Yoshitaka Kawaraguchi

Cardiac-Specific Overexpression of Caveolin-3 Attenuates Cardiac Hypertrophy and Increases Natriuretic Peptide Expression and Signaling

OBJECTIVES We hypothesized that cardiac myocyte-specific overexpression of caveolin-3 (Cav-3), a muscle-specific caveolin, would alter natriuretic peptide signaling and attenuate cardiac hypertrophy. BACKGROUND Natriuretic peptides modulate cardiac hypertrophy and are potential therapeutic options for patients with heart failure. Caveolae, microdomains in the plasma membrane that contain caveolin proteins and natriuretic peptide receptors, have been implicated in cardiac hypertrophy and natriuretic peptide localization. METHODS We generated transgenic mice with cardiac myocyte-specific overexpression of caveolin-3 (Cav-3 OE) and also used an adenoviral construct to increase Cav-3 in cardiac myocytes. RESULTS The Cav-3 OE mice subjected to transverse aortic constriction had increased survival, reduced cardiac hypertrophy, and maintenance of cardiac function compared with control mice. In left ventricle at baseline, messenger ribonucleic acid for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were increased 7- and 3-fold, respectively, in Cav-3 OE mice compared with control subjects and were accompanied by increased protein expression for ANP and BNP. In addition, ventricles from Cav-3 OE mice had greater cyclic guanosine monophosphate levels, less nuclear factor of activated T-cell nuclear translocation, and more nuclear Akt phosphorylation than ventricles from control subjects. Cardiac myocytes incubated with Cav-3 adenovirus showed increased expression of Cav-3, ANP, and Akt phosphorylation. Incubation with methyl-β-cyclodextrin, which disrupts caveolae, or with wortmannin, a PI3K inhibitor, blocked the increase in ANP expression. CONCLUSIONS These results imply that cardiac myocyte-specific Cav-3 OE is a novel strategy to enhance natriuretic peptide expression, attenuate hypertrophy, and possibly exploit the therapeutic benefits of natriuretic peptides in cardiac hypertrophy and heart failure.

Mitochondria-localized caveolin in adaptation to cellular stress and injury

We show here that the apposition of plasma membrane caveolae and mitochondria (first noted in electron micrographs >50 yr ago) and caveolae‐mitochondria interaction regulates adaptation to cellular stress by modulating the structure and function of mitochondria. In C57Bl/6 mice engineered to overexpress caveolin specifically in cardiac myocytes (Cav‐3 OE), localization of caveolin to mitochondria increases membrane rigidity (4.2%; P<0.05), tolerance to calcium, and respiratory function (72% increase in state 3 and 23% increase in complex IV activity; P<0.05), while reducing stress‐induced generation of reactive oxygen species (by 20% in cellular superoxide and 41 and 28% in mitochondrial superoxide under states 4 and 3, respectively; P<0.05) in Cav‐3 OE vs. TGneg. By contrast, mitochondrial function is abnormal in caveolin‐knockout mice and Caenorhabditis elegans with null mutations in caveolin (60% increase free radical in Cav‐2 C. elegans mutants; P<0.05). In human colon cancer cells, mitochondria with increased caveolin have a 30% decrease in apoptotic stress (P<0.05), but cells with disrupted mitochondria‐caveolin interaction have a 30% increase in stress response (P<0.05). Targeted gene transfer of caveolin to mitochondria in C57Bl/6 mice increases cardiac mitochondria tolerance to calcium, enhances respiratory function (increases of 90% state 4, 220% state 3, 88% complex IV activity; P<0.05), and decreases (by 33%) cardiac damage (P<0.05). Physical association and apparently the transfer of caveolin between caveolae and mitochondria is thus a conserved cellular response that confers protection from cellular damage in a variety of tissues and settings.—Fridolfsson, H. N., Kawaraguchi, Y., Ali, S. S., Panneerselvam, M., Niesman, I. R., Finley, J. C., Kellerhals, S. E., Migita, M. Y., Okada, H., Moreno, A. L., Jennings, M., Kidd, M. W., Bonds, J. A., Balijepalli, R. C., Ross, R. S., Patel, P. M., Miyanohara, A., Chen, Q., Lesnefsky, E. J., Head, B. P., Roth, D. M., Insel, P. A., Patel, H. H. Mitochondria‐localized caveolin in adaptation to cellular stress and injury. FASEB J. 26, 4637–4649 (2012). www.fasebj.org

Disruption of protein kinase A localization using a trans-activator of transcription (TAT)-conjugated A-kinase-anchoring peptide reduces cardiac function.

Localization of protein kinase A (PKA) via A-kinase-anchoring proteins (AKAPs) is important for cAMP responsiveness in many cellular systems, and evidence suggests that AKAPs play an important role in cardiac signaling. To test the importance of AKAP-mediated targeting of PKA on cardiac function, we designed a cell-permeable peptide, which we termed trans-activator of transcription (TAT)-AKAD for TAT-conjugated A-kinase-anchoring disruptor, using the PKA binding region of AKAP10 and tested the effects of this peptide in isolated cardiac myocytes and in Langendorff-perfused mouse hearts. We initially validated TAT-AKAD as a PKA localization inhibitor in cardiac myocytes by the use of confocal microscopy and cellular fractionation to show that treatment with the peptide disrupts type I and type II PKA regulatory subunits. Knockdown of PKA activity was demonstrated by decrease in phosphorylation of phospholamban and troponin I after β-adrenergic stimulation in isolated myocytes. Treatment with TAT-AKAD reduced myocyte shortening and rates of contraction and relaxation. Injection of TAT-AKAD (1 μm), but not scrambled control peptide, into the coronary circulation of isolated perfused hearts rapidly (<1 min) and reversibly decreased heart rate and peak left ventricular developed pressure. TAT-AKAD also had a pronounced effect on developed pressure (−dP/dt), consistent with a delayed relaxation of the heart. The effects of TAT-AKAD on heart rate and contractility persisted in hearts pretreated with isoproterenol. Disruption of PKA localization with TAT-AKAD thus had negative effects on chronotropy, inotropy, and lusitropy, thereby indicating a key role for AKAP-targeted PKA in control of heart rate and contractile function.

Role of Caveolin-3 and Glucose Transporter-4 in Isoflurane-induced Delayed Cardiac Protection

Background:Caveolae are small, flask-like invaginations of the plasma membrane. Caveolins are structural proteins found in caveolae that have scaffolding properties to allow organization of signaling. The authors tested the hypothesis that delayed cardiac protection induced by volatile anesthetics is caveolae or caveolin dependent. Methods:An in vivo mouse model of ischemia–reperfusion injury with delayed anesthetic preconditioning (APC) was tested in wild-type, caveolin-1 knockout, and caveolin-3 knockout mice. Mice were exposed to 30 min of oxygen or isoflurane and allowed to recover for 24 h. After 24 h recovery, mice underwent 30-min coronary artery occlusion followed by 2 h of reperfusion at which time infarct size was determined. Biochemical assays were also performed in excised hearts. Results:Infarct size as a percent of the area at risk was reduced by isoflurane in wild-type (24.0 ± 8.8% vs. 45.1 ± 10.1%) and caveolin-1 knockout mice (27.2 ± 12.5%). Caveolin-3 knockout mice did not show delayed APC (41.5 ± 5.0%). Microscopically distinct caveolae were observed in wild-type and caveolin-1 knockout mice but not in caveolin-3 knockout mice. Delayed APC increased the amount of caveolin-3 protein but not caveolin-1 protein in discontinuous sucrose-gradient buoyant fractions. In addition, glucose transporter-4 was increased in buoyant fractions, and caveolin-3/glucose transporter-4 colocalization was observed in wild-type and caveolin-1 knockout mice after APC. Conclusions:These results show that delayed APC involves translocation of caveolin-3 and glucose transporter-4 to caveolae, resulting in delayed protection in the myocardium.

Volatile Anesthetics Protect Cancer Cells against Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis via Caveolins

Background: Volatile anesthetics have a dual effect on cell survival dependent on caveolin expression. The effect of volatile anesthetics on cancer cell survival and death after anesthetic exposure has not been well investigated. The authors examined the effects of isoflurane exposure on apoptosis and its regulation by caveolin-1 (Cav-1). Methods: The authors exposed human colon cancer cell lines to isoflurane and proapoptotic stimuli and assessed what role Cav-1 plays in cell protection. They evaluated apoptosis using assays for nucleosomal fragmentation, cleaved caspase 3 expression, and caspase activity assays. To test the mechanism, they used pharmacologic inhibitors (i.e., pertussis toxin) and assessed changes in glycolysis. Results: Apoptosis as measured by nucleosomal fragmentation was enhanced by isoflurane (1.2% in air) in HT29 (by 64% relative to control, P < 0.001) and decreased in HCT116 (by 23% relative to control, P < 0.001) cells. Knockdown of Cav-1 in HCT116 cells increased the sensitivity to apoptotic stimuli but not with scrambled small interfering RNA (siRNA) treatment (19.7 ± 0.4 vs. 20.0 ± 0.6, P = 0.7786 and 19.7 ± 0.5 vs. 16.3 ± 0.4, P = 0.0012, isoflurane vs. control in Cav-1 small interfering RNA vs. scrambled small interfering RNA treated cells, respectively). The protective effect of isoflurane with various exposure times on apoptosis was enhanced in HT29 cells overexpressing Cav-1 (P < 0.001 by two-way ANOVA). Pertussis toxin effectively blocked the antiapoptotic effect of isoflurane exhibited by Cav-1 in all cell lines. Cav-1 cells had increased glycolysis with isoflurane exposure; however, in the presence of tumor necrosis factor-related apoptosis-inducing ligand, this increase in glycolysis was maintained in HT29-Cav-1 but not control cells. Conclusion: Brief isoflurane exposure leads to resistance against apoptosis via a Cav-1–dependent mechanism.

Integrins protect cardiomyocytes from ischemia/reperfusion injury

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165-175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury.

Opioid-induced preconditioning is dependent on caveolin-3 expression.

We tested the hypothesis that caveolin-3 (Cav-3) is essential for opioid-induced preconditioning in vivo. Cav-3 overexpressing mice, Cav-3 knockout mice, and controls were exposed to myocardial ischemia/reperfusion (I/R) in the presence of SNC-121 (SNC), a &dgr;-selective opioid agonist, or naloxone, a nonselective opioid antagonist. Controls were protected from I/R injury by SNC. No protection was produced by SNC in Cav-3 knockout mice. Cav-3 overexpressing mice showed innate protection from I/R compared with controls that was abolished by naloxone. Our results show that opioid-induced preconditioning is dependent on Cav-3 expression and that endogenous protection in Cav-3 overexpressing mice is opioid dependent.

The effects of prostaglandin E1 on intraoperative temperature changes and the incidence of postoperative shivering during deliberate mild hypothermia for neurosurgical procedures.

UNLABELLED We investigated the effects of i.v. prostaglandin E1 (PGE1) on intraoperative changes of core temperature and the incidence of postoperative shivering in neurosurgical patients undergoing deliberate mild hypothermia. Eighty-three patients were randomly assigned to one of three groups: patients in the control group did not receive PGE1, whereas patients in the PG20 group and PG50 group received PGE1 at a dose of 0.02 and 0.05 microg x kg(-1) x min(-1), respectively. The administration of PGE1 was started just after the induction of anesthesia and continued until the end of anesthesia. Anesthesia was maintained with nitrous oxide in oxygen, sevoflurane, and fentanyl. After the induction of anesthesia, patients were cooled using a water blanket and a convective device blanket. Tympanic membrane temperature was maintained at 34.5 degrees C. During surgical wound closure, patients were rewarmed. Intraoperative changes in tympanic membrane and skin temperatures and the incidence of postoperative shivering were compared among groups. Demographic and intraoperative variables were similar among groups. There were no significant differences in tympanic temperatures among groups at each point during the operation. Skin temperature 30 min after rewarming and just after tracheal extubation was significantly lower in the PG20 group than in the PG50 group. Postoperative shivering was more frequent in the PG20 group (43%) than in the control (13%) and PG50 (17%) groups. These results suggest that the intraoperative administration of PGE1 does not affect changes in core temperature during deliberate mild hypothermia and that PGE1 at a dose of 0.02 microg x kg(-1) x min(-1) may increase the occurrence of postoperative shivering. IMPLICATIONS Deliberate mild hypothermia has been proposed as a means of providing cerebral protection during neurosurgical procedures. Vasodilating drugs may be used during deliberate mild hypothermia to maintain peripheral circulation and to enhance the cooling and rewarming rate. In the present study, however, we found no benefit from i.v. prostaglandin E1 administration during deliberate mild hypothermia in neurosurgical patients.

From local to global: lipid emulsion (intralipid) makes a move.

I N clinical practice, lipid emulsion is used commonly as a component of parenteral nutrition. Lipid emulsion also is used as therapy for severe cardiotoxicity secondary to accidental overdose of local anesthetics, an effect that has been confirmed in animals and humans. Because patients with local anesthetic-induced cardiac arrest are considered to be less responsive for standard resuscitation methods, this finding is striking, and currently infusion of lipid emulsion is considered the primary treatment for local anesthetic toxicity. In this issue of ANESTHESIOLOGY, Rahman et al. report a new and potentially clinically relevant benefit of lipid emulsion therapy. Classic preconditioning produces in the heart a state profoundly protected from ischemia–reperfusion injury; however, the prerequisite for knowing when the ischemic injury will occur has limited the clinical translation and relevance of preconditioning. Recently, a potentially more clinically relevant form of cardiac protection, termed postconditioning, has been described. Postconditioning involves the application of protective interventions (e.g., brief ischemia–reperfusion, volatile anesthetics, opioids, and other classic cardioprotective agents) after ischemia but before reperfusion. Rahman et al. extend the list of postconditioning agents to include Intralipid (Sigma, St. Louis, MO). They show that lipid emulsion infusion just before reperfusion (i.e., postconditioning) protects from myocardial ischemia–reperfusion injury. Subsequent mechanistic studies define a role for glycogen synthase kinase-3 and the mitochondrial permeability transition pore (mPTP) in this response. Mitochondria, a source of cellular adenosine triphosphate, are increasingly implicated in cell survival and death signaling in the heart. In particular, the mPTP has been suggested as the final effector in cardiac myocyte protection. The mPTP opening in response to stress leads to an increase in mitochondrial membrane permeability to small molecules, resulting in cellular apoptosis and necrosis. Thus, regulating the mPTP opening has been considered to be a promising target for cardiac protection, and others have suggested the involvement of mPTP in postconditioning. Postconditioning is mediated via a complex molecular signaling cascade involving the reperfusion injury salvage kinase and the survivor activating factor enhancement pathways. Rahman et al. implicate common survival pathways, such as PI3K-Akt and ERK, in lipid emulsion-induced protection. In addition, they link these upstream survival kinases to downstream regulation of glycogen synthase kinase-3 . Recent work has shown that increased phosphorylation of glycogen synthase kinase-3 reduces the affinity of the adenine nucleotide translocase for cyclophilin D, suggesting that assembly of the complex is targeted by protective signals to limit mPTP opening. Thus, there appears to be a direct link between survival kinase regulation and mPTP end effector function. These intriguing findings by Rahman et al. leave us with some important questions to be addressed in future investigation. First, how does lipid emulsion activate survival kinases—via a direct effect or secondary to receptor stimulation? General protective interventions appear to signal through G-protein–coupled receptor pathways, including preconditioning and postconditioning via opioids, volatile anesthetics, and other cardioprotective agents. It would be important to determine whether lipid emulsion is sensitive to Illustration: J. P. Rathmell/A. Johnson.