Yasuo M. Tsutsumi

Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1

Caveolae, small invaginations in the plasma membrane, contain caveolins (Cav) that scaffold signaling molecules including the tyrosine kinase Src. We tested the hypothesis that cardiac protection involves a caveolin‐dependent mechanism. We used in vitro and in vivo models of ischemia‐reperfusion injury, electron microscopy (EM), transgenic mice, and biochemical assays to address this hypothesis. We found that Cav‐1 mRNA and protein were expressed in mouse adult cardiac myocytes (ACM). The volatile anesthetic, isoflurane, protected ACM from hypoxia‐induced cell death and increased sarcolemmal caveolae. Hearts of wild‐type (WT) mice showed rapid phosphorylation of Src and Cav‐1 after isoflurane and ischemic preconditioning. The Src inhibitor PP2 reduced phosphorylation of Src (Y416) and Cav‐1 in the heart and abolished isoflurane‐induced cardiac protection in WT mice. Infarct size (percent area at risk) was reduced by isoflurane in WT (30.5±4 vs. 44.2±3, n=7, P<0.05) but not Cav‐1−/− mice (46.6±5 vs. 41.7±3, n=7). Cav‐1−/−mice exposed to isoflurane showed significant alterations in Src phosphorylation and recruitment of C‐terminal Src kinase, a negative regulator of Src, when compared to WT mice. The results indicate that isoflurane modifies cardiac myocyte sarcolemmal membrane structure and composition and that activation of Src and phosphorylation of Cav‐1 contribute to cardiac protection. Accordingly, therapies targeted to post‐translational modification of Src and Cav‐1 may provide a novel approach for such protection.—Patel, H. H., Tsutsumi, Y. M., Head, B. P., Niesman, I. R., Jennings, M., Horikawa, Y. Huang, D., Moreno, A. L., Patel, P. M., Insel, P. A., Roth, D. M. Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin‐1. FASEB J. 21, 1565–1574 (2007)

Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning

Background— Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte–specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo. Methods and Results— Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte–specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3β phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3β and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion. Conclusions— Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.

Caveolin-1 expression is essential for N-methyl-D-aspartate receptor-mediated Src and extracellular signal-regulated kinase 1/2 activation and protection of primary neurons from ischemic cell death

N‐Methyl‐d‐aspartate (NMDA) receptor (NMDAR) activation and downstream signaling are important for neuronal function. Activation of prosur‐vival Src family kinases and extracellular signal‐regulated kinase (ERK) 1/2 is initiated by NMDAR activation, but the cellular organization of these kinases in relation to NMDARs is not entirely clear. We hypothesized that caveolin‐1 scaffolds and coordinates protein complexes involved in NMDAR signaling and that this organization is necessary for neuronal preconditioning, whereby NMDAR activation protects neurons from subsequent ischemic cell death. We found that suble‐thal ischemia (SLI) or preconditioning via NMDA treatment of primary cortical neurons from neonatal rats or mice increases expression of phosphorylated (P) caveo‐lin‐1, P‐Src, and P‐ERK1/2. The NMDAR antagonist, MK801, or the Src inhibitor, PP2, attenuated SLI‐induced preconditioning. NMDAR2B distributed to buoyant fractions and heavy fractions, partially colocalized with caveo‐lin‐1 and the membrane raft marker, cholera toxin B. Cultures of primary neurons treated with caveolin‐1 small interfering RNA or from caveolin‐1−/− mice lacked the NMDA‐mediated increase in P‐Src and P‐ERK, as well as SLI‐ and NMDA‐induced preconditioning. Adenovirally mediated expression of caveolin‐1 in neurons from caveo‐lin‐1−/− mice restored NMDA‐mediated enhancement of P‐Src and P‐ERK1/2, redistributed NMDAR2B to buoyant fractions, and enhanced NMDAR2B localization to membrane rafts. We conclude that caveolin‐1, perhaps via its ability to scaffold key signaling components, is essential for NMDAR localization to neuronal membrane rafts, NMDAR/Src tyrosine kinase family/ERK signaling, and protection of neurons from ischemic injury and cell death.—Head, B. P., Patel, H. H., Tsutsumi, Y. M., Hu, Y., Mejia, T., Mora, R. C., Insel, P. A., Roth, D. M. Drummond, J. C., Patel, P. M. Caveolin‐1 expression is essential for N‐methyl‐D‐aspartate receptor‐mediated Src and ERK 1/2 activation and protection of primary neurons from ischemic cell death. FASEB J. 22, 828–840 (2008)

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

OBJECTIVESnWe hypothesized that cardiac myocyte-specific overexpression of caveolin-3 (Cav-3), a muscle-specific caveolin, would alter natriuretic peptide signaling and attenuate cardiac hypertrophy.nnnBACKGROUNDnNatriuretic 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.nnnMETHODSnWe 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnThese 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.

Isoflurane Produces Sustained Cardiac Protection after Ischemia–Reperfusion Injury in Mice

Background: Isoflurane reduces myocardial ischemia–reperfusion injury within hours to days of reperfusion. Whether isoflurane produces sustained cardiac protection has never been examined. The authors studied isoflurane-induced cardiac protection in the intact mouse after 2 h and 2 weeks of reperfusion and determined the dependence of this protection on adenosine triphosphate–dependent potassium channels and the relevance of this protection to myocardial function and apoptosis. Methods: Mice were randomly assigned to receive oxygen or isoflurane for 30 min with 15 min of washout. Some mice received mitochondrial (5-hydroxydecanoic acid) or sarcolemmal (HMR-1098) adenosine triphosphate–dependent potassium channel blockers with or without isoflurane. Mice were then subjected to a 30-min coronary artery occlusion followed by 2 h or 2 weeks of reperfusion. Infarct size was determined at 2 h and 2 weeks of reperfusion. Cardiac function and apoptosis were determined 2 weeks after reperfusion. Results: Isoflurane did not change hemodynamics. Isoflurane reduced infarct size after reperfusion when compared with the control groups (27.7 ± 6.3 vs. 41.7 ± 6.4% at 2 h and 19.6 ± 5.9 vs. 28.8 ± 9.0% at 2 weeks). Previous administration of 5-hydroxydecanoic acid, but not HMR-1098, abolished isoflurane-induced cardiac protection. At 2 weeks, left ventricular end-diastolic diameter was decreased significantly and end-systolic pressure and maximum and minimum dP/dt were improved by isoflurane. Isoflurane-treated mice subjected to ischemia and 2 weeks of reperfusion showed less expression of proapoptotic genes, significantly decreased expression of cleaved caspase-3, and significantly decreased deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling–positive nuclei compared with the control group. Conclusions: Cardiac protection induced by isoflurane against necrotic and apoptotic cell death is associated with an acute memory period that is sustained and functionally relevant 2 weeks after ischemia–reperfusion injury in mice in vivo.

Molecular mechanisms of the inhibitory effects of propofol and thiamylal on sarcolemmal adenosine triphosphate-sensitive potassium channels

BackgroundBoth propofol and thiamylal inhibit adenosine triphosphate–sensitive potassium (KATP) channels. In the current study, the authors investigated the effects of these anesthetics on the activity of recombinant sarcolemmal KATP channels encoded by inwardly rectifying potassium channel (Kir6.1 or Kir6.2) genes and sulfonylurea receptor (SUR1, SUR2A, or SUR2B) genes. MethodsThe authors used inside-out patch clamp configurations to investigate the effects of propofol and thiamylal on the activity of recombinant KATP channels using COS-7 cells transfected with various types of KATP channel subunits. ResultsPropofol inhibited the activities of the SUR1/Kir6.2 (EC50 = 77 &mgr;m), SUR2A/Kir6.2 (EC50 = 72 &mgr;m), and SUR2B/Kir6.2 (EC50 = 71 &mgr;m) channels but had no significant effects on the SUR2B/Kir6.1 channels. Propofol inhibited the truncated isoform of Kir6.2 (Kir6.2&Dgr;C36) channels (EC50 = 78 &mgr;m) that can form functional KATP channels in the absence of SUR molecules. Furthermore, the authors identified two distinct mutations R31E (arginine residue at position 31 to glutamic acid) and K185Q (lysine residue at position 185 to glutamine) of the Kir6.2&Dgr;C36 channel that significantly reduce the inhibition of propofol. In contrast, thiamylal inhibited the SUR1/Kir6.2 (EC50 = 541 &mgr;m), SUR2A/Kir6.2 (EC50 = 248 &mgr;m), SUR2B/Kir6.2 (EC50 = 183 &mgr;m), SUR2B/Kir6.1 (EC50 = 170 &mgr;m), and Kir6.2&Dgr;C36 channels (EC50 = 719 &mgr;m). None of the mutants significantly affects the sensitivity of thiamylal. ConclusionsThese results suggest that the major effects of both propofol and thiamylal on KATP channel activity are mediated via the Kir6.2 subunit. Site-directed mutagenesis study suggests that propofol and thiamylal may influence Kir6.2 activity by different molecular mechanisms; in thiamylal, the SUR subunit seems to modulate anesthetic sensitivity.

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.

Molecular mechanisms underlying ketamine-mediated inhibition of sarcolemmal adenosine triphosphate-sensitive potassium channels

Background: Ketamine inhibits adenosine triphosphate-sensitive potassium (KATP) channels, which results in the blocking of ischemic preconditioning in the heart and inhibition of vasorelaxation induced by KATP channel openers. In the current study, the authors investigated the molecular mechanisms of ketamine’s actions on sarcolemmal KATP channels that are reassociated by expressed subunits, inwardly rectifying potassium channels (Kir6.1 or Kir6.2) and sulfonylurea receptors (SUR1, SUR2A, or SUR2B). Methods: The authors used inside-out patch clamp configurations to investigate the effects of ketamine on the activities of reassociated Kir6.0/SUR channels containing wild-type, mutant, or chimeric SURs expressed in COS-7 cells. Results: Ketamine racemate inhibited the activities of the reassociated KATP channels in a SUR subtype-dependent manner: SUR2A/Kir6.2 (IC50 = 83 &mgr;m), SUR2B/Kir6.1 (IC50 = 77 &mgr;m), SUR2B/Kir6.2 (IC50 = 89 &mgr;m), and SUR1/Kir6.2 (IC50 = 1487 &mgr;m). S-(+)-ketamine was significantly less potent than ketamine racemate in blocking all types of reassociated KATP channels. The ketamine racemate and S-(+)-ketamine both inhibited channel currents of the truncated isoform of Kir6.2 (Kir6.2&Dgr;C36) with very low affinity. Application of 100 &mgr;m magnesium adenosine diphosphate significantly enhanced the inhibitory potency of ketamine racemate. The last transmembrane domain of SUR2 was essential for the full inhibitory effect of ketamine racemate. Conclusions: These results suggest that ketamine-induced inhibition of sarcolemmal KATP channels is mediated by the SUR subunit. These inhibitory effects of ketamine exhibit specificity for cardiovascular KATP channels, at least some degree of stereoselectivity, and interaction with intracellular magnesium adenosine diphosphate.

Molecular mechanisms of the inhibitory effects of bupivacaine, levobupivacaine, and ropivacaine on sarcolemmal adenosine triphosphate-sensitive potassium channels in the cardiovascular system

Background:Sarcolemmal adenosine triphosphate–sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. Methods:The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Results:Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 &mgr;m) stereoselectively (levobupivacaine, IC50 = 168 &mgr;m; ropivacaine, IC50 = 249 &mgr;m). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2&Dgr;C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics’ affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Conclusions:Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.

Clinically relevant concentrations of propofol have no effect on adenosine triphosphate-sensitive potassium channels in rat ventricular myocytes

Background Activation of adenosine triphosphate–sensitive potassium (KATP) channels produces cardioprotective effects during ischemia. Because propofol is often used in patients who have coronary artery disease undergoing a wide variety of surgical procedures, it is important to evaluate the direct effects of propofol on KATP channel activities in ventricular myocardium during ischemia. Methods The effects of propofol (0.4–60.1 &mgr;g/ml) on both sarcolemmal and mitochondrial KATP channel activities were investigated in single, quiescent rat ventricular myocytes. Membrane currents were recorded using cell-attached and inside-out patch clamp configurations. Flavoprotein fluorescence was measured to evaluate mitochondrial oxidation mediated by mitochondrial KATP channels. Results In the cell-attached configuration, open probability of KATP channels was reduced by propofol in a concentration-dependent manner (EC50 = 14.2 &mgr;g/ml). In the inside-out configurations, propofol inhibited KATP channel activities without changing the single-channel conductance (EC50 = 11.4 &mgr;g/ml). Propofol reduced mitochondrial oxidation in a concentration-dependent manner with an EC50 of 14.6 &mgr;g/ml. Conclusions Propofol had no effect on the sarcolemmal KATP channel activities in patch clamp configurations and the mitochondrial flavoprotein fluorescence induced by diazoxide at clinically relevant concentrations (< 2 &mgr;m), whereas it significantly inhibited both KATP channel activities at very high, nonclinical concentrations (> 5.6 &mgr;g/ml; 31 &mgr;m).