Jean Chrisostome Bopassa

A novel estrogen receptor GPER inhibits mitochondria permeability transition pore opening and protects the heart against ischemia-reperfusion injury

Several studies have recently demonstrated that G protein-coupled receptor 30 (GPER) can directly bind to estrogen and mediate its action. We investigated the role and the mechanism of estrogen-induced cardioprotection after ischemia-reperfusion using a specific GPER agonist G1. Isolated hearts from male mice were perfused using Langendorff technique with oxygenated (95% O(2) and 5% CO(2)) Krebs Henseleit buffer (control), with G1 (1 microM), and G1 (1 microM) together with extracellular signal-regulated kinase (Erk) inhibitor PD-98059 (5 microM). After 20 min of perfusion, hearts were subjected to 20 min global normothermic (37 degrees C) ischemia followed by 40 min reperfusion. Cardiac function was measured, and myocardial necrosis was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Mitochondria were isolated after 10 min of reperfusion to assess the Ca(2+) load required to induce mitochondria permeability transition pore (mPTP) opening. G1-treated hearts developed better functional recovery with higher rate pressure product (RPP, 6140 +/- 264 vs. 2,640 +/- 334 beats mmHg(-1) min(-1), P < 0.05). The infarct size decreased significantly in G1-treated hearts (21 +/- 2 vs. 46 +/- 3%, P < 0.001), and the Ca(2+) load required to induce mPTP opening increased (2.4 +/- 0.06 vs. 1.6 +/- 0.11 microM/mg mitochondrial protein, P < 0.05) compared with the controls. The protective effect of G1 was abolished in the presence of PD-98059 [RPP: 4,120 +/- 46 beats mmHg(-1) min(-1), infarct size: 53 +/- 2%, and Ca(2+) retention capacity: 1.4 +/- 0.11 microM/mg mitochondrial protein (P < 0.05)]. These results suggest that GPER activation provides a cardioprotective effect after ischemia-reperfusion by inhibiting the mPTP opening, and this effect is mediated by the Erk pathway.

mitoBKCa is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location

The large-conductance Ca2+- and voltage-activated K+ channel (BKCa, MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BKCa-like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BKCa agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BKCa (mitoBKCa) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBKCa is the Kcnma1 gene, which produces a protein that migrates at ∼140 kDa and arranges in clusters of ∼50 nm in purified mitochondria. Physiological experiments further support the origin of mitoBKCa as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BKCa transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBKCa.

Phosphorylation of GSK-3β Mediates Intralipid-induced Cardioprotection against Ischemia/Reperfusion Injury

Background:Intralipid (Sigma, St. Louis, MO), a brand name for the first safe fat emulsion for human use, has been shown to be cardioprotective. However, the mechanism of this protection is not known. The authors investigated the molecular mechanism(s) of Intralipid-induced cardioprotection against ischemia/reperfusion injury, particularly the role of glycogen synthase kinase-3&bgr; (GSK-3&bgr;) and mitochondrial permeability transition pore in this protective action. Methods:In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with Intralipid (1% in ex vivo and one bolus of 20% in in vivo) or vehicle. The hemodynamic function, infarct size, threshold for the opening of mitochondrial permeability transition pore, and phosphorylation levels of protein kinase B (Akt)/extracellular signal regulating kinase (ERK)/GSK-3&bgr; were measured. Results:Administration of Intralipid at the onset of reperfusion resulted in approximately 70% reduction in infarct size in the in vivo rat model. Intralipid also significantly improved functional recovery of isolated Langendorff-perfused mouse hearts as the rate pressure product was increased from 2,999 ± 863 mmHg*beats/min in the control group to 13,676 ± 611 mmHg*beats/min (mean±SEM) and the infarct size was markedly smaller (18.3 ± 2.4% vs. 54.8 ± 2.9% in the control group, P < 0.01). The Intralipid-induced cardioprotection was fully abolished by LY294002, a specific inhibitor of PI3K, but only partially by PD98059, a specific ERK inhibitor. Intralipid also increased the phosphorylation levels of Akt/ERK1/glycogen synthase kinase-3&bgr; by eightfold, threefold, and ninefold, respectively. The opening of mitochondrial permeability transition pore was inhibited by Intralipid because calcium retention capacity was higher in the Intralipid group (274.3 ± 8.4 nM/mg vs. 168.6 ± 9.6 nM/mg in the control group). Conclusions:Postischemic treatment with Intralipid inhibits the opening of mitochondiral permeability transition pore and protects the heart through glycogen synthase kinase-3&bgr; via PI3K/Akt/ERK pathways.

Estrogen Contributes to Gender Differences in Mouse Ventricular Repolarization

Rationale: Fast-transient outward K+ (Ito,f) and ultrarapid delayed rectifier K+ currents (IK,slow, also known as IKur) contribute to mouse cardiac repolarization. Gender studies on these currents have reported conflicting results. Objective: Key missing information in these studies is the estral stage of the animals. We revisited gender-related differences in K+ currents, taking into consideration the females’ estral stage. We hypothesized that changes in estrogen levels during the estral cycle could play a role in determining the densities of K+ currents underlying ventricular repolarization. Methods and Results: Peak total K+ current (IK,total) densities (pA/pF, at +40 mV) were much higher in males (48.6±3.0) versus females at estrus (27.2±2.3) but not at diestrus-2 (39.1±3.4). Underlying this change, Ito,f and IK,slow were lower in females at estrus versus males and diestrus-2 (IK,slow: male 21.9±1.8, estrus 14.6±0.6, diestrus-2 20.3±1.4; Ito,f: male 26.8±1.9, estrus 14.9±1.6, diestrus-2 22.1±2.1). Lower IK,slow in estrus was attributable to only IK,slow1 reduction, without changes in IK,slow2. Estrogen treatment of ovariectomized mice decreased IK,total (46.4±3.0 to 28.4±1.6), Ito,f (26.6±1.6 to 12.8±1.0) and IK,slow (22.2±1.6 to 17.2±1.4). Transcript levels of Kv4.3 and Kv1.5 (underlying Ito,f and IK,slow, respectively) were lower in estrus versus diestrus-2 and male. In ovariectomized mice, estrogen treatment resulted in downregulation of Kv4.3 and Kv1.5 but not Kv4.2, KChIP2, or Kv2.1 transcripts. K+ current reduction in high estrogenic conditions were associated with prolongation of the action potential duration and corrected QT interval. Conclusion: Downregulation of Kv4.3 and Kv1.5 transcripts by estrogen are one mechanism defining gender-related differences in mouse ventricular repolarization.

Skeletal muscle action of estrogen receptor α is critical for the maintenance of mitochondrial function and metabolic homeostasis in females

ERα action in skeletal muscle is involved in the preservation of mitochondrial health and insulin sensitivity in female mice and can serve as a defense against metabolic disease in women. Postmenopausal muscle and mitochondrial mayhem Menopause ushers in a host of changes that range from unpleasant to undesirable. One undesirable shift is a loss of protection against insulin resistance, which brings with it a constellation of consequences in the form of chronic disease associated with metabolic dysfunction. Now, Ribas et al. investigate the mechanism underlying the postmenopausal chinks in a woman’s energy homeostasis armor. The estrogen receptor (ER) is known to participate in the preservation of mitochondrial health and insulin sensitivity in mice, but the precise tissue-specific mechanisms remain unclear. Because skeletal muscle is a main tissue responsible for insulin-stimulated glucose disposal, the authors first showed that ERα expression in muscle correlated with metabolic health in human females. They then created a muscle-specific ERα knockout (MERKO) mouse and found that glucose homeostasis was disrupted, fat accumulation increased, and mitochondrial function impaired. These findings imply that ERα action in skeletal muscle helps maintain mitochondrial function and metabolic homeostasis in females. Impaired estrogen receptor α (ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERα expression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERα knockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A–regulator of calcineurin 1–calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERα deficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERα in the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women.

Visualization and quantification of cardiac mitochondrial protein clusters with STED microscopy

The visualization and quantification of mitochondria-associated proteins with high power microscopy methods is of particular interest to investigate protein architecture in this organelle. We report the usage of a custom-made STimulated Emission Depletion (STED) fluorescence nanoscope with ~30nm lateral resolution for protein mapping of Percoll-purified viable mitochondria from murine heart. Using this approach, we were able to quantify and resolve distinct protein clusters within mitochondria; specifically, cytochrome c oxidase subunit 2 is distributed in clusters of ~28nm; whereas the voltage dependent anion channel 1 displays three size distributions of ~33, ~55 and ~83nm.

Rescue of Pressure Overload‐Induced Heart Failure by Estrogen Therapy

Background Estrogen pretreatment has been shown to attenuate the development of heart hypertrophy, but it is not known whether estrogen could also rescue heart failure (HF). Furthermore, the heart has all the machinery to locally biosynthesize estrogen via aromatase, but the role of local cardiac estrogen synthesis in HF has not yet been studied. Here we hypothesized that cardiac estrogen is reduced in HF and examined whether exogenous estrogen therapy can rescue HF. Methods and Results HF was induced by transaortic constriction in mice, and once mice reached an ejection fraction (EF) of ≈35%, they were treated with estrogen for 10 days. Cardiac structure and function, angiogenesis, and fibrosis were assessed, and estrogen was measured in plasma and in heart. Cardiac estrogen concentrations (6.18±1.12 pg/160 mg heart in HF versus 17.79±1.28 pg/mL in control) and aromatase transcripts (0.19±0.04, normalized to control, P<0.05) were significantly reduced in HF. Estrogen therapy increased cardiac estrogen 3‐fold and restored aromatase transcripts. Estrogen also rescued HF by restoring ejection fraction to 53.1±1.3% (P<0.001) and improving cardiac hemodynamics both in male and female mice. Estrogen therapy stimulated angiogenesis as capillary density increased from 0.66±0.07 in HF to 2.83±0.14 (P<0.001, normalized to control) and reversed the fibrotic scarring observed in HF (45.5±2.8% in HF versus 5.3±1.0%, P<0.001). Stimulation of angiogenesis by estrogen seems to be one of the key mechanisms, since in the presence of an angiogenesis inhibitor estrogen failed to rescue HF (ejection fraction=29.3±2.1%, P<0.001 versus E2). Conclusions Estrogen rescues pre‐existing HF by restoring cardiac estrogen and aromatase, stimulating angiogenesis, and suppressing fibrosis.

Gsk Mediates Rapid Estrogen-Induced Cardioprotection Without Akt Activation After Ischemia/Reperfusion

We investigated the role of Glycogen Synthase Kinase (GSK-3β), Erk and Akt activation in rapid 17s-estradiol (E2)-induced cardioprotection after ischemia/reperfusion.Isolated mice hearts were retrograde-perfused using the Langendorff system at 37oC. Hearts were perfused with oxygenated (95% O2 and 5% CO2) Krebs Henseleit solution (control) or with E2 (40 nM). After 20 min perfusion, hearts were subjected to 20 min global normothermic ischemia followed by 40 min reperfusion. Cardiac function was recorded throughout the experiment and at the end of the reperfusion (60 min) infarct size was evaluated by TTC staining. After 10 min of reperfusion, mitochondria were isolated to assess the calcium load required to induce the opening of mitochondria permeability transition pore (mPTP) referred as Calcium Retention Capacity (CRC), and whole heart lysates were prepared for Western blot analysis of pGSK-3β, pErk, pAkt and vinculin.The E2-treated group had significantly increased CRC (287±17 vs. 180±12 nM/mg mitochondrial protein p<0.05), reduced infarct size (26±3% vs. 54±2.8% p<0.001) and improved heart functional recovery (RPP, 11900±467 vs. 5911±318 mmHgxbeats/min, p<0.001) when compared to control. GSK-3β and Erk1/2 phosphorylation levels were significantly increased in E2-treated hearts (∼3 and ∼2 fold, respectively) without significant changes in Akt phosphorylation. These results indicate that rapid E2-induced cardioprotection inhibits the mPTP opening resulting in a reduction of the infarct size and improvement of heart function recovery via GSK-3β and Erk phosphorylation independently of Akt phosphorylation. Supported by NIH.

Superresolution Optical Microscopy of Isolated Cardiac Mitochondrial Proteins

To study the structural organization of mitochondrial proteins, we applied Stimulated Emission Depletion (STED) microscopy in isolated mitochondria. In STED microscopy, two laser beams are used: one for excitation of fluorophores and the other, with doughnut shape, to deplete them in order to allow fluorescence emission only from the excited volume located at the doughnuts center. With STED a lateral resolution of ∼45 nm was achieved in images of isolated mitochondria. We investigated the localization pattern and distribution of MaxiKα, COX4 and VDAC1. After a combined analysis of classical confocal and STED images, we found distinct distributions for VDAC1, MaxiKα and COX4. COX4 distribution was consistent with localization in the cristae. We established that there are 7-15 clusters of MaxiKα, 10-15 clusters of VDAC1, and 15-20 clusters of COX4 per mitochondria. We have demonstrated that protein clusters in the mitochondria can be resolved with a separation power of ∼45 nm, and that it is possible to retrieve quantitative information about the number of clusters and density of proteins in mitochondria. This approach can be extended to eins in mitochondria and subcellular organelles. Supported by NIH.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Quantification of Fluorescence Signals in Purified Mitochondria

Studies of localization and transient association of proteins with mitochondria are limited by the lack of appropriate purification, high resolution imaging and quantification techniques. Here, we investigated the localization of mitochondrial proteins and quantified them in isolated mitochondria from murine heart. Mitochondria were rapidly isolated and purified using a Percoll gradient. The degree of purity of three fractions (M1-M3) was verified by Western blots using markers of different cellular components along with mitochondrial markers. M3 fraction was the purest with negligible amounts of contaminating proteins from the plasma membrane (0.2%), nucleus (6.5%), Golgi complex (1.8%), and endoplasmic reticulum (2.2%) as compared to the whole cell lysate fractions, but was enriched with mitochondrial markers VDAC1 (126%) and COX4 (139%). Thus, M3 fraction was used for sequential labeling with Mitotracker (100 µM) and specific antibodies for mitochondrial proteins. Images were obtained using confocal microscopy. To extract signal information of distinct mitochondria, we utilized the Statistical Region Merging (SRM) and Robust Automatic Threshold Selection (RATS) algorithms to minimize the human bias introduced by the commonly used empiric thresholding. The criteria to positively identify isolated mitochondria were: Mitotracker labeling, average intensity and size. Once mitochondria were selected, the degree of protein co-labeling was quantified. As a proof of concept, we show that VDAC1 and COX4 highly colocalize with Mitotracker labeled mitochondria but not alpha 1C Ca channel. These techniques pave the way to further study mitochondrial proteins and their temporal association with non-mitochondria proteins. Supported by NIH.