Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes.

Statins, which reduce LDL-cholesterol by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are among the most widely prescribed drugs. Skeletal myopathy is a known statin-induced adverse effect associated with mitochondrial changes. We hypothesized that similar effects would occur in cardiac myocytes in a lipophilicity-dependent manner between 2 common statins: atorvastatin (lipophilic) and pravastatin (hydrophilic). Neonatal cardiac ventricular myocytes were treated with atorvastatin and pravastatin for 48 h. Both statins induced endoplasmic reticular (ER) stress, but only atorvastatin inhibited ERK1/2T202/Y204, AktSer473, and mammalian target of rapamycin signaling; reduced protein abundance of caveolin-1, dystrophin, epidermal growth factor receptor, and insulin receptor-ß; decreased Ras homolog gene family member A activation; and induced apoptosis. In cardiomyocyte-equivalent HL-1 cells, atorvastatin, but not pravastatin, reduced mitochondrial oxygen consumption. When male mice underwent atorvastatin and pravastatin administration per os for up to 7 mo, only long-term atorvastatin, but not pravastatin, induced elevated serum creatine kinase; swollen, misaligned, size-variable, and disconnected cardiac mitochondria; alteration of ER structure; repression of mitochondria- and endoplasmic reticulum-related genes; and a 21% increase in mortality in cardiac-specific vinculin-knockout mice during the first 2 months of administration. To our knowledge, we are the first to demonstrate in vivo that long-term atorvastatin administration alters cardiac ultrastructure, a finding with important clinical implications.-Godoy, J. C., Niesman, I. R., Busija, A. R., Kassan, A., Schilling, J. M., Schwarz, A., Alvarez, E. A., Dalton, N. D., Drummond, J. C., Roth, D. M., Kararigas, G., Patel, H. H., Zemljic-Harpf, A. E. Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes.

Caveolins and cavins in the trafficking, maturation, and degradation of caveolae: implications for cell physiology.

Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae ("little caves") require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring of Cav oligomers, ubiquitination, internalization, and degradation. In this review, we provide a perspective of the life cycle (biogenesis, degradation), composition, and physiologic roles of Cavs and caveolae and identify unanswered questions regarding the roles of Cavs and cavins in caveolae and in regulating cell physiology.1.

The plasma membrane as a capacitor for energy and metabolism.

When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as "capacitors for energy and metabolism." Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell.

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin-43-containing gap junctions in cardiac myocytes.

Vinculin (Vcl) links actin filaments to integrin- and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl co-immunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and ß1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection.

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-ß-cyclodextrin (MßCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MßCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MßCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 µM MßCD, whereas SLP was more robust and only inhibited with ≥200 µM MßCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

ROS-independent preconditioning in neurons via activation of mitoK(ATP) channels by BMS-191095.

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3beta. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.

Neuron-Targeted Caveolin-1 Improves Molecular Signaling, Plasticity, and Behavior Dependent on the Hippocampus in Adult and Aged Mice.

BACKGROUND: Studies in vitro demonstrate that neuronal membrane/lipid rafts (MLRs) establish cell polarity by clustering progrowth receptors and tethering cytoskeletal machinery necessary for neuronal sprouting. However, the effect of MLR and MLR-associated proteins on neuronal aging is unknown. METHODS: Here, we assessed the impact of neuron-targeted overexpression of an MLR scaffold protein, caveolin-1 (Cav-1) (via a synapsin promoter, SynCav1), in the hippocampus in vivo in adult (6-month-old) and aged (20-month-old) mice on biochemical, morphologic, and behavioral changes. RESULTS: SynCav1 resulted in increased expression of Cav-1, MLRs, and MLR-localization of Cav-1 and tropomyosin-related kinase B receptor independent of age and time post gene transfer. Cav-1 overexpression in adult mice enhanced dendritic arborization within the apical dendrites of hippocampal cornu ammonis 1 and granule cell neurons, effects that were also observed in aged mice, albeit to a lesser extent, indicating preserved impact of Cav-1 on structural plasticity of hippocampal neurons with age. Cav-1 overexpression enhanced contextual fear memory in adult and aged mice demonstrating improved hippocampal function. CONCLUSIONS: Neuron-targeted overexpression of Cav-1 in the adult and aged hippocampus enhances functional MLRs with corresponding roles in cell signaling and protein trafficking. The resultant structural alterations in hippocampal neurons in vivo are associated with improvements in hippocampal-dependent learning and memory. Our findings suggest Cav-1 as a novel therapeutic strategy in disorders involving impaired hippocampal function.

Chronic ß1-adrenoceptor blockade impairs ischaemic tolerance and preconditioning in murine myocardium.

ß-adrenoceptor antagonists are commonly used in ischaemic heart disease (IHD) patients, yet may impair signalling and efficacy of 'cardioprotective' interventions. We assessed effects of chronic ß1-adrenoceptor antagonism on myocardial resistance to ischaemia-reperfusion (IR) injury and the ability of cardioprotective interventions [classic ischaemic preconditioning (IPC); novel sustained ligand-activated preconditioning (SLP)] to reduce IR injury in murine hearts. Young male C57Bl/6 mice were untreated or received atenolol (0.5g/l in drinking water) for 4 weeks. Subsequently, two cardioprotective stimuli were evaluated: morphine pellets implanted (to induce SLP, controls received placebo) 5 days prior to Langendorff heart perfusion, and IPC in perfused hearts (3×1.5min ischaemia/2min reperfusion). Atenolol significantly reduced in vivo heart rate. Untreated control hearts exhibited substantial left ventricular dysfunction (~50% pressure development recovery, ~20mmHg diastolic pressure rise) with significant release of lactate dehydrogenase (LDH, tissue injury indicator) after 25min ischaemia/45min reperfusion. Contractile dysfunction and elevated LDH were reduced >50% with IPC and SLP. While atenolol treatment did not modify baseline contractile function, post-ischaemic function was significantly depressed compared to untreated hearts. Atenolol pre-treatment abolished beneficial effects of IPC, whereas SLP protection was preserved. These data indicate that chronic ß1-adrenoceptor blockade can exert negative effects on functional IR tolerance and negate conventional IPC (implicating ß1-adrenoceptors in IR injury and IPC signalling). However, novel morphine-induced SLP is resistant to inhibition by ß1-adrenoceptor antagonism.

Impaired mitochondria-dependent vasodilation in cerebral arteries of Zucker obese rats with insulin resistance.

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP-sensitive K+ (K ATP) channels and generation of reactive oxygen species (ROS). Insulin resistance accompanying obesity causes mitochondrial dysfunction, but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitochondrial K ATP channel activator, on cerebral arteries of Zucker obese (ZO) rats with insulin resistance and lean (ZL) controls. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared with ZL rats. Maximal relaxation was 38 +/- 3% in ZL vs. 21 +/- 4% in ZO rats (P < 0.05). Iberiotoxin, a Ca2+-activated K+ channel inhibitor, or manganese(III) tetrakis(4-benzoic acid)porphyrin chloride, an SOD mimetic, or endothelial denudation diminished vasodilation to diazoxide, implicating Ca2+-activated K+ channels, ROS, and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL rats diminished diazoxide-induced vasodilation in intact arteries, but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO rats enhanced vasodilation in endothelium-denuded arteries, but intact arteries were unaffected, suggesting that activity of endothelial NOS was abolished, whereas factors derived from nonendothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production, and nitric oxide generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of endothelial NOS and SODs in ZO arteries. Thus, cerebrovascular dilation to mitochondria-derived factors involves integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitochondrial K(ATP) channel activation, diminished mitochondrial ROS generation, increased ROS scavenging, and abnormal NOS activity.