SNAREs are expressed in cardiac myocytes
We first characterized the expression of SNARE proteins in neonatal cardiac myocytes. Neonatal cardiac myocytes were selected for this study for two reasons: (1) neonatal rat hearts express equal levels of ANP in both atria and ventricles between 0–3 d after birth [29
]; (2) adult cardiac myocytes are difficult to transfect and culture for more than 48 h. Accordingly, cardiac myocytes were harvested from 1–2 d old neonatal rats, plated for 48 h, then lysed, and cell lysates were fractionated by PAGE and analyzed by immunoblotting. Mouse brain was used as a positive control. SNAP-23, Syntaxin-4, VAMP-1, VAMP-2 and VAMP-3 are expressed in cardiac myocytes (). However, other SNAREs such as SNAP-25 and syntaxin-1 are absent from myocytes ().
Cardiac myocytes express SNAREs and other proteins involved in vesicle trafficking
We found that other proteins involved in vesicle trafficking are also expressed in myocytes. Cardiac myocytes express NSF and its co-factor α-SNAP, the calcium sensor synaptotagmin, and the GTPases Rab8 and Rab4 ().
ANP is expressed in isolated neonatal cardiac myocytes
Neonatal myocytes were analyzed for ANP expression by Western blotting analysis and by immunostaining where ANP appears in a granular pattern (). To validate the use of neonatal cardiac myocytes as an experimental model, we also compared the expression of ANP and SNAREs between adult atria and adult ventricles. The same amount of proteins were loaded for atria and ventricle. Only atria express ANP (). Atria express more VAMP-1 and VAMP-2 than ventricles; both atria and ventricles express roughly equivalent levels of syntaxin-4 ().
ANP is expressed in primary cardiac myocytes in a perinuclear granular pattern
Co-localization of ANP and SNAREs
To analyze the subcellular distribution of ANP and SNARE proteins, we performed ultracentrifugation. Cardiac myocyte lysates were fractionated by ultracentrifugation into three main fractions: 1) granules (Gran), 2) plasma membrane (PM), 3) cytoplasm (Cyto). Fractions were collected and immunoblotted.
ANP is found in the granular compartment, but not in the plasma membrane or cytoplasm (). VAMP-1 and VAMP-2 are also found entirely in the granular fraction. In addition, syntaxin-4 is present in part in the granular fraction with ANP and in part in the plasma membrane fraction. None of these SNAREs are found in the cytoplasm. Cadherin and 14-3-3 proteins were used as markers of plasma membrane and cytosol fractions respectively.
Co-localization of ANP and SNAREs
We also performed confocal immunocytochemistry to visualize the distribution of ANP and SNARE proteins (). ANP is distributed in cardiac myocytes in a punctate perinuclear pattern, matching prior reports.[26
]. VAMP-1 partially overlaps with ANP, with a Pearson’s correlation coefficient of 0.52 ± 0.07 (, top). VAMP-2 also co-localizes with ANP, with a Pearson’s coefficient is 0.81 ± 0.04 (, second row). The presence and localization of VAMP-3 was also examined. VAMP-3 appears in a punctuate fashion around the nuclei and in the cytoplasm. Only a small portion of VAMP-3 localizes with ANP with a Pearson’s coefficient of 0.14 ±0.05 (, third row).
SNAP-23 is also expressed in cardiac myocytes and localizes in the periphery of cells with the plasma membrane. SNAP-23 is also visible also within the cardiac myocyte associated with linear structures of the cells ( fourth row). Although SNAP-23 is a plasma membrane protein, it has also been shown to associate with the cytoskeleton in adipocytes [32
]. Syntaxin-4 is distributed mainly at the periphery of the cardiac myocyte to the plasma membrane with some presence in the perinuclear region; the majority of syntaxin-4 does not overlap with ANP, with a Pearson’s correlation coefficient of 0.35 ± 0.01 (, bottom).
Taken together these sedimentation and imaging data suggest that ANP co-localizes with a subset of SNARE molecules.
SNARE interactions in cardiac myocytes
SNARE molecules interact to form SNARE complexes composed of three SNAREs necessary for membrane fusion. We hypothesized that syntaxin-4, VAMP-1, and VAMP-2 form a ternary SNARE complex inside cardiac myocytes. To test this idea, we immunoprecipitated syntaxin-4, and then probed precipitants for VAMP-1 and VAMP-2. Our initial studies showed that syntaxin-4 does not interact with VAMP-1 and VAMP-2 (, lane B). Although SNARE molecules can assemble into a complex, the protein N-ethylmaleimide sensitive factor (NSF) can associate with SNAREs and rapidly disassemble the complex into separate SNARE components. We therefore treated cardiac myocytes with N-ethylmaleimide to block NSF, and then searched again for an interaction between SNAREs. After NSF inhibition, syntaxin-4 forms a complex containing both VAMP-1 and VAMP-2 (, lanes C and D). Thus three specific SNAREs form a complex inside cardiac myocytes.
Endothelin Induces ANP Release
We confirmed that endothelin activates cardiac myocyte exocytosis of ANP in vitro
] We added increasing concentrations of endothelin-1 (ET-1) for 1 h to cardiac myocytes and measured ANP release by an ELISA. ET-1 induces ANP release in a dose-dependent manner (). Cardiac myocytes continue to release ANP after exposure to ET-1 over 60 min ().
Endothelin-1 induces ANP release
SNAREs Regulate ANP Release
Syntaxin-4 is expressed mostly on the plasma membrane of cardiac myocytes, as well as the perinuclear region (). In other cells, syntaxin-4 interacts with vesicle associated SNARE proteins, leading to exocytosis of the granules. We used RNA silencing to assess the role of syntaxin-4 in ANP exocytosis. We transfected cardiac myocytes with oligonucleotides directed against syntaxin-4. In cardiac myocytes, increasing amounts of siRNA against syntaxin-4 decreases syntaxin-4 expression without affecting ANP intracellular expression (). We then stimulated these transfected cardiac myocytes with ET-1. Silencing of syntaxin-4 decreases ANP release in a dose-dependent manner (). These data support the idea that syntaxin-4 regulates ANP release from cardiac myocytes.
Syntaxin-4 regulates ANP release
In other cell types, VAMP-1 or VAMP-2 or VAMP-3 is present on the surface of granules and interact with specific SNARE proteins on the plasma membrane, such as syntaxin-4, leading to membrane fusion. Since we found that VAMP-1, VAMP-2 and VAMP-3 are expressed in cardiac myocytes, we next explored the role of these proteins in the release of ANP from cardiac myocytes. We transfected cardiac myocytes with a control scrambled oligonucleotide or with an oligonucleotide directed against VAMP-1 (). RNA silencing decreases VAMP-1 expression only in the cells transfected with the specific siRNA and does not affect the expression of VAMP-2 nor the expression of intracellular content of ANP (). We then stimulated these transfected cells with ET-1. Silencing of VAMP1 decreases stimulated ANP release (). These data suggest that VAMP1 mediates cardiac myocytes exocytosis.
VAMP-1 and VAMP-2 regulate ANP release
The same approach was used to study the role of VAMP-2 and VAMP-3 in ANP release from stimulated cardiac myocytes. Transfection of cardiac myocytes with siRNA directed against VAMP-2 decreased VAMP-2 expression but not VAMP-1 or ANP expression (), and control siRNA did not have any effect on expression of these proteins. Cells were then stimulated with ET-1 and ANP release in the extracellular medium assessed by ELISA. Knock-down of VAMP-2 in cardiac myocytes resulted in reduced ANP release from stimulated cells to basal level ().
In contrast to VAMP-1 and VAMP-2, the SNARE protein VAMP-3 does not play any role in stimulated exocytosis of ANP in cardiac myocytes (). Instead, silencing of VAMP-3 did not change the levels of ANP released from cardiac myocytes after stimulation with ET-1.