Pim-1 kinase protects mitochondrial integrity in cardiomyocytes

Abstract

Rationale: Cardioprotective signaling mediates antiapoptotic actions through multiple mechanisms including maintenance of mitochondrial integrity. Pim-1 kinase is an essential downstream effector of AKT-mediated cardioprotection but the mechanistic basis for maintenance of mitochondrial integrity by Pim-1 remains unexplored. This study details antiapoptotic actions responsible for enhanced cell survival in cardiomyocytes with elevated Pim-1 activity.

Objective: The purpose of this study is to demonstrate that the cardioprotective kinase Pim-1 acts to inhibit cell death by preserving mitochondrial integrity in cardiomyocytes.

Methods and results: A combination of biochemical, molecular, and microscopic analyses demonstrate beneficial effects of Pim-1 on mitochondrial integrity. Pim-1 protein level increases in the mitochondrial fraction with a corresponding decrease in the cytosolic fraction of myocardial lysates from hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Cardiac-specific overexpression of Pim-1 results in higher levels of antiapoptotic Bcl-X(L) and Bcl-2 compared to samples from normal hearts. In response to oxidative stress challenge, Pim-1 preserves the inner mitochondrial membrane potential. Ultrastructure of the mitochondria is maintained by Pim-1 activity, which prevents swelling induced by calcium overload. Finally, mitochondria isolated from hearts created with cardiac-specific overexpression of Pim-1 show inhibition of cytochrome c release triggered by a truncated form of proapoptotic Bid.

Conclusion: Cardioprotective action of Pim-1 kinase includes preservation of mitochondrial integrity during cardiomyopathic challenge conditions, thereby raising the potential for Pim-1 kinase activation as a therapeutic interventional approach to inhibit cell death by antagonizing proapoptotic Bcl-2 family members that regulate the intrinsic apoptotic pathway.

Figure 1. Induction of Pim-1 in response to ex vivo I/R injury

Mitochondrial (A) and cytosolic (B) fractions from NTG whole hearts challenged by ischemia (I), ischemia/reperfusion (I/R), or perfusion (P) as control probed for Pim-1 levels by immunoblot analyses. Corresponding quantitative graphs represent Pim-1 induction (fold change) determined by comparing Pim-1 levels (normalized to loading control, mitochondrial fraction = VDAC and cytosolic fraction = GAPDH) to corresponding perfusion control. Results represented as mean ± SEM, N = 3. Student's T test: **P<0.02 for 30I vs. 30I/30R in the mitochondrial fraction and 30I vs. 30I/10R in the cytosolic fraction.

Figure 2. Anti-apoptotic Bcl-2 family members are upregulated in Pim WT hearts

Bcl-X_L (A) and Bcl-2 (B) from mitochondrial and cytosolic fractions of NTG mouse hearts probed by immunoblot analyses to determine protein expression levels. Quantitative graphs represent relative Bcl-X_L and Bcl-2 levels normalized to VDAC (mitochondrial fraction) or GAPDH (cytosolic fraction). Results are represented as mean ± SEM, n = at least 3 hearts. Student's T test: **P<0.02 for NTG vs. Pim WT Bcl-X_L levels in the mitochondria fraction. *P<0.05 for NTG vs. Pim WT Bcl-2 levels in the cytosolic fraction.

Figure 3. Mitochondrial inner membrane depolarization in cardiomyocytes induced by H₂O₂ is delayed by Pim-1 expression

TMRE fluorescence of NRCMs under control conditions (untreated) versus oxidative stress challenge (H₂O₂; 100 μmol/L). NRCM (NI) = non-infected NRCM (A), GFP = NRCM infected with GFP adenovirus (B), Pim WT = NRCM infected with Pim-1 adenovirus (C), and combined plot comparing results of A, B, and C is shown in D. Quantitative graphs comparing H₂O₂ treated NRCM, GFP, and Pim WT at 2, 6, and 10 minutes (E). Results are represented as mean ± SEM. N = at least 3 independent experiments. One-way ANOVA: *P< 0.001 for NRCM (NI) vs. Pim WT at 2 minutes. #P<0.001 for Pim WT vs. GFP at 2 minutes. **P<0.001 for NRCM (NI) vs. GFP vs. Pim WT at 6 and 10 minutes.

Figure 4. Pim-1 overexpression attenuates calcium-induced mitochondrial swelling

Rate of mitochondrial swelling of untreated, calcium treated (150 μmol/L CaCl₂), and CsA treated (1 μmol/L CsA) isolated NTG mitochondria (A). Rate of mitochondrial swelling of untreated and calcium treated mitochondria from Pim DN (B) or Pim WT (C). Quantitative graphs comparing calcium treated NTG, Pim DN, and Pim WT's percentage decrease of 520 nm absorbance reading at 10 minutes and 20 minutes (D). Results represented as mean ± SEM. N = at least 5. One-way ANOVA: *P<0.01 for NTG vs. Pim WT at 10 minutes. **P<0.001 for NTG vs. Pim DN and Pim DN vs. Pim WT at 10 minutes; NTG vs. Pim DN v. Pim WT at 20 minutes.

Figure 5. Representative electron micrographs of mitochondria under normal or calcium overload conditions

Untreated mitochondrial preparations from NTG (A), Pim WT (C), or Pim DN (E) compared to calcium challenged NTG (B), Pim WT (D), or Pim DN (F). Magnification at 2700X, scale bar set at 2000 nm.

Figure 6. Morphometric comparison of mitochondrial diameter resulting from exposure to calcium overload

Quantitative graphs displaying the percentage of total untreated and calcium treated mitochondria with corresponding diameter in nm (A-C). The median of each sample is listed in the upper right corner of each graph with reported diameter shift (D) and significance (P) value using KS test for NTG (A), Pim WT (B), and Pim DN (C) mitochondrial preparations from 2 separate hearts. Average diameter length represented as mean ± SEM with corresponding percentage increase when treated with calcium (D). One-way ANOVA: ***P<0.001 for untreated NTG vs. calcium treated NTG, ###P<0.001 for untreated Pim DN vs. calcium treated Pim DN, +++P<0.001 for untreated Pim WT vs. calcium treated Pim WT.

Figure 7. Pim-1 overexpression decreases cytochrome c release from mitochondria

Preparations of NTG versus Pim WT under normal conditions (- tBid) or challenged with tBid (+ tBid) and subsequently separated into mitochondrial pellet (A) or supernatant (B) fractions for immunoblot analysis to quantitate cytochrome c levels. Corresponding quantitative graph is normalized to VDAC as a loading control. Results are represented as mean ± SEM. N = 5 hearts. Two-way ANOVA: *P<0.05 for untreated NTG vs. tBid treated NTG in mitochondrial and supernatant fraction, #P<0.01 for tBid treated NTG vs. tBid treated Pim WT in supernatant fraction, ##P<0.001 for tBid treated NTG vs. untreated Pim WT in supernatant fraction. (C) Corresponding percentage of total cytochrome c in mitochondrial and supernatant fractions of untreated and tBid treated isolated NTG and Pim WT mitochondria. Two-way ANOVA: ***P<0.001 for untreated NTG vs. tBid treated NTG in mitochondrial fraction, +++P<0.001 for untreated NTG vs. tBid treated in supernatant fraction, ns = not significant for untreated Pim WT vs. tBid treated Pim WT in mitochondrial and supernatant fraction.