WT myoblasts by qPCR. Expression levels were normalized to Actb as an internal control. Data points represent mean ±S.D. (error bars) for each individual biological replicate of each group. ∗∗∗ <p < 0.001. B, the indicated CL species, phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylserine (PS), were serially diluted and spotted onto a PVDF membrane that was incubated overnight in buffer containing 25 μg of the indicated recombinant proteins PDP1. Interactions were detected by immunoblotting with an antibody against the His tag.
Figure 3
Figure 3
PDP1 activity is required for TLCL-mediated rescue of PDH activity.A, phosphorylated PDH (p-PDH) levels and PDH activity (B) were evaluated in mitochondria from TAZ-KO myoblasts treated with or without phosphatase inhibitor and supplemented for 2.5 h with or without C(18:2)4 CL. Data points represent mean ±S.D. (error bars) for each individual biological replicate of each group. ∗ 0.01<p < 0.05, ∗∗ 0.001<p < 0.01.
Figure 4
Figure 4
The binding of PDP1 to PDH is reduced in TAZ-KO cells.A, WB analysis of the bait-prey pull-down assay. B, cell lysates from WT and TAZ-KO cells were subjected to WB analysis using the indicated antibodies. C, co-immunoprecipitation assay for PDP1, PDH-E1, and PDH-E2 protein interaction. The indicated myoblast lysates were immunoprecipitated using the antibodies specified on the left side of the figure, and eluted protein was subjected to WB analysis by probing for the specific proteins listed on the right side. The negative control lane represents WT lysate passed through empty beads lacking primary antibodies.
Figure 5
Figure 5
PDP1 activity is decreased in TAZ-KO cells. Increasing amounts of recombinant PDP1 protein were incubated with WT or TAZ-KO lysates. PDP1 eluted from this mixture was subsequently incubated with PDH complex for 1 h. The dephosphorylation activity of PDP1 was evaluated by assaying phosphorylated PDH (p-PDH) using WB analysis. p-PDH levels were quantified by dividing the amount of p-PDH signal by total PDH-E1 in each sample, and values were normalized relative to the first lane in each group (containing the lowest concentration of eluted PDP1).
Figure 6
Figure 6
TAZ-KO mitochondria exhibit decreased calcium levels. WT and TAZ-KO cells were preloaded with Fluo-4 AM and TMRM before permeabilization with 50 μg/ml (w/v) digitonin and 1 μM thapsigargin. Steady-state quantification of Fluo-4 AM (A) and TMRM (B) signals in WT and TAZ-KO mitochondria was performed using Image J software. n = 31. Subsequently, WT and TAZ-KO mitochondria were injected with 5 mM CaCl2 (100 μl) into the 250 μl imaging solution at the indicated time points (indicated as each ∗, with the resulting effective [Ca2+] listed below). Fluo-4 AM (C) and TMRM (D) density were quantified over time. Data points represent mean ±S.D. (error bars) for each individual biological replicate of each group. ∗∗ 0.001<p < 0.01, ∗∗∗ <p < 0.001.
Figure 7
Figure 7
CaLac treatment restores PDH activity and rescues OCR in TAZ-KO cells. Phosphorylated PDH (p-PDH) levels were measured by WB following treatment with calcium lactate (CaLac) at the indicated concentrations (A) and with 10 mM CaLac (B). Oxygen consumption rates (OCR) of myoblasts subjected to 2.5 mM CaLac (C) and 12.5 mM CaLac (D) treatments. n = 5. Data points represent mean ±S.D. (error bars) for each individual biological replicate of each group. ∗ 0.01<p < 0.05, ∗∗ 0.001<p < 0.01, ∗∗∗ <p < 0.001.
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