Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria

Abstract

Although functional coupling between protein kinase Cepsilon (PKCepsilon) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKCepsilon remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKCepsilon and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKCepsilon with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKCepsilon complex at approximately 2%, approximately 0.2%, and approximately 1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKCepsilon can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKCepsilon resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKCepsilon in mice, which is cardioprotective, greatly increased interaction of PKCepsilon with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKCepsilon did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKCepsilon transgenesis. Collectively, these data demonstrate that PKCepsilon forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKCepsilon-induced cardioprotection.

Figure 1

Expression of individual mitochondrial pore proteins in mouse cardiac mitochondria. Mitochondrial lysates from control mouse hearts were subjected to SDS-PAGE followed by Western immunoblotting with (from top to bottom) anti-VDAC1, ANT1, HKII, CypD, and PKCε. Each lane represents an individual heart (n=8). IB indicates immunoblotting.

Figure 2

PKCε interacts with mitochondrial pore proteins in vivo. Mitochondrial lysates from control mouse hearts were immunoprecipitated with anti-PKCε antibody. The complexes were subjected to SDS-PAGE followed by Western immunoblotting with (from top to bottom) anti-VDAC1, ANT1, HKII, CypD, and PKCε antibodies. Lanes 1 through 3 contain PKCε immunoprecipitates from 3 individual hearts; lane 4 is blank; and lane 5 contains cardiac mitochondrial lysate as positive control (n=8). IP indicates immunoprecipitation; IB, immunoblotting.

Figure 3

PKCε interacts with and phosphorylates VDAC in vitro. A, Equal amounts of recombinant GST or GST-PKCε fusion proteins conjugated to glutathione-Sepharose beads were incubated with mixes containing [³⁵S]-labeled VDAC1, ANT1, HKII, or CypD produced by in vitro translation (IVT). The complexes were pulled down and subjected to SDS-PAGE and either autoradiography (top 4 panels) or immunoblotting with anti-GST antibody (bottom panel). Lane 1 contains GST alone plus the respective in vitro translation product; lane 2 contains GST-PKCε plus the respective in vitro translation product; lane 3 is blank; and lane 4 contains the respective in vitro translation product as positive control (n=3). B, Recombinant GST-VDAC1 fusion protein was incubated with γ[³²P]-ATP plus increasing amounts of recombinant PKCε and subjected to SDS-PAGE and either autoradiography (top panel) or immunoblotting with anti-GST antibody (bottom panel). Lanes 1 through 4 contain radio-labeled GST-VDAC1 after incubation with 0, 10, 50, and 100 ng of recombinant PKCε, respectively (n=3). IB indicates immunoblotting.

Figure 4

Recombinant PKCε inhibits swelling in mouse cardiac mitochondria. Mitochondria were isolated from nontransgenic mouse hearts and swelling was induced by 200 μmol/L CaCl₂ and measured spectrophotometrically as a decrease in A₅₂₀. In some experiments, mitochondria were incubated with either the pore inhibitors cyclosporin A (30 nmol/L) or MgCl₂ (10 μmol/L) 5 minutes before calcium was added, or with recombinant PKCε (2 μg) plus PMA (1 μmol/L) 15 minutes before calcium was added. Figure shows representative traces for control, cyclosporin A–, Mg²⁺-, and PKCε-treated mitochondria (n=3).

Figure 5

Expression of PKCε and pore proteins in mitochondria from mice with cardiac-specific expression of active and inactive PKCε. Mitochondrial lysates from NTG, AE, and DN mouse hearts were subjected to SDS-PAGE followed by Western immunoblotting with (from top to bottom) anti-VDAC1, ANT1, HKII, CypD, and PKCε antibodies (n=6). IB indicates immunoblotting.

Figure 6

PKCε interaction with pore proteins is enhanced in mitochondria from mice with cardiac-specific expression of active and inactive PKCε. A, Mitochondrial lysates from NTG and AE mouse hearts were immunoprecipitated with anti-VDAC1 (top), ANT1 (middle), or HKII (bottom) antibodies. The complexes were subjected to SDS-PAGE followed by Western immunoblotting with anti-VDAC1 (top), ANT1 (middle), and HKII (bottom) antibodies (n=6). B, Mitochondrial lysates from NTG, AE, and DN mouse hearts were immunoprecipitated with anti-VDAC1 (top), ANT1 (middle), or HKII (bottom) antibodies. The complexes were subjected to SDS-PAGE followed by Western immunoblotting with anti-PKCε antibody (n=6). IP indicates immunoprecipitation; IB, immunoblotting.

Figure 7

Mitochondrial swelling is inhibited in mice with cardiac-specific expression of active but not inactive PKCε. Mitochondria were isolated from NTG, AE, and DN hearts, and swelling was induced by 200 μmol/L CaCl₂ and measured spectrophotometrically as a decrease in A₅₂₀. Figure shows representative traces for NTG, AE, and DN mitochondria (n=4).

Figure 8

Atractyloside inhibits cardioprotection in mice with cardiac-specific expression of active PKCε. Anesthetized NTG and AE mice were subjected to 30 minutes of coronary occlusion followed by 4 hours reperfusion, and the resultant infarct size was determined by postmortem tetrazolium staining. Atractyloside (ATR) was administered as a bolus injection (25 mg/kg, IV) 15 minutes before the coronary occlusion. Data are represented as mean±SEM. *P<0.05 compared with NTG (n=5 to 6).