Factor Associated With Neutral Sphingomyelinase Activation and Its Role in Cardiac Cell Death
Generation of proapoptotic sphingolipids by neutral sphingomyelinase activation is an early response to hypoxia/reoxygenation (HR) in cardiomyocytes. Factor associated with neutral sphingomyelinase activation (FAN) mediates activation of sphingomyelinase and subsequent apoptosis. However, the participation of FAN in HR-induced cardiomyocyte cell death has not been elucidated. We therefore investigated the expression and role of FAN in rat cardiomyocytes. A cDNA was isolated from rat heart encoding putative rat FAN. Reverse transcriptase–polymerase chain reaction, immunoelectron microscopy, and immunofluorescence demonstrated for the first time the expression of FAN specifically in rat cardiomyocytes. FAN expression was confirmed by the finding that expression of a dominant-negative FAN almost completely abrogated HR-induced cell death, whereas overexpression of wild-type FAN led to an increase. Treatment of FAN and dominant-negative FAN–expressing cells with C2-ceramide produced substantial cell death, indicating dominant-negative FAN exerts its protective action by interfering with the activation of the sphingolipid cascade. Taking these results together, we conclude that FAN is a previously undescribed and important HR signaling component in the heart and that inhibition of FAN may provide a novel intervention point for reducing ischemia/reperfusion injury.
The identification of signaling components involved in ischemia/reperfusion (IR) injury is a critical step in designing strategies for mitigating cell death and improving patient outcomes. The sphingolipid-signaling pathway has recently been appreciated as playing a role in apoptotic cell death in cardiomyocytes and cardiovascular disease in general.1 Activation of neutral sphingomyelinase (nSMase)2 and generation of ceramide3 and sphingosine4 may be one of the earliest events in IR injury.
Factor associated with neutral sphingomyelinase activation (FAN) is an adapter protein that is necessary for activation of nSMase by receptors such as tumor necrosis factor receptor type 1 (TNFR1)5 and CD40.6 For example, cell death in TNFα-treated fibroblasts from FAN-deficient mice was impaired but could be restored by transfection of FAN.7
Considering the central role of FAN in the sphingolipid pathway and the potential role of nSMase in cardiac IR injury, we hypothesized that FAN might also play a role in activating the sphingolipid pathway in the heart. In this study, we report for the first time that FAN is expressed in cardiac cells. We also demonstrate its functional role in HR-induced cell death by providing evidence that cell death is abrogated if FAN is functionally repressed.
Materials and Methods
For Materials and Methods, see the online data supplement, available at http://www.circresaha.org.
Results and Discussion
To determine FAN expression in rat heart, total RNA from adult rat heart was used for reverse transcriptase–polymerase chain reaction (RT-PCR) cloning. A novel 2787-bp clone was found, encoding a putative protein of 920 residues with 95% and 91% identity to human and mouse FAN, respectively (Figure 1A). This putative protein of 104.5 kDa was confirmed by Western analysis in both adult and neonatal cardiomyocytes (data not shown).
In common with human and mouse, rat FAN possesses conserved WD-40 repeat, BEACH, and GRAM domains5 (Figure 1A). A search of the PROSITE database revealed consensus protein kinase C (PKC), PKA, and tyrosine kinase phosphorylation sites. Their presence implies FAN may be regulated by kinases and that phosphorylation of FAN may play a role in activation nSMase. Identification of consensus myristylation sites indicates that FAN may be membrane-associated.
Figure 1C shows FAN expression in various tissues by RT-PCR and demonstrates that FAN is expressed not only in the rat heart but also in kidney, brain, and skeletal muscle, while not appreciably in lung.
To elucidate FAN expression specifically in cardiomyocytes, RNA from cultured cardiomyocytes was isolated and used for RT-PCR of FAN mRNA. To ensure these cells were free of fibroblasts, RT-PCR of ANF and col1a1 from cardiomyocytes and COS-7 fibroblast cells was also performed. ANF and col1a1 are cardiomyocyte- and fibroblast-specific, respectively. cDNAs were cloned and sequenced to verify their identity. A FAN product was obtained from both cell types, whereas only cardiomyocytes expressed ANF (Figure 1C). The cardiomyocytes seemed to be a pure population, determined by a lack of col1a1 product, whereas col1a1 was obtained from fibroblasts, as expected.
Coimmunofluorescence was performed to additionally evaluate expression of FAN in cardiomyocytes and its subcellular location. A polyclonal antibody was produced against a peptide (amino acids 537 to 550 of rat FAN). Neonatal cardiomyocytes were coincubated with FAN and caveolin-3 (cav3) antibodies. Cav3 is a sarcolemmal muscle-specific isoform of caveolin used as a marker to identify cardiomyocytes.8 To verify the specificity of the FAN antibody, antibodies were preincubated with increasing amounts of FAN peptide. FAN, but not cav3 staining, was substantially reduced when antibodies were preincubated with FAN peptide (Figure 2A), indicating FAN antibodies were specific.
Importantly, FAN staining was seen at the edge of neonatal cardiomyocytes (Figures 2A and 2B, arrows), which was partially colocalized with cav3 at the sarcolemma (yellow in Figure 2B overlay). FAN staining also appeared to be intracellular, possibly vesicular. These data suggest that FAN is expressed at the plasma membrane, consistent with previous reports on human FAN.9 Immunoelectron microscopy was used to additionally elucidate the localization of FAN in adult cardiomyocytes. Gold particles were localized principally to the cytoplasmic surfaces of the sarcolemma (Figure 2C, arrows), which was not seen using preimmune serum, additionally supporting the novel finding that FAN is expressed in cardiomyocytes at the sarcolemma.
FAN couples various receptors to nSMase activation in other cell types5,6,10 to mediate apoptosis.6,7 Considering that nSMase activation is one of the first responses to HR in cardiomyocytes,2 we determined if FAN is necessary for HR-induced cell death using neonatal rat cardiomyocytes and a well-characterized model simulating IR injury.2,11,12 Cells were cotransfected with GFP and empty-vector, wild-type, or dominant-negative FAN (DN-FAN) V5-tagged constructs. The DN-FAN was designed to encode only the WD-40 repeats. Others have shown that, although still capable of interacting with upstream proteins, the DN-FAN renders wild-type FAN incapable of nSMase activation.5–7,10 The action of this construct was confirmed by demonstration that the DN-FAN abrogated TNFα-induced nSMase activation in COS-7 cells (online Figure 1). Gene transfer and V5 expression were confirmed by Western analysis (Figure 3A).
HR elicited cell death in 13.5±3.2% of control GFP-expressing cells, corresponding to a 7.5-fold activation of cell death over nontreatment (Figure 3B). HR-treated cells overexpressing wild-type FAN exhibited a 45% increase in cell death compared with HR control cells, with levels reaching 19.7±1.2%. This increase was not seen in untreated cells overexpressing FAN, indicating FAN may be playing a role in cell death in response to HR, but not basally. Moreover, HR-treated cells expressing DN-FAN exhibited 3.1±2.7% death, corresponding to a 5-fold decrease in cell death from empty vector HR-treated cells, indicating interruption of FAN signaling can reduce cell death induced by HR.
To determine whether a downstream effector of the nSMase pathway could overcome the inhibitory effects of DN-FAN on cell death, FAN or DN-FAN expressing cells were treated with cell-permeable C2-ceramide and analyzed by TUNEL. Treatment resulted in 19.4±4.5% cell death, a 7.3-fold increase over nontreated cells (Figure 3C). This level of cell death was not significantly affected by expression of FAN-V5 or DN-FAN-V5, which resulted in 28.5±0.1% and 20.1±6.5% cell death, respectively. Together, these data indicate that ceramide acts downstream of FAN and confirms that DN-FAN inhibits cell death by blocking activation of nSMase.
The presented data suggest FAN may be a critical gatekeeper in the sphingolipid pathway, leading to cell death in the cardiomyocyte. Results implying nSMase involvement in the heart’s response to IR are consistent with previous work demonstrating a central role of this signaling cascade in IR injury.2–4 The novel discovery that FAN may be playing a crucial role in the molecular mechanism of HR-induced cell death suggests TNFR1 or another receptor may be involved in the FAN/nSMase signaling system in the heart. TNFα is released by cardiomyocytes during hypoxia4 and may play a central role in heart failure.13 However, although TNF-α can induce cardiomyocyte apoptosis,14 it can also be protective.15 Additional studies are needed to evaluate whether the role of FAN in HR-induced cell death is through TNFR1 or a novel mechanism. Furthermore, identifying the specific nSMase isoform activated by FAN during IR signaling will be crucial to understanding the molecular pathways underlying IR injury.
This work was supported by grants from NIH-HLBI63975 and NIH-NS-HLBI-25037 (to C.C.G.) and Medlyte, Inc (to R.A.S.).
- Received January 7, 2003.
- Revision received February 11, 2003.
- Accepted March 3, 2003.
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