Heat shock protein 60 and cardiovascular diseases: An intricate love-hate story.

Cardiovascular diseases (CVDs) are the result of complex pathophysiological processes in the tissues comprising the heart and blood vessels. Inflammation is the main culprit for the development of cardiovascular dysfunction, and it may be traced to cellular stress events including apoptosis, oxidative and shear stress, and cellular and humoral immune responses, all of which impair the system's structure and function. An intracellular chaperone, heat shock protein 60 (HSP60) is an intriguing example of a protein that may both be an ally and a foe for cardiovascular homeostasis; on one hand providing protection against cellular injury, and on the other triggering damaging responses through innate and adaptive immunity. In this review we will discuss the functions of HSP60 and its effects on cells and the immune system regulation, only to later address its implications in the development and progression of CVD. Lastly, we summarize the outcome of various studies targeting HSP60 as a potential therapeutic strategy for cardiovascular and other diseases.

Exosomes in disease and regeneration: biological functions, diagnostics, and beneficial effects.

Exosomes are a subtype of extracellular vesicles. They range from 30 to 150 nm in diameter and originate from intraluminal vesicles. Exosomes were first identified as the mechanism for releasing unnecessary molecules from reticulocytes as they matured to red blood cells. Since then, exosomes have been shown to be secreted by a broad spectrum of cells and play an important role in the cardiovascular system. Different stimuli are associated with increased exosome release and result in different exosome content. The release of harmful DNA and other molecules via exosomes has been proposed as a mechanism to maintain cellular homeostasis. Because exosomes contain parent cell-specific proteins on the membrane and in the cargo that is delivered to recipient cells, exosomes are potential diagnostic biomarkers of various types of diseases, including cardiovascular disease. As exosomes are readily taken up by other cells, stem cell-derived exosomes have been recognized as a potential cell-free regenerative therapy to repair not only the injured heart but other tissues as well. The objective of this review is to provide an overview of the biological functions of exosomes in heart disease and tissue regeneration. Therefore, state-of-the-art methods for exosome isolation and characterization, as well as approaches to assess exosome functional properties, are reviewed. Investigation of exosomes provides a new approach to the study of disease and biological processes. Exosomes provide a potential "liquid biopsy," as they are present in most, if not all, biological fluids that are released by a wide range of cell types.

Impaired proteostasis in senescent vascular endothelial cells: a perspective on estrogen and oxidative stress in the aging vasculature.

The heat shock response is an important cytoprotective mechanism for protein homeostasis and is an essential protective response to cellular stress and injury. Studies on changes in the heat shock response with aging have been mixed with regard to whether it is inhibited, and this, at least in part, reflects different tissues and different models. Cellular senescence is a key feature in aging, but work on the heat shock response in cultured senescent (SEN) cells has largely been limited to fibroblasts. Given the prevalence of oxidative injury in the aging cardiovascular system, we investigated whether SEN primary human coronary artery endothelial cells have a diminished heat shock response and impaired proteostasis. In addition, we tested whether this downregulation of heat shock response can be mitigated by 17ß-estradiol (E2), which has a critical cardioprotective role in women, as we have previously reported that E2 improves the heat shock response in endothelial cells (Hamilton KL, Mbai FN, Gupta S, Knowlton AA. Arterioscler Thromb Vasc Biol 24: 1628-1633, 2004). We found that SEN endothelial cells, despite their unexpectedly increased proteasome activity, had a diminished heat shock response and had more protein aggregation than early passage cells. SEN cells had increased oxidative stress, which promoted protein aggregation. E2 treatment did not decrease protein aggregation or improve the heat shock response in either early passage or SEN cells. In summary, cellular senescence in adult human endothelial cells is accompanied by increased oxidative stress and a blunting of proteostasis, and E2 did not mitigate these changes. NEW & NOTEWORTHY Senescent human endothelial cells have a diminished heat shock response and increased protein aggregates. Senescent human endothelial cells have increased basal oxidative stress, which increases protein aggregates. Physiological level of 17ß-estradiol did not improve proteostasis in endothelial cells.

Distinct subcellular mechanisms for the enhancement of the surface membrane expression of SK2 channel by its interacting proteins, α-actinin2 and filamin A.

KEY POINTS: Ion channels are transmembrane proteins that are synthesized within the cells but need to be trafficked to the cell membrane for the channels to function. Small-conductance, Ca2+ -activated K+ channels (SK, KCa 2) are unique subclasses of K+ channels that are regulated by Ca2+ inside the cells; they are expressed in human atrial myocytes and responsible for shaping atrial action potentials. We have previously shown that interacting proteins of SK2 channels are important for channel trafficking to the membrane. Using total internal reflection fluorescence (TIRF) and confocal microscopy, we studied the mechanisms by which the surface membrane localization of SK2 (KCa 2.2) channels is regulated by their interacting proteins. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. ABSTRACT: The normal function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane. Small-conductance, Ca2+ -activated K+ channels (SK, KCa 2) are expressed in human atrial myocytes and are responsible for shaping atrial action potentials. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. We have previously demonstrated that the C- and N-termini of SK2 channels interact with the actin-binding proteins α-actinin2 and filamin A, respectively. However, the roles of the interacting proteins on SK2 channel trafficking remain incompletely understood. Using total internal reflection fluorescence (TIRF) microscopy, we studied the mechanisms of surface membrane localization of SK2 (KCa 2.2) channels. When SK2 channels were co-expressed with filamin A or α-actinin2, the membrane fluorescence intensity of SK2 channels increased significantly. We next tested the effects of primaquine and dynasore on SK2 channels expression. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. In contrast, treatment with dynasore failed to alter the surface membrane expression of SK2 channels. Further investigations using constitutively active or dominant-negative forms of Rab GTPases provided additional insights into the distinct roles of the two cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α-Actinin2 facilitated recycling of SK2 channels from both early and recycling endosomes while filamin A probably aids the recycling of SK2 channels from recycling endosomes.

Paying for the Tolls: The High Cost of the Innate Immune System for the Cardiac Myocyte.

The cardiac myocyte differs strikingly from the specialized cells of the immune system, which has two different responses to invading organisms and tissue damage. Adaptive or acquired immunity generates highly specific antibodies in response to threats and is an essential component of immunity; however, adaptive immunity can take 4-7 days to mobilize, and a more primitive response, innate immunity, fills the gap. Innate immunity is expressed in complex and in primitive life forms. Specialized receptors, Toll-like receptors (TLRs), which are widely distributed throughout different tissues recognize danger signals and rapidly respond with the release of noxious substances, such as TNFα. The problem is that many endogenous molecules have been found to act as ligands for specific TLRs, and when these molecules are released into the extracellular environment, they can cause problems by activating innate immunity and an inflammatory response. In cardiac myocytes heat shock protein (HSP)60 can activate TLR4, as can HMGB1, and this type of response can amplify the response to ischemia/reperfusion leading to increased cell and tissue injury. Activation of TLRs can potentially amplify chronic, inflammatory diseases, such as ischemic heart failure. Thus, it is important to understand the regulation of the TLRs and their downstream effects. This chapter will focus on the TLRs and cardiac myocytes.

Functional interaction with filamin A and intracellular Ca2+ enhance the surface membrane expression of a small-conductance Ca2+-activated K+ (SK2) channel.

For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca(2+)-activated K(+) channels (KCa2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca(2+)-dependent. Further, the Ca(2+) dependence relies on another channel-interacting protein, α-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.

11,12-Epoxyecosatrienoic acids mitigate endothelial dysfunction associated with estrogen loss and aging: Role of membrane depolarization.

OBJECTIVE: Endothelial dysfunction, including upregulation of inflammatory adhesion molecules and impaired vasodilatation, is a key element in cardiovascular disease. Aging and estrogen withdrawal in women are associated with endothelial inflammation, vascular stiffness and increased cardiovascular disease. Epoxyecosatrienoic acids (EETs), the products of arachidonic acid metabolism mediated by cytochrome P450 (CYP) 2J, 2C and other isoforms, are regulated by soluble epoxide hydrolase (sEH)-catalyzed conversion into less active diols. We hypothesized that 11,12-EETs would reduce the endothelial dysfunction associated with aging and estrogen loss. APPROACH/RESULTS: When stabilized by an sEH inhibitor (seHi), 11,12-EET at a physiologically low dose (0.1nM) reduced cytokine-stimulated upregulation of adhesion molecules on human aorta endothelial cells (HAEC) and monocyte adhesion under shear flow through marked depolarization of the HAEC when combined with TNFα. Mechanistically, neither 11,12-EETs nor 17ß-estradiol (E2) at physiologic concentrations prevented activation of NFκB by TNFα. E2 at physiological concentrations reduced sEH expression in HAEC, but did not alter CYP expression, and when combined with TNFα depolarized the cell. We also examined vascular dysfunction in adult and aged ovariectomized Norway brown rats (with and without E2 replacement) using an ex-vivo model to analyze endothelial function in an intact segment of artery. sEHi and 11,12-EET with or without E2 attenuated phenylephrine induced constriction and increased endothelial-dependent dilation of aortic rings from ovariectomized rats. CONCLUSIONS: Increasing 11,12-EETs through sEH inhibition effectively attenuates inflammation and may provide an effective strategy to preserve endothelial function and prevent atherosclerotic heart disease in postmenopausal women.

Regulation of gene transcription by voltage-gated L-type calcium channel, Cav1.3.

Cav1.3 L-type Ca(2+) channel is known to be highly expressed in neurons and neuroendocrine cells. However, we have previously demonstrated that the Cav1.3 channel is also expressed in atria and pacemaking cells in the heart. The significance of the tissue-specific expression of the channel is underpinned by our previous demonstration of atrial fibrillation in a Cav1.3 null mutant mouse model. Indeed, a recent study has confirmed the critical roles of Cav1.3 in the human heart (Baig, S. M., Koschak, A., Lieb, A., Gebhart, M., Dafinger, C., Nürnberg, G., Ali, A., Ahmad, I., Sinnegger-Brauns, M. J., Brandt, N., Engel, J., Mangoni, M. E., Farooq, M., Khan, H. U., Nürnberg, P., Striessnig, J., and Bolz, H. J. (2011) Nat. Neurosci. 14, 77-84). These studies suggest that detailed knowledge of Cav1.3 may have broad therapeutic ramifications in the treatment of cardiac arrhythmias. Here, we tested the hypothesis that there is a functional cross-talk between the Cav1.3 channel and a small conductance Ca(2+)-activated K(+) channel (SK2), which we have documented to be highly expressed in human and mouse atrial myocytes. Specifically, we tested the hypothesis that the C terminus of Cav1.3 may translocate to the nucleus where it functions as a transcriptional factor. Here, we reported for the first time that the C terminus of Cav1.3 translocates to the nucleus where it functions as a transcriptional regulator to modulate the function of Ca(2+)-activated K(+) channels in atrial myocytes. Nuclear translocation of the C-terminal domain of Cav1.3 is directly regulated by intracellular Ca(2+). Utilizing a Cav1.3 null mutant mouse model, we demonstrate that ablation of Cav1.3 results in a decrease in the protein expression of myosin light chain 2, which interacts and increases the membrane localization of SK2 channels.

Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure.

RATIONALE: Calmodulin (CaM) associates with cardiac ryanodine receptor type-2 (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure, cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia. However, the in situ binding properties for CaM-RyR2 are unknown. OBJECTIVE: We sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and heart failure ventricular myocytes, estimate the percentage of Z-line-localized CaM that is RyR2-bound, and test cellular function of defective CaM-RyR2 interaction. METHODS AND RESULTS: Using fluorescence resonance energy transfer in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets and measured CaM-RyR2 binding affinity in situ (Kd=10-20 nmol/L). Using RyR2(ADA/+) knock-in mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ wave production and stress-induced ventricular arrhythmia in RyR2(ADA/+) mice. In a post-myocardial infarction rat heart failure model, we detected a decrease in the CaM-RyR2 binding affinity (Kd≈51 nmol/L; ≈3-fold increase) and unaltered RyR2 affinity for the FK506-binding protein FKBP12.6 (Kd~0.8 nmol/L). CONCLUSIONS: CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits sarcoplasmic reticulum Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes, and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.

Hyperamylinemia contributes to cardiac dysfunction in obesity and diabetes: a study in humans and rats.

RATIONALE: Hyperamylinemia is common in patients with obesity and insulin resistance, coincides with hyperinsulinemia, and results in amyloid deposition. Amylin amyloids are generally considered a pancreatic disorder in type 2 diabetes. However, elevated circulating levels of amylin may also lead to amylin accumulation and proteotoxicity in peripheral organs, including the heart. OBJECTIVE: To test whether amylin accumulates in the heart of obese and type 2 diabetic patients and to uncover the effects of amylin accumulation on cardiac morphology and function. METHODS AND RESULTS: We compared amylin deposition in failing and nonfailing hearts from lean, obese, and type 2 diabetic humans using immunohistochemistry and Western blots. We found significant accumulation of large amylin oligomers, fibrils, and plaques in failing hearts from obese and diabetic patients but not in normal hearts and failing hearts from lean, nondiabetic humans. Small amylin oligomers were even elevated in nonfailing hearts from overweight/obese patients, suggesting an early state of accumulation. Using a rat model of hyperamylinemia transgenic for human amylin, we observed that amylin oligomers attach to the sarcolemma, leading to myocyte Ca(2+) dysregulation, pathological myocyte remodeling, and diastolic dysfunction, starting from prediabetes. In contrast, prediabetic rats expressing the same level of wild-type rat amylin, a nonamyloidogenic isoform, exhibited normal heart structure and function. CONCLUSIONS: Hyperamylinemia promotes amylin deposition in the heart, causing alterations of cardiac myocyte structure and function. We propose that detection and disruption of cardiac amylin buildup may be both a predictor of heart dysfunction and a novel therapeutic strategy in diabetic cardiomyopathy.

IRF-1 and miRNA126 modulate VCAM-1 expression in response to a high-fat meal.

RATIONALE: A high-fat diet accompanied by hypertriglyceridemia increases an individual's risk for development of atherosclerosis. An early event in this process is monocyte recruitment through binding to vascular cell adhesion molecule 1 (VCAM-1) upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect. OBJECTIVE: We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects after consumption of a high-fat meal. METHODS AND RESULTS: Postprandial TGRL isolated from 38 subjects were categorized as proatherogenic or antiatherogenic according to their capacity to alter the inflammatory response of HAEC. Proatherogenic TGRL increased expression of VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and E-selectin by ≈20% compared with stimulation with tumor necrosis factor-α alone, whereas antiatherogenic TGRL decreased VCAM-1 expression by ≈20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, apolipoprotein (Apo)CIII, ApoE, and cholesterol. Ω3-PUFA mimicked the effect of antiatherogenic TGRL by downregulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor interferon regulatory factor 1 (IRF-1) and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the proatherogenic or antiatherogenic regulation of VCAM-1. CONCLUSIONS: In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and posttranscriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a meal produced TGRL that was enriched in nonesterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.

Heart failure and mitochondrial dysfunction: the role of mitochondrial fission/fusion abnormalities and new therapeutic strategies.

The treatment of heart failure (HF) has evolved during the past 30 years with the recognition of neurohormonal activation and the effectiveness of its inhibition in improving the quality of life and survival. Over the past 20 years, there has been a revolution in the investigation of the mitochondrion with the development of new techniques and the finding that mitochondria are connected in networks and undergo constant division (fission) and fusion, even in cardiac myocytes. This has led to new molecular and cellular discoveries in HF, which offer the potential for the development of new molecular-based therapies. Reactive oxygen species are an important cause of mitochondrial and cellular injury in HF, but there are other abnormalities, such as depressed mitochondrial fusion, that may eventually become the targets of at least episodic treatment. The overall need for mitochondrial fission/fusion balance may preclude sustained change in either fission or fusion. In this review, we will discuss the current HF therapy and its impact on the mitochondria. In addition, we will review some of the new drug targets under development. There is potential for effective, novel therapies for HF to arise from new molecular understanding.

Mitochondrial dynamic changes in health and genetic diseases.

Mitochondria are highly specialized in function, but mitochondrial and, therefore, cellular integrity is maintained through their dynamic nature. Through the frequent processes of fusion and fission, mitochondria continuously change in shape and adjust function to meet cellular requirements. Abnormalities in fusion/fission dynamics generate cellular dysfunction that may lead to diseases. Mutations in the genes encoding mitochondrial fusion/fission proteins, such as MFN2 and OPA1, have been associated with an increasing number of genetic disorders, including Charcot-Marie-Tooth disease type 2A (CMT2A) and autosomal dominant optic atrophy. In this review, we address the mitochondrial dynamic changes in several important genetic diseases, which will bring the new insight of clinical relevance of mitochondrial genetics.

Tocotrienols confer resistance to ischemia in hypercholesterolemic hearts: insight with genomics.

Most clinical trials with vitamin E could not lower cholesterol and thus, have been deemed unsuccessful. Recently, tocotrienols, isomers of vitamin E have been found to lower LDL levels. To explore if tocotrienols could be the drug target for vitamin E, rabbits were kept on cholesterol diet for 60 days supplemented with tocotrienol-α, tocotrienol-δ, and tocotrienol-γ for the last 30 days. The serum cholesterol levels (in mmol/l) were 24.4 (tocotrienol-α), 34.9 (tocotrienol-δ), 19.8 (tocotrienol-γ) vs. 39.7 (control). Left ventricular function including aortic flow and developed pressure exhibited significantly improved recovery with tocotrienol-γ and -α, but not with tocotrienol-δ. The myocardial infarct size showed a similar pattern: 33% (tocotrienol-α), 23% (tocotrienol-γ), and 47% (tocotrienol-δ). To examine the molecular mechanisms of cardioprotective effects, gene expression profile was determined using Atlas 1.2/1.2II followed by determination of gene profiles using PedQuest 8.3 software. Based on genomic profiles, the following cholesterol-related proteins were examined: FABP, TGF-ß (cholesterol suppresses TGF-ß), ET-1 (increased by hypercholesterolemia), SPOT 14 (linked with hypercholesterolemia), and matrix metalloproteinase (MMP) 2 and MMP9 (cholesterol regulates MMP2 and MMP9 expression) in the heart. Consistent with the cardioprotective effects of tocotrienol-α and -γ, these two isomers reduced ET-1, decreased MMP2 and MM9, increased TGF-ß and reduced SPOT 14, while tocotrienol-δ had no effects. The results of the present study demonstrate that the two isomers of tocotrienols, α and γ, render the hypercholesterolemic hearts resistant to ischemic reperfusion injury by lowering several hypercholesterolemic proteins including MMP2, MMP9, ET-1, and SPOT 14 and upregulating TGF-ß.

Cardiac small conductance Ca2+-activated K+ channel subunits form heteromultimers via the coiled-coil domains in the C termini of the channels.

RATIONALE: Ca(2+)-activated K(+) channels are present in a wide variety of cells. We have previously reported the presence of small conductance Ca(2+)-activated K(+) (SK or K(Ca)) channels in human and mouse cardiac myocytes that contribute functionally toward the shape and duration of cardiac action potentials. Three isoforms of SK channel subunits (SK1, SK2, and SK3) are found to be expressed. Moreover, there is differential expression with more abundant SK channels in the atria and pacemaking tissues compared with the ventricles. SK channels are proposed to be assembled as tetramers similar to other K(+) channels, but the molecular determinants driving their subunit interaction and assembly are not defined in cardiac tissues. OBJECTIVE: To investigate the heteromultimeric formation and the domain necessary for the assembly of 3 SK channel subunits (SK1, SK2, and SK3) into complexes in human and mouse hearts. METHODS AND RESULTS: Here, we provide evidence to support the formation of heteromultimeric complexes among different SK channel subunits in native cardiac tissues. SK1, SK2, and SK3 subunits contain coiled-coil domains (CCDs) in the C termini. In vitro interaction assay supports the direct interaction between CCDs of the channel subunits. Moreover, specific inhibitory peptides derived from CCDs block the Ca(2+)-activated K(+) current in atrial myocytes, which is important for cardiac repolarization. CONCLUSIONS: The data provide evidence for the formation of heteromultimeric complexes among different SK channel subunits in atrial myocytes. Because SK channels are predominantly expressed in atrial myocytes, specific ligands of the different isoforms of SK channel subunits may offer a unique therapeutic opportunity to directly modify atrial cells without interfering with ventricular myocytes.

CD11c/CD18 expression is upregulated on blood monocytes during hypertriglyceridemia and enhances adhesion to vascular cell adhesion molecule-1.

OBJECTIVE: Atherosclerosis is associated with monocyte adhesion to the arterial wall that involves integrin activation and emigration across inflamed endothelium. Involvement of ß(2)-integrin CD11c/CD18 in atherogenesis was recently shown in dyslipidemic mice, which motivates our study of its inflammatory function during hypertriglyceridemia in humans. METHODS AND RESULTS: Flow cytometry of blood from healthy subjects fed a standardized high-fat meal revealed that at 3.5 hours postprandial, monocyte CD11c surface expression was elevated, and the extent of upregulation correlated with blood triglycerides. Monocytes from postprandial blood exhibited an increased light scatter profile, which correlated with elevated CD11c expression and uptake of lipid particles. Purified monocytes internalized triglyceride-rich lipoproteins isolated from postprandial blood through low-density lipoprotein-receptor-related protein-1, and this also elicited CD11c upregulation. Laboratory-on-a-chip analysis of whole blood showed that monocyte arrest on a vascular cell adhesion molecule-1 (VCAM-1) substrate under shear flow was elevated at 3.5 hours and correlated with blood triglyceride and CD11c expression. At 7 hours postprandial, blood triglycerides decreased and monocyte CD11c expression and arrest on VCAM-1 returned to fasting levels. CONCLUSIONS: During hypertriglyceridemia, monocytes internalize lipids, upregulate CD11c, and increase adhesion to VCAM-1. These data suggest that analysis of monocyte inflammation may provide an additional framework for evaluating individual susceptibility to cardiovascular disease.

Neutral sphingomyelinase 2 (nSMase2) is a phosphoprotein regulated by calcineurin (PP2B).

We previously reported that exposure of human airway epithelial cells to oxidative stress increased ceramide generation via specific activation of neutral sphingomyelinase2 (nSMase2). Here we show that nSMase2 is a phosphoprotein exclusively phosphorylated at serine residues. The level of nSMase2 phosphorylation can be modulated by treatment with anisomycin or phorbol 12-myristate 13-acetate (PMA/12-O-tetradecanoylphorbol-13-acetate), suggesting that p38 mitogen-activated protein kinase (MAPK) and protein kinases Cs are upstream of nSMase2 phosphorylation. Oxidative stress enhances both the activity and phosphorylation of nSMase2. Strikingly, we show here that nSMase2 is bound directly by the phosphatase calcineurin (CaN), which acts as an on/off switch for nSMase2 phosphorylation in the presence or absence of oxidative stress. Specifically, CaN is being inhibited/degraded and therefore does not bind nSMase2 under oxidative stress, and a mutant nSMase2 that lacks the CaN binding site exhibits constitutively elevated phosphorylation and increased activity relative to wild type nSMase2. Importantly, the phosphorylation and activity of the mutant no longer responds to oxidative stress, confirming that CaN is the critical link that allows oxidative stress to modulate nSMase2 phosphorylation and function.

Extracellular heat shock protein 60, cardiac myocytes, and apoptosis.

RATIONALE: Previously, we have found that changes in the location of intracellular heat shock protein (HSP)60 are associated with apoptosis. HSP60 has been reported to be a ligand of toll-like receptor (TLR)-4. OBJECTIVE: We hypothesized that extracellular HSP60 (exHSP60) would mediate apoptosis via TLR4. METHODS AND RESULTS: Adult rat cardiac myocytes were treated with HSP60, either recombinant human or with HSP60 purified from the media of injured rat cardiac myocytes. ExHSP60 induced apoptosis in cardiac myocytes, as detected by increased caspase 3 activity and increased DNA fragmentation. Apoptosis could be reduced by blocking antibodies to TLR4 and by nuclear factor kappaB binding decoys, but not completely inhibited, even though similar treatment blocked lipopolysaccharide-induced apoptosis. Three distinct controls showed no evidence for involvement of a ligand other than exHSP60 in the mediation of apoptosis. CONCLUSIONS: This is the first report of HSP60-induced apoptosis via the TLRs. HSP60-mediated activation of TLR4 may be a mechanism of myocyte loss in heart failure, where HSP60 has been detected in the plasma.

Molecular coupling of a Ca2+-activated K+ channel to L-type Ca2+ channels via alpha-actinin2.

Cytoskeletal proteins are known to sculpt the structural architecture of cells. However, their role as bridges linking the functional crosstalk of different ion channels is unknown. Here, we demonstrate that a small conductance Ca(2+)-activated K(+) channels (SK2 channel), present in a variety of cells, where they integrate changes in intracellular Ca(2+) concentration [Ca(2+)(i)] with changes in K(+) conductance and membrane potential, associate with L-type Ca(2+) channels; Ca(v)1.3 and Ca(v)1.2 through a physical bridge, alpha-actinin2 in cardiac myocytes. SK2 channels do not physically interact with L-type Ca(2+) channels, instead, the 2 channels colocalize via their interaction with alpha-actinin2 cytoskeletal protein. The association of SK2 channel with alpha-actinin2 localizes the channel to the entry of external Ca(2+) source, which regulate the channel function. Furthermore, we demonstrated that the functions of SK2 channels in atrial myocytes are critically dependent on the normal expression of Ca(v)1.3 Ca(2+) channels. Null deletion of Ca(v)1.3 channel results in abnormal function of SK2 channel and prolongation of repolarization and atrial arrhythmias. Our study provides insight into the molecular mechanisms of the coupling of SK2 channel with voltage-gated Ca(2+) channel, and represents the first report linking the coupling of 2 different types of ion channels via cytoskeletal proteins.

Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-alpha. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-kappaB, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.