Cardioprotective trafficking of caveolin to mitochondria is Gi-protein dependent

Abstract

Background: Caveolae are a nexus for protective signaling. Trafficking of caveolin to mitochondria is essential for adaptation to cellular stress though the trafficking mechanisms remain unknown. The authors hypothesized that G protein-coupled receptor/inhibitory G protein (Gi) activation leads to caveolin trafficking to mitochondria.

Methods: Mice were exposed to isoflurane or oxygen vehicle (30 min, ± 36 h pertussis toxin pretreatment, an irreversible Gi inhibitor). Caveolin trafficking, cardioprotective "survival kinase" signaling, mitochondrial function, and ultrastructure were assessed.

Results: Isoflurane increased cardiac caveolae (n = 8 per group; data presented as mean ± SD for Ctrl versus isoflurane; [caveolin-1: 1.78 ± 0.12 vs. 3.53 ± 0.77; P < 0.05]; [caveolin-3: 1.68 ± 0.29 vs. 2.67 ± 0.46; P < 0.05]) and mitochondrial caveolin levels (n = 16 per group; [caveolin-1: 0.87 ± 0.18 vs. 1.89 ± .19; P < 0.05]; [caveolin-3: 1.10 ± 0.29 vs. 2.26 ± 0.28; P < 0.05]), and caveolin-enriched mitochondria exhibited improved respiratory function (n = 4 per group; [state 3/complex I: 10.67 ± 1.54 vs. 37.6 ± 7.34; P < 0.05]; [state 3/complex II: 37.19 ± 4.61 vs. 71.48 ± 15.28; P < 0.05]). Isoflurane increased phosphorylation of survival kinases (n = 8 per group; [protein kinase B: 0.63 ± 0.20 vs. 1.47 ± 0.18; P < 0.05]; [glycogen synthase kinase 3β: 1.23 ± 0.20 vs. 2.35 ± 0.20; P < 0.05]). The beneficial effects were blocked by pertussis toxin.

Conclusions: Gi proteins are involved in trafficking caveolin to mitochondria to enhance stress resistance. Agents that target Gi activation and caveolin trafficking may be viable cardioprotective agents.

Figure 1. Flow diagram of the experimental protocol

Mice were treated with pertussis toxin (PTX, 100 µg/kg intraperitoneal) or vehicle and allowed to recover for 36 hrs. After recovery, animals were exposed to isoflurane (1 MAC, minimum alveolar concentration) or oxygen for 30 min. Hearts were excised at 15 or 45 minutes following isoflurane for biochemical, mitochondrial or electron microscopy (EM) analysis.

Figure 2. Pertussis toxin (PTX) inhibits isoflurane-dependent upregulation of caveolin/caveolae

Mice were exposed to isoflurane or oxygen for 30 min ± PTX treatment. After 15 min washout, excised hearts underwent sucrose density fractionation and buoyant (BF) and heavy (HF) membrane fractions were pooled and analyzed for caveolin-3 (Cav-3) (A) and caveolin-1 (Cav- 1) (B) expression. Cav-3 and Cav- 1 was increased in BFs and HFs after Iso exposure, while this effect was blocked by PTX. Data are from 8 mice per group. **P<0.01, ***P<0.001 relative to respective control. Ctrl = control; Iso = isoflurane; Ctrl-P = control with PTX treatment; Iso-P = isoflurane with PTX treatment; GAPDH=glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. Pertussis toxin (PTX) inhibits isoflurane-dependent kinase signaling

A. Whole-heart homogenates showed elevated phosphorylation of Akt and GSK3β after 30 min isoflurane exposure and 15 min washout. Phosphorylation of Akt (B, Ser 473) and GSK3β (C, Ser 9) was attenuated by PTX (n=8 each group). ***P<0.001 relative to respective control. Ctrl = control; Iso = isoflurane; Ctrl-P = control with PTX treatment; Iso-P = isoflurane with PTX treatment; pAKT = phosphorylated protein kinase B; tAKT = total protein kinase B; pGSK-3β = phosphorylated glycongen synthase kinase 3 beta; tGSK-3β = total glycongen synthase kinase 3 beta; GAPDH=glyceraldehyde 3-phosphate dehydrogenase.

Figure 4. Pertussis toxin (PTX) attenuates isoflurane-dependent caveolae formation and impacts mitochondrial morphology

Control (A), isoflurane (B), PTX+control (C), and PTX+isoflurane (D) treated mice were allowed 15 min washout following isoflurane/oxygen exposure. Animals were perfusion fixed and hearts processed for electron microscopy (EM) analysis. Isoflurane increased membrane invaginations consistent with caveolae (white arrowheads, B), and this was blocked with PTX. PTX treatment resulted in loss of mitochondrial structure in control and isoflurane groups (* and arrow represent altered mitochondrial morphology and potential mitophagy, respectively).

Figure 5. Pertussis toxin (PTX) inhibits isoflurane-dependent caveolin trafficking to mitochondria

Mitochondria were isolated after 30 min isoflurane and 45 min washout (controls were exposed to oxygen). Whole heart homogenate (H) and pure mitochondria (M) were probed for cell compartment markers. Purified mitochondria (A, high enrichment of mitochondrial markers with only trace contamination of nuclei and clathrin) were enriched in Cav- 1 and -3 following isoflurane, and trafficking was inhibited by PTX (B-D). Data are from 16 mice per group. ***P<0.001 relative to respective control. Ctrl = control; Iso = isoflurane; Ctrl-P or Ctrl-PTX = control with PTX treatment; Iso-P or Iso-PTX = isoflurane with PTX treatment.

Figure 6. Pertussis toxin (PTX) impacts mitochondrial structure and function

A. Control cardiac tissue showing both subsarcolemmal (SSM) and interfibrillary (IFM) populations and normal ultrastructural morphology. B. Isoflurane treated hearts showing the two distinct healthy mitochondrial populations. Inset localizes increased internalized caveolae (arrows) near IFM mitochondria, indicating trafficking from the PM to internal compartments. C-D. PTX treated control and isoflurane heart ultrastructure, respectively. Arrowheads and * mark an atypical shaped SSM, which appears to “block” a Z-line transport zone. Limited internalized caveolae are seen in either C or D. The blockade effect of PTX treatment is seen dramatically in D where vesicles appear trapped between the PM and SSM. Insets demonstrate the clumping of SSM and irregular shape and distribution of IFM. E. Higher magnification of the PM and SSM following PTX treatment. Internalized putative caveolae vesicles (*) are trapped by the smaller mitochondria (arrows). Mag bars A-D = 2 μm. Insets = 500nM E = 200nM. F. Mice were exposed to O₂ or isoflurane (30 min) ± PTX treatment, and hearts were excised after 45 min washout for preparation of crude mitochondria for respiratory function studies. State 4 respiration was assessed with complex 1 substrates. State 3 was assessed with complex I (malate/pyruvate, mal/pyr) and II (succinate, succ) substrates. Isoflurane enhanced state 3 respiration was attenuated with PTX treatment. Data are from 4 mice per group. *P<0.05, relative to respective control. Ctrl = control; Iso = isoflurane; Ctrl-PTX = control with PTX treatment; Iso-PTX = isoflurane with PTX treatment.

Figure 7. Pertussis toxin (PTX) blunts intrinsic IR tolerance with cardiac specific Cav-3 overexpression

Hearts from Cav-3 OE mice treated with PTX (3OE PTX) show higher LVEDP (A, left ventricular end diastolic pressure) over 45 min reperfusion and lower LVDP (B, left ventricular developed pressure) compared to hearts from untreated Cav-3 OE (3OE) mice. Data were analyzed with repeated measure 2-Way ANOVA and Bonferroni post-hoc comparison. Significance was accepted when *p<0.05; #p<0.05. Data presented as mean±SD, n = 11-13/group, C57 = C57Bl/6 mice; Pre-Isch = pre-ischemia.

Figure 8. Summary diagram

Cartoon depicting homeostatic, isoflurane-induced, and pertussis toxin (PTX)-sensitive trafficking of caveolin/caveolae and alterations in membrane/mitochondrial dynamics.