Yousuke T. Horikawa

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.

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.

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.

Dark chocolate receptors: epicatechin-induced cardiac protection is dependent on δ-opioid receptor stimulation

Epicatechin, a flavonoid, is a well-known antioxidant linked to a variety of protective effects in both humans and animals. In particular, its role in protection against cardiovascular disease has been demonstrated by epidemiologic studies. Low-dose epicatechin, which does not have significant antioxidant activity, is also protective; however, the mechanism by which low-dose epicatechin induces this effect is unknown. Our laboratory tested the hypothesis that low-dose epicatechin mediates cardiac protection via opioid receptor activation. C57BL/6 mice were randomly assigned to 1 of 10 groups: control, epicatechin, naloxone (nonselective opioid receptor antagonist), epicatechin + naloxone, naltrindole (δ-specific opioid receptor antagonist), epicatechin + naltrindole, norbinaltorphimine (nor-BNI, κ-specific opioid receptor antagonist), epicatechin + nor-BNI, 5-hydroxydecanoic acid [5-HD, ATP-sensitive potassium channel antagonist], and epicatechin + 5-HD. Epicatechin (1 mg/kg) or other inhibitors (5 mg/kg) were administered by oral gavage or intraperitoneal injection, respectively, daily for 10 days. Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion, and infarct size was determined via planimetry. Whole heart homogenates were assayed for downstream opioid receptor signaling targets. Infarct size was significantly reduced in epicatechin- and epicatechin + nor-BNI-treated mice compared with control mice. This protection was blocked by naloxone, naltrindole, and 5-HD. Epicatechin and epicatechin + nor-BNI increased the phosphorylation of Src, Akt, and IκBα, while simultaneously decreasing the expression of c-Jun NH(2)-terminal kinase and caspase-activated DNase. All signaling effects are consistent with opioid receptor stimulation and subsequent cardiac protection. Naloxone, naltrindole, and 5-HD attenuated these effects. In conclusion, epicatechin acts via opioid receptors and more specifically through the δ-opioid receptor to produce cardiac protection from ischemia-reperfusion injury.

Electrophysiology and metabolism of caveolin-3-overexpressing mice

Caveolin-3 (Cav-3) plays a critical role in organizing signaling molecules and ion channels involved in cardiac conduction and metabolism. Mutations in Cav-3 are implicated in cardiac conduction abnormalities and myopathies. Additionally, cardiac-specific overexpression of Cav-3 (Cav-3 OE) is protective against ischemic and hypertensive injury, suggesting a potential role for Cav-3 in basal cardiac electrophysiology and metabolism involved in stress adaptation. We hypothesized that overexpression of Cav-3 may alter baseline cardiac conduction and metabolism. We examined: (1) ECG telemetry recordings at baseline and during pharmacological interventions, (2) ion channels involved in cardiac conduction with immunoblotting and computational modeling, and (3) baseline metabolism in Cav-3 OE and transgene-negative littermate control mice. Cav-3 OE mice had decreased heart rates, prolonged PR intervals, and shortened QTc intervals with no difference in activity compared to control mice. Dobutamine or propranolol did not cause significant changes between experimental groups in maximal (dobutamine) or minimal (propranolol) heart rate. Cav-3 OE mice had an overall lower chronotropic response to atropine. The expression of Kv1.4 and Kv4.3 channels, Nav1.5 channels, and connexin 43 were increased in Cav-3 OE mice. A computational model integrating the immunoblotting results indicated shortened action potential duration in Cav-3 OE mice linking the change in channel expression to the observed electrophysiology phenotype. Metabolic profiling showed no gross differences in VO2, VCO2, respiratory exchange ratio, heat generation, and feeding or drinking. In conclusion, Cav-3 OE mice have changes in ECG intervals, heart rates, and cardiac ion channel expression. These findings give novel mechanistic insights into previously reported Cav-3 dependent cardioprotection.

Geranylgeranylacetone protects the heart via caveolae and caveolin-3

AIMS Geranylgeranylacetone (GGA) is commonly utilized to protect the gastric mucosa in peptic ulcer disease. Recently GGA has been shown to protect the myocardium from ischemia/reperfusion by activating heat shock proteins. However, the exact mechanism as to how GGA activates these protective proteins is unknown. Caveolae and caveolin-3 (Cav-3) have been implicated in ischemia, anesthetic, and opioid induced cardiac protection. Given the lipophilic nature of GGA it is our hypothesis that GGA induced cardiac protection requires caveolae and Cav-3. MAIN METHODS We used an in vivo mouse model of ischemia-reperfusion injury and performed biochemical assays in excised hearts. KEY FINDINGS GGA treated control mice revealed increased caveolae formation and caveolin-3 in buoyant fractions, mediating heat shock protein 70 activation. Furthermore, control mice treated with GGA were protected against ischemia/reperfusion injury whereas Cav-3 knockout (Cav-3 KO) mice were not. Troponin levels confirmed myocardial damage. Finally, Cav-3 KO mice treated with GGA were not protected against mitochondrial swelling whereas control mice had significant protection. SIGNIFICANCE This study showed that caveolae and caveolin-3 are essential in facilitating GGA induced cardiac protection by optimizing spatial and temporal signaling to the mitochondria.

Whey Peptide–Based Formulas With ω-3 Fatty Acids Are Protective in Lipopolysaccharide-Mediated Sepsis

BACKGROUND Sepsis and septic shock syndrome are among the leading causes of death in critically ill patients. Lipopolysaccharide (LPS) released by bacteria within the colon may translocate across a compromised epithelium, leading to oxidative stress, inflammation, sepsis, and eventually death. METHODS We examined the effects of a whey-based enteral formula high in cysteine (antioxidant precursor) and the addition of ω-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), against a mouse model of LPS-induced sepsis. Mice were fed either a whey-based diet with EPA-DHA (PAF), a whey-based diet without EPA-DHA (PSTD), or a casein-based control diet (CONT). RESULTS Mice fed PAF or PSTD were protected against LPS-induced weight loss. Whey-based diets suppressed inflammatory cytokine release and oxidative stress damage. Furthermore, PAF and PSTD were able to inhibit autophagy, a mechanism in which the cell recycles damaged organelles. These anti-inflammatory and antioxidative effects of PSTD and PAF resulted in decreased liver inflammation and intestinal damage and promoted protective microbiota within the intestines. CONCLUSIONS These data suggest a clinical role for whey peptide-based diets in promoting healing and recovery in critically ill patients.

Signaling epicenters: the role of caveolae and caveolins in volatile anesthetic induced cardiac protection.

Caveolae are flask-like invaginations of the cell surface that have been identified as signaling epicenters. Within these microdomains, caveolins are structural proteins of caveolae, which are able to interact with numerous signaling molecules affecting temporal and spatial dimensions required in cardiac protection. This complex moiety is essential to the mechanisms involved in volatile anesthetics. In this review we will outline a general overview of caveolae and caveolins and their role in protective signaling with a focus on the effects of volatile anesthetics. These recent developments have allowed us to better understand the mechanistic effect of volatile anesthetics and their potential in cardiac protection.